# Patent

## Title
Polynucleotide encoding a novel TRP channel family member, LTRPC3, and splice variants thereof

## Abstract

The present invention provides novel polynucleotides encoding LTRPC3 polypeptides, fragments and homologues thereof. The present invention also provides polynucleotides encoding variants and splice variants of LTRPC3 polypeptides, LTRPC3b, LTRPC3c, LTRPC3d, LTRPC3e, and LTRPC3f, respectively. Also provided are vectors, host cells, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel LTRPC3, LTRPC3b, LTRPC3c, LTRPC3d, LTRPC3e, and LTRPC3f polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

## Background
This application is a continuation-in-part application of non-provisional application U.S. Ser. No. 10/210,152, filed Aug. 1, 2002 now abandoned, which claims benefit to provisional application U.S. Ser. No. 60/309,544 filed Aug. 2, 2001, under 35 U.S.C. 119(e).


FIELD OF THE INVENTION
The present invention provides novel polynucleotides encoding LTRPC3 polypeptides, fragments and homologues thereof. The present invention also provides polynucleotides encoding variants and splice variants of LTRPC3 polypeptides, LTRPC3b, LTRPC3c, and LTRPC3e, respectively. Also provided are vectors, host cells, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel LTRPC3, LTRPC3b, LTRPC3c, and LTRPC3e polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.
BACKGROUND OF THE INVENTION
Intracellular Ca2+ plays a pivotal role in various cell functions, ranging from exocytosis and contraction to gene expression and cell differentiation, proliferation and apoptosis. Ca2+ entry into cells, particularly in non-excitable cells, can be mediated via store-operated Ca2+ channel (s) (SOC). Following Ca2+ release from the intracellular stores, SOC mediate Ca2+ influx from the extracellular space to generate sustained increases in intracellular Ca2+ concentration and replenish the internal Ca2+ stores. The molecular mechanism of SOC activation and the molecular identity of SOC remains elusive. Members of TRP (Transient Receptor Potential) channels, an emerging class of Ca2+-permeable cation channel superfamily, are likely candidates for SOC (reviewed in Trends Neurosci, 23, 159-166, (2000)).
Human mutations in the genes involved in intracellular Ca2+ handling result in visual defects, diabetes mellitus, disorders in the skin, skeletal-muscle, nervous, cardiac and vascular systems (reviewed by Missiaen et al., 2000). In addition to the well characterized voltage-dependent Ca2+ channels, Ca2+ pumps and Ca2+-permeable ligand-gated channels, TRPC (Transient Receptor Potential Channels) is an emerging class of Ca2+-permeable cation channel superfamily. All of the channels in this family contain a six-trans-membrane domain although various cellular mechanisms have been implicated in their functions.
Following the identification of the founding member of this family, dTRP, from the Drosophila mutants trp whose photoreceptors failed to generate a sustained receptor potential in response to intense sustained light (Neuron 8, 643-651, (1992)), mammalian homologues have been cloned and all of them contain a six-trans-membrane domain followed by a TRP motif (XWKFXR, SEQ ID NO:), the diagnosed feature of the TRP family of proteins. The mutant fly showed a reduced Ca2+ selectivity of the light response and the channel activity of DTRP depended on PLC activation was also demonstrated.
Based on their homology, they are divided into three subfamilies: short (s), osm (o) and long (l). New nomenclature for each subfamily has recently been proposed and is as follows: TRPC (canonical), TRPV (vanilloid), and TRPM (melastatin) (Mol. Cell. 9, 229-231, (2002)). The sTRPC subfamily includes TRP1-7. Although the specific physiological function of each isoform remains to be assigned, it is generally believed that they may be involved in Ca2+ entry after activation of receptors coupling to PLC. The TRP2 is specifically expressed in vomeronasal organ and involved in pheromone sensory signaling (Liman, et al., 1999). TRP1 and TRP6 are functioned in vascular smooth muscle cells and may play a role in controlling smooth muscle tone, arteriosclerosis and neointimal hypoerplasia (Inoue et al., 2001; Xu & Beech, 2001). It has been shown that TRP4−/− mice lack an endothelial store-operated Ca2+ current, which leads to reduced agonist-dependent vasorelaxation (Freichel et al., 2001).
The first member of oTRPC Subfamily is OSM-9 cloned from C. elegans. It is involved in responses to odorants, high osmotic strength, and mechanical stimulation. Recently, several mammalian homologues including vanilloid receptor (VR1) and vanilloid receptor-like receptor (VRL-1), which may have functions in pain and heat perception (Caterina, 1999; Caterina et al., 2000). VR1 has also been shown to be the receptor of anandamide and mediating its vasodilation effect (Zygmunt et al., 1999). OTRPC4 is an osmotically activated channel and a candidate osmoreceptor, may be involved in regulation of cellular volume (Strotmann et al., 2000). CaT1 & ECaC1 may be the calcium-release-activated calcium channel and involved in Ca2+ reabsorption in intestine and kidney (Peng, et al, 1999; Yu et al., 2001).
The function of the lTRPC is less clear. The cloned mammalian lTRPC includes melastatin1/MLSN1/LTRPC1, MTR1/LTRPC5, TRPC7/LTRPC2 and TRP-P8. It is known that melastatin 1 is down regulated in metastatic melanomas (Duncan et al., 1998) and MTR1 is associated with Beckwith-Wiedemann syndrome and a predisposition to neoplasias (Prawitt et al., 2000). TRPC7 is mapped to the chromosome region linked to bipolar affective disorder, nonsyndromic hereditary deafness, Knobloch syndrome and holosencephaly (Nagamine et al., 1998). TRP-P8 is a prostate-specific gene and up-regulated in prostate cancer and other malignancies (Tsavaler et al., 2001). A recently cloned TRP-PLIK/hSOC-2/hCRAC-1 exhibits a very interesting feature in that it is a bi-functional protein with kinase and ion channel activities (Runnels et al., 2001). Additionally, a very long TRPC homologue NOMPC was found in Drosophila and C. elegans. NOMPC was identified as a mechanosensitive channel that can detect sound, pressure or movement changes (Walker et al., 2000).
Members of the TRPM subfamily are characteristic of their unusually long cytoplasmic tails at both ends of the channel domain and some of the family members contain an enzyme domain at the C-terminal region. Despite their similarities of structure, TRPMs have been implicated in a variety of biological functions. TRPM1 is found to be down-regulated in metastatic melanomas (Cancer Res. 58, 1515-1520, (1998)). TRPM2 is a Ca2+-permeable channel that contains an ADP-ribose pyrophosphatase domain and can be activated by ADP-ribose, NAD (Nature 411, 595-599, (2001); and Science 293, 1327-1330, (2001)) and changes in redox status (Mol. Cell. 9, 163-173, (2002)). TRPM2 is mapped to the chromosome region linked to bipolar affective disorder, nonsyndromic hereditary deafness, Knobloch syndrome and holosencephaly (Genomics 54, 124-131, (1998)). Two splice variants of TRPM4 have been described. TRPM4a is predominantly a Ca2+ permeable channel (Proc. Natl. Acad. Sci. U.S.A. 98, 10692-10697, (2001); whereas TRPM4b conducts monovalent cations upon activation by changes in intracellular Ca2+ (Cell 109, 397-401, (2002)). TRPM5 is associated with Beckwith-Wiedemann syndrome and a predisposition to neoplasias (Mol. Genet. 9, 203-216, (2001)). TRPM7, another bi-functional protein, has kinase activity in additional to its ion channel activity. TRPM7 is regulated by Mg2+-ATP and/or PIP2, and required for cell viability (Science 291, 1043-1047, (2001); Nature 411, 690-695, (2001); and Nat. Cell Biol. 4, 329-36, (2002)). TRPM8 is up-regulated in prostate cancer and other malignancies (Cancer Res. 61, 3760-3769, (2001)). Recently, it has also been shown to be a receptor that senses cold stimuli (Nature 416, 52-58, (2002); and Cell 108, 705-715, (2002)).
Characterization of the LTRPC3 polypeptide of the present invention led to the determination that it is involved in the modulation of the FEN1 DNA base-excision repair/proliferation modulating protein, either directly or indirectly.
In mammalian cells, single-base lesions, such as uracil and abasic sites, appear to be repaired by at least two base excision repair (BER) subpathways: “single-nucleotide BER” requiring DNA synthesis of just one nucleotide and “long patch BER” requiring multi-nucleotide DNA synthesis. In single-nucleotide BER, DNA polymerase beta (beta-pol) accounts for both gap filling DNA synthesis and removal of the 5′-deoxyribose phosphate (dRP) of the abasic site, whereas the involvement of various DNA polymerases in long patch BER is less well understood.
Flap endonuclease 1 (Fen1) is a structure-specific metallonuclease that plays an essential function in DNA replication and DNA repair (Tom, S., Henricksen, L, A., Bambara, R, A, J. Biol, Chem., 275(14):10498-505, (2000)). It interacts like many other proteins involved in DNA metabolic events with proliferating cell nuclear antigen (PCNA), and its enzymatic activity is stimulated by PCNA in vitro by as much as 5 to 50 fold (Stucki, M., Jonsson, Z, O., Hubscher, U, J. Biol, Chem., 276(11):7843-9, (2001)). Recently, immunodepletion experiments in human lymphoid cell extracts have shown long-patch BER to be dependent upon FEN1 (Prasad, R., Dia, G, L., Bohr, V, A., Wilson, S, H, J. Biol, Chem., 275(6):4460-6, (2000)). In addition, FEN1 has also been shown to cooperate with beta-pol in long patch BER excision and is involved in determining the predominant excision product seen in cell extracts. The substrate for FEN1 is a flap formed by natural 5′-end displacement of the short intermediates of lagging strand replication. FEN1 binds to the 5′-end of the flap, tracks to the point of annealing at the base of the flap, and then cleaves the substrate (Tom, S., Henricksen, L, A., Bambara, R, A, J. Biol, Chem., 275(14):10498-505, (2000)).
The FEN1 is also referred to as Rad27. FEN1 plays a critical role in base-excision repair as evidenced by Saccharomyces cerevisiae FEN1 null mutants displaying an enhancement in recombination that increases as sequence length decreases (Negritto, M, C., Qiu, J., Ratay, D, O., Shen, B., Bailis, A, M, Mol, Cell, Biol., 21(7):2349-58, (2001)). The latter suggests that Rad27 preferentially restricts recombination between short sequences. Since wild-type alleles of both RAD27 and its human homologue FEN1 complement the elevated short-sequence recombination (SSR) phenotype of a rad27-null mutant, this function may be conserved from yeast to humans. Furthermore, mutant Rad27 and FEN-1 enzymes with partial flap endonuclease activity but without nick-specific exonuclease activity were shown to partially complement the SSR phenotype of the rad27-null mutant suggesting that the endonuclease activity of Rad27 (FEN-1) plays a role in limiting recombination between short sequences in eukaryotic cells. In addition, preliminary data from yeast suggests the FEN-1 deficiencies may result in genomic instability (Ma, X., Jin, Q., Forsti, A., Hemminki, K., Ku, R, Int, J. Cancer., 88(6):938-42, (2000)). More recently, FEN 1 null mutants results in the expansion of repetitive sequences (Henricksen, L, A., Tom, S., Liu, Y., Bambara, R, A, J. Biol, Chem., 275(22):16420-7, (2000)).
Aside from the role of FEN1 in base-excision repair, FEN1 has also been shown to play a significant role in modulating signal transduction in proliferating cells. This role is intricately associated with the role of FEN1 in DNA replication. Of particular significance is the observation that FEN1 is a nuclear antigen, that it is expressed by cycling cells, and that it co-localizes with PCNA and polymerase alpha during S phase. Fen1 expression is topologically regulated in vivo and is associated with proliferative populations (Warbrick, E., Coates, P, J., Hall, P, A, J. Pathol., 186(3):319-24, (1998)). Antibodies have been described by Warbrick et al. that specifically bind FEN1, the assays of which are hereby incorporated herein by reference.
In addition, experiments in S. cerevisiae using the novel immunosuppressant agent SR 31747 have shown that SR 31747 arrests cell proliferation by directly targeting sterol isomerase and that FEN 1 is required to mediate the proliferation arrest induced by ergosterol depletion (Silve, S., Leplatois, P., Josse, A., Dupuy, P, H., Lanau, C., Kaghad, M., Dhers, C., Picard, C., Rahier, A., Taton, M., Le, Fur, G., Caput, D., Ferrara, P., Loison, G, Mol, Cell, Biol., 16(6):2719-27, (1996)).
Using the above examples, it is clear the availability of a novel cloned transient receptor potential channel family provides an opportunity for adjunct or replacement therapy, and are useful for the identification of transient receptor potential channel agonists, or stimulators (which might stimulate and/or bias transient receptor potential channel function), as well as, in the identification of transient receptor potential channel inhibitors. All of which might be therapeutically useful under different circumstances.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of LTRPC3, LTRPC3b, LTRPC3c, and LTRPC3e polypeptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the LTRPC3, LTRPC3b, LTRPC3c, and LTRPC3e polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.
BRIEF SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the LTRPC3b protein having the amino acid sequence shown in FIGS. 2A-F (SEQ ID NO:4) or the amino acid sequence encoded by the cDNA clone, LTRPC3b (also referred to as AL358786, NT—008306, clone 86, TRPM3, and/or LTRPC6 splice variant).
The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the LTRPC3c protein having the amino acid sequence shown in FIGS. 3A-F (SEQ ID NO:6) or the amino acid sequence encoded by the cDNA clone, LTRPC3c (also referred to as AL358786, NT—008306, clone 86, TRPM3, and/or LTRPC6 splice variant).
The present invention provides isolated nucleic acid molecules, that comprise, or alternatively consist of, a polynucleotide encoding the LTRPC3e protein having the amino acid sequence shown in FIGS. 5A-F (SEQ ID NO:9) or the amino acid sequence encoded by the cDNA clone, LTRPC3e (also referred to as AL358786, NT—008306, clone 86, TRPM3, and/or LTRPC6 splice variant).
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells, in addition to their use in the production of LTRPC3, LTRPC3b, LTRPC3c, and LTRPC3e polynucleotides or polypeptides using recombinant techniques. Synthetic methods for producing the polypeptides and polynucleotides of the present invention are provided. Also provided are diagnostic methods for detecting diseases, disorders, and/or conditions related to the LTRPC3, LTRPC3b, LTRPC3c, and LTRPC3e polypeptides and polynucleotides, and therapeutic methods for treating such diseases, disorders, and/or conditions. The invention further relates to screening methods for identifying binding partners of the polypeptides.
The invention further provides an isolated LTRPC3b polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
The invention further provides an isolated LTRPC3c polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
The invention further provides an isolated LTRPC3e polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
The invention further relates to a polynucleotide encoding a polypeptide fragment of SEQ ID NO:4, 6, 7, 9, which is hybridizable to SEQ ID NO: 3, 5, and/or 8.
The invention further relates to a polynucleotide encoding a polypeptide domain of SEQ ID NO: 4, 6, 7, 9, which is hybridizable to SEQ ID NO: 3, 5, and/or 8.
The invention further relates to a polynucleotide encoding a polypeptide epitope of SEQ ID NO: 4, 6, 7, 9, and/or 10 or a polypeptide epitope encoded by the cDNA sequence included in the deposited clone, which is hybridizable to SEQ ID NO: 3, 5, and/or 8.
The invention further relates to a polynucleotide encoding a polypeptide of SEQ ID NO:4, 6, 7, 9, which is hybridizable to SEQ ID NO: 3, 5, and/or 8, having biological activity.
The invention further relates to a polynucleotide which is a variant of SEQ ID NO: 8.
The invention further relates to a polynucleotide which is an allelic variant of SEQ ID NO: 8.
The invention further relates to a polynucleotide which encodes a species homologue of the SEQ ID NO: 9.
The invention further relates to an isolated nucleic acid molecule of SEQ ID NO:8 wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NO: 9.
The invention further relates to an isolated nucleic acid molecule of SEQ ID NO:8, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO: 8.
The invention further relates to an isolated nucleic acid molecule of SEQ ID NO: 8, wherein the nucleotide sequence comprises sequential nucleotide deletions from either the C-terminus or the N-terminus.
The invention further relates to an isolated polypeptide comprising an amino acid sequence that comprises a polypeptide fragment of SEQ ID NO: 9
The invention further relates to a polypeptide fragment of SEQ ID NO: 9, having biological activity.
The invention further relates to a polypeptide domain of SEQ ID NO: 9.
The invention further relates to a full length protein of SEQ ID NO: 9.
The invention further relates to a variant of SEQ ID NO: 9.
The invention further relates to an allelic variant of SEQ ID NO: 9. The invention further relates to a species homologue of SEQ ID NO: 9.
The invention further relates to the isolated polypeptide of SEQ ID NO: 9, wherein the full length protein comprises sequential amino acid deletions from either the C-terminus or the N-terminus.
The invention further relates to an isolated antibody that binds specifically to the isolated polypeptide of SEQ ID NO: 9.



BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS
The file of this patent contains at least one Figure executed in color. Copies of this patent with color Figure(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.”
FIGS. 1A-F show the polynucleotide sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) of the novel human transient receptor potential channel member, LTRPC3, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence contains a sequence of 4665 nucleotides (SEQ ID NO:1), encoding a polypeptide of 1554 amino acids (SEQ ID NO:2). An analysis of the LTRPC3 polypeptide determined that it comprised the following features: six transmembrane domains (TM1 thru TM6) located from about amino acid 620 to about amino acid 637 (TM1), from about amino acid 717 to about amino acid 734 (TM2), from about amino acid 790 to about amino acid 803 (TM3), from about amino acid 817 to about amino acid 834 (TM4), from about amino acid 851 to about amino acid 868 (TM5), and/or from about amino acid 938 to about amino acid 958 (TM6) of SEQ ID NO:2 represented by double underlining; a predicted TRP domain (EWKFAR) located from about amino acid 973 to about amino acid 978 of SEQ ID NO:2 represented by light shading; a predicted ion transport signature domain located at about amino acid 748 to about amino acid 959 of SEQ ID NO:2 represented by dark shading; a predicted coiled-coil domain located at about amino acid 1063 to about amino acid 1117 of SEQ ID NO:2 represented by italics; and conserved cysteine residues located at amino acid 94, 209, 265, 419, 517, 582, 598, 821, 912, 929, 942, 1045, 1136, and 1402 of SEQ ID NO:2 represented in bold. Conservation of cysteines at key amino acid residues is indicative of conserved structural features, which may correlate with conservation of protein function and/or activity.
FIGS. 2A-F show the polynucleotide sequence (SEQ ID NO:3) and deduced amino acid sequence (SEQ ID NO:4) of the novel human transient receptor potential channel member splice variant, LTRPC3b, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence contains a sequence of 4701 nucleotides (SEQ ID NO:3), encoding a polypeptide of 1566 amino acids (SEQ ID NO:4). An analysis of the LTRPC3b polypeptide determined that it comprised the following features: six transmembrane domains (TM1 thru TM6) located from about amino acid 632 to about amino acid 649 (TM1), from about amino acid 729 to about amino acid 746 (TM2), from about amino acid 802 to about amino acid 815 (TM3), from about amino acid 829 to about amino acid 846 (TM4), from about amino acid 863 to about amino acid 880 (TM5), and/or from about amino acid 950 to about amino acid 970 (TM6) of SEQ ID NO:4 represented by double underlining; a predicted TRP domain (EWKFAR) located from about amino acid 985 to about amino acid 990 of SEQ ID NO:4 represented by light shading; a predicted ion transport signature domain located at about amino acid 760 to about amino acid 971 of SEQ ID NO:4 represented by dark shading; a predicted coiled-coil domain located at about amino acid 1075 to about amino acid 1129 of SEQ ID NO:4 represented by italics; and conserved cysteine residues located at amino acid 94, 209, 265, 431, 529, 594, 611, 833, 924, 941, 954, 1057, 1148, and 1114 of SEQ ID NO:4 represented in bold. Conservation of cysteines at key amino acid residues is indicative of conserved structural features, which may correlate with conservation of protein function and/or activity.
FIGS. 3A-F show the polynucleotide sequence (SEQ ID NO:5) and deduced amino acid sequence (SEQ ID NO:6) of the novel human transient receptor potential channel member splice variant, LTRPC3c, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence contains a sequence of 4701 nucleotides (SEQ ID NO:5), encoding a polypeptide of 1566 amino acids (SEQ ID NO:6). An analysis of the LTRPC3c polypeptide determined that it comprised the following features: six transmembrane domains (TM1 thru TM6) located from about amino acid 620 to about amino acid 637 (TM1), from about amino acid 717 to about amino acid 734 (TM2), from about amino acid 790 to about amino acid 803 (TM3), from about amino acid 817 to about amino acid 834 (TM4), from about amino acid 851 to about amino acid 868 (TM5), and/or from about amino acid 950 to about amino acid 970 (TM6) of SEQ ID NO:6 represented by double underlining; a predicted TRP domain (EWKFAR) located from about amino acid 985 to about amino acid 990 of SEQ ID NO:6 represented by light shading; a predicted ion transport signature domain located at about amino acid 760 to about amino acid 971 of SEQ ID NO:6 represented by dark shading; a predicted coiled-coil domain located at about amino acid 1075 to about amino acid 1129 of SEQ ID NO:6 represented by italics; and conserved cysteine residues located at amino acid 94, 210, 265, 419, 517, 582, 599. 821, 941, 1057, 1148, and 1414 of SEQ ID NO:6 represented in bold. Conservation of cysteines at key amino acid residues is indicative of conserved structural features, which may correlate with conservation of protein function and/or activity.
FIGS. 4A-F show the polynucleotide sequence (SEQ ID NO:9) and deduced amino acid sequence (SEQ ID NO:10) of the novel human transient receptor potential channel member splice variant, LTRPC3e, of the present invention. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence. The polynucleotide sequence contains a sequence of 4671 nucleotides (SEQ ID NO:9), encoding a polypeptide of 1556 amino acids (SEQ ID NO:10). An analysis of the LTRPC3e polypeptide determined that it comprised the following features: six transmembrane domains (TM1 thru TM6) located from about amino acid 622 to about amino acid 639 (TM1), from about amino acid 719 to about amino acid 736 (TM2), from about amino acid 792 to about amino acid 805 (TM3), from about amino acid 819 to about amino acid 836 (TM4), from about amino acid 853 to about amino acid 870 (TM5), and/or from about amino acid 940 to about amino acid 960 (TM6) of SEQ ID NO:10 represented by double underlining; a predicted TRP domain (EWKFAR) located from about amino acid 975 to about amino acid 980 of SEQ ID NO:10 represented by light shading; a predicted ion transport signature domain located at about amino acid 750 to about amino acid 961 of SEQ ID NO:10 represented by dark shading; a predicted coiled-coil domain located at about amino acid 1065 to about amino acid 1119 of SEQ ID NO:10 represented by italics; and conserved cysteine residues located at amino acid 94, 209, 265, 431, 519, 584, 601, 823, 914, 931, 944, 1047, 1138, and 1404 of SEQ ID NO:10 represented in bold. Conservation of cysteines at key amino acid residues is indicative of conserved structural features, which may correlate with conservation of protein function and/or activity.
FIGS. 5A-D show the regions of identity and similarity between the LTRPC3 (SEQ ID NO:2), LTRPC3b (SEQ ID NO:4), LTRPC3c (SEQ ID NO:6), and LTRPC3e (SEQ ID NO:10) polypeptides of the present invention to another member of human transient receptor potential channel family, specifically, the human melastatin 1 protein (Melastatin1; Genbank Accession No. gi|3243075; SEQ ID NO:11). The alignment was created using the CLUSTALW algorithm described elsewhere herein using default parameters (CLUSTALW parameters: gap opening penalty: 10; gap extension penalty: 0.5; gap separation penalty range: 8; percent identity for alignment delay: 40%; and transition, weighting: 0). The darkly shaded amino acids represent regions of matching identity. The lightly shaded amino acids represent regions of matching similarity. Dots between residues indicate gapped regions for the aligned polypeptides.
FIG. 6 shows an expression profile of the novel human transient receptor potential channel family member, LTRPC3 (SEQ ID NO:2). The figure illustrates the relative expression level of LTRPC3 amongst various mRNA tissue sources. As shown, transcripts corresponding to LTRPC3 expressed predominately in kidney tissue. The LTRPC3 polypeptide was also expressed significantly in spinal cord, testis, and brain. Expression data was obtained by measuring the steady state LTRPC3 mRNA levels by RT-PCR using the PCR primer pair provided as SEQ ID NO: 375 and 376 as described herein.
FIG. 7 shows an expression profile of the novel human transient receptor potential channel family member, LTRPC3 (SEQ ID NO:2). The figure illustrates the relative expression level of LTRPC3 amongst various mRNA tissue, and cell sources. As shown, transcripts corresponding to LTRPC3 expressed predominately in kidney tissue. The LTRPC3 polypeptide was also expressed significantly in brain, and testis. Expression data was obtained by probing a Northern blot using a LTRPC3 645-bp PCR amplified fragment as described herein.
FIG. 8 shows a table illustrating the percent identity and percent similarity between the LTRPC3, LTRPC3b, LTRPC3c, and LTRPC3e polypeptides of the present invention with the human melastatin 1 protein (Melastatin1; Genbank Accession No. gi|3243075; SEQ ID NO:11). The percent identity and percent similarity values were determined based upon the GAP algorithm (GCG suite of programs; and Henikoff, S, and Henikoff, J. G., Proc. Natl. Acad. Sci. USA 89: 10915-10919 (1992)).
FIG. 9A-E shows the regions of identity between the LTRPC3 polypeptide (SEQ ID NO:2) of the present invention to its predicted splice variants LTRPC3b (SEQ ID NO:4), LTRPC3c (SEQ ID NO:6), LTRPC3d (SEQ ID NO:7), LTRPC3e (SEQ ID NO:9), and LTRPC3f (SEQ ID NO:10). The alignment was created using the CLUSTALW algorithm described elsewhere herein using default parameters (CLUSTALW parameters: gap opening penalty: 10; gap extension penalty: 0.5; gap separation penalty range: 8; percent identity for alignment delay: 40%; and transition, weighting: 0). The darkly shaded amino acids represent regions of matching identity. The lightly shaded amino acids represent regions of matching similarity. Dots between residues indicate gapped regions for the aligned polypeptides.
FIG. 10 shows three internal encoding polynucleotide sequences from the human bac AL358786 genomic sequence (Genbank Accession No. gi|AL358786; SEQ ID NOS:12, 13, and 14), in addition to, the human chromosome 9 genomic sequence (Genbank Accession No. gi|NT—008306; SEQ ID NO:15) used to design primers for cloning the LTRPC3, LTRPC3b, LTRPC3c, LTRPC3d, LTRPC3e, and LTRPC3f polynucleotides of the present invention as described herein. Both the BAC AL358786 and human chromosome 9 genomic sequence were predicted to encode portions of the LTRPC3 sequence as described herein. The initiating start codon of LTRPC3 within the human chromosome 9 genomic sequence is denoted in bold and underlined.
FIGS. 11A-C show the polypeptide sequences of several known transient potential receptors that were used to identify the LTRPC3 polypeptide of the present invention (SEQ ID NOS:16-25) via BLAST analysis against the genomic database, as described more specifically herein. The standard one-letter abbreviation for amino acids is used to illustrate the deduced amino acid sequence.
FIG. 12 shows an expanded expression profile of the novel human transient receptor potential channel member, LTRPC3. The figure illustrates the relative expression level of LTRPC3 amongst various mRNA tissue sources. As shown, the LTRPC3 polypeptide was expressed predominately in the brain, specifically the cerebellum, choroid plexus, the locus coeruleus, the posterior hypothalamus and the substantia nigra. Expression of LTRPC3 was also significantly expressed in the kidney, with higher levels observed in the cortex than in the medulla or pelvis. LTRPC3 was also significantly expressed in the spinal cord, testis, and to a lesser extent in other tissues as shown. Expression data was obtained by measuring the steady state LTRPC3 mRNA levels by quantitative PCR using the PCR primer pair provided as SEQ ID NO:270 and 271, and Taqman probe (SEQ ID NO:272) as described in Example 4 herein.
FIG. 13 shows an expanded expression profile of the novel human transient receptor potential channel member, LTRPC3. The figure illustrates the relative expression level of LTRPC3 amongst various mRNA tissue sources isolated from normal and tumor tissues. As shown, the LTRPC3 polypeptide was differentially expressed in renal, testicular, and ovarian cancers compared to each respective normal tissue. Expression data was obtained by measuring the steady state LTRPC3 mRNA levels by quantitative PCR using the PCR primer pair provided as SEQ ID NO:270 and 307, and Taqman probe (SEQ ID NO:272) as described in Example 4 herein.
FIGS. 14A-B show the polynucleotide sequence (SEQ ID NO:281) of the novel human transient receptor potential channel splice variant, LTRPC3d, of the present invention.
FIGS. 15A-B show the polynucleotide sequence (SEQ ID NO:282) of the novel human transient receptor potential channel splice variant, LTRPC3f, of the present invention.
FIG. 16A shows the genomic organization of the novel human transient receptor potential channel, LTRPC3, in addition to LTRPC3 splice variants b, c, d, e, and f, and a known partial sequence of the LTRPC3 gene, KIAA1616 (Genbank Accession No. gi|14743666; SEQ ID NO:283). Differences within the LTRPC3 splice variants b thru f are underlined. The coding exons are shown schematically as boxes, with the exon number indicated as numerals above each exon box. As shown, LTRPC3b contains an extra exon 11; LTRPC3c contains an additional 12 amino acids at exon 20, LTRPC3d does not contain exon 13; LTRPC3e contains an extra exon 11 and does not contain exon 13; and LTRPC3f contains an extra exon 4.
FIG. 16B shows a phylogenetic tree of the TRPM (melastatin) subfamily. As shown, LTRPC3 is most closely related to TRPM1. The phylogenetic tree was created using the CLUSTALW algorithm as provided in the Vector NTI AlignX program.
FIG. 17 shows a confocal-image of demonstrating that LTRPC3 is expressed as an integral membrane protein. The left top panel shows the image obtained with a Fluorescein filter; the left bottom panel shows the image obtained with a DAPI filter; the right top panel shows the image obtained using transmitted light; and the right bottom panel is the merged picture of the above three images. One of cells expressing LTRPC3 on the plasma membrane regions is indicated by an arrow. Forty-eight hours after transfection, LTRPC3 expressing HEK 293 cells were fixed with 4% paraformaldehyde, permeabilized with 1% Triton, and labeled with fluorescein conjugated anti-HA (3F10) 48 hours post transfection. The cell nuclei were stained with DAPI. Microscopy was performed as described in Example 6 herein.
FIG. 18 shows that LTRPC3 mediates concentration-dependent permeability to Ca2+. Transfected HEK 293 cells loaded with Fluo-4 were incubated in 1 mM Ca2+ solution (A), or a nominally Ca2+-free solution (B), and 0, 1, 3 and 10 mM Ca2+ solutions were added respectively to cells as indicated. The left panels represent vector-transfected control cells, and the right panels represent LTRPC3-transfected cells. Physiometric methods were performed as described in Example 7 herein.
FIG. 19 shows Ca2+ entry currents are induced upon store depletion in LTRPC3 expressing HEK 293 cells. 2 mM thapsigargin (A), or 50 μM carbachol (B) was first added to LTRPC3 expressing HEK 293 cells in the absence of external Ca2+. After 9 minutes, 2 mM Ca2+ solution was added to the cell medium at the indicated times. Currents for vector transfected HEK 293 cells, and LTRPC3 expressing HEK 293 cells are labeled accordingly. Panel (C) shows LTRPC3 mediated Ca2+ currents are inhibited by La3+ and Gd3+. Cells loaded with Fluo-4 were incubated in 1 mM Ca2+ solution and treated with 100 μM of La3+ (“w/ La3+”), Gd3+ (“w/ Gd3+”) or without blockers (“w/o blocker”) for 10 min prior to the addition of 10 mM Ca2+ to the cell medium as indicated. Curves represent averages of three independent experiments, each involving at least 8 wells per condition. Physiometric methods were performed as described in Example 7 herein.
FIG. 20 shows In situ Hybridization (ISH) results of LTRPC3. LTRPC3 mRNA expression was localized in the collecting tubular epithelium of human kidney medulla, medullary rays, and periglomerular regions, with the highest expression localized in medulary tubules. Bright field (i and iv) and dark field (ii and v) photomicrographs show the hybridization signal from antisense hTRMP3 riboprobe as aggregates of fine granules in the cytoplasm of the collecting tubular epithelial cells (arrows) in longitudinal (i and ii) and transverse (iv and v) sections. Low background signal is shown by the control LTRPC3 sense riboprobe in photographs iii and vi (the collecting tubules are indicated by arrowheads). Scale bar denotes 50 μm. ISH methods were performed as described in Example 8 herein.
FIG. 21 shows an expanded confocal-image demonstrating that LTRPC3 is expressed as an integral membrane protein. The left top panel (i) shows the image obtained with anti-HA staining (green) which is specific for expressed HA-tagged LTRPC3; the right top panel (ii) shows the image obtained with CM-DiI staining (red), a lipophilic cell membrane marker; the right bottom panel (iii) shows a merged image showing anti-HA, CM-DiI, and DAPI (nuclear stain, blue), with co-localization of LTRPC3 and CM-DiI appearing as yellow; and the left bottom panel (iv) showing the intensity profiles for anti-HA staining (green) and CM-DiI staining (red) along the path indicated by the white line in panel iii, showing overlap of anti-HA staining with CM-DiI staining in a quantitative manner. Scale bar denotes 10 μm. Microscopy was performed as described in Example 9 herein.
FIG. 22 shows that TRPM3-mediated Ca2+ entry can be inhibited by Gd3+. Panels A, C and E show results obtained for vector-transfected cells, while Panels B, D and F show results obtained for TRPM3-transfected cells. Panels A and B, show TRPM3-mediated concentration-dependent Ca2+ entry was partially inhibited by Gd3+. Cells loaded with Fluo-4 were incubated in 1 mM Ca2+ solution and treated with 100 μM of Gd3+ (red) or without blockers (black) for 10 min prior to the addition of 10 mM Ca2+ to the bath solution as indicated. Panels C-F, show that both Tg (Panels C and D) and CCh (Panels E and F) induced LTRPC3-mediated Ca2+ entry can be inhibited by Gd3+. Cells loaded with Fluo-4 were incubated in a nominally Ca2+-free media and treated without blockers (black) or with 100 μM of Gd3+ (red) for 10 min prior to the addition of 2 μM TG (Panels C and D, first arrow) or 50 μM CCh (Panels E and F, first arrow), Ca2+ entry was stimulated by the bath addition of 10 mM Ca2+ (second arrow). Representative traces from one of three independent experiments are shown; each trace is the mean of 12 wells per condition. Physiometric methods were performed as described in Example 10 herein.



Table I provides a summary of the novel polypeptides and their encoding polynucleotides of the present invention.
Table II illustrates the preferred hybridization conditions for the polynucleotides of the present invention. Other hybridization conditions may be known in the art or are described elsewhere herein.
Table III provides a summary of various conservative substitutions encompassed by the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included herein. All references to “LTRPC3” shall be construed to apply to “LTRPC3”, “LTRPC3b”, “LTRPC3c”, “LTRPC3d”, “LTRPC3e”, and/or “LTRPC3f” unless otherwise specified herein.
The invention provides a novel human sequence that potentially encodes a novel human transient receptor potential channel family member called LTRPC3, in addition to, its splice variants LTRPC3b, LTRPC3c, LTRPC3d, LTRPC3e, and LTRPC3f, which all belong to the lTRPC subfamily. LTRPC3 shares significant homologue with other transient receptor potential channel family members, such as human melatonin receptor 1. Transcripts for LTRPC3 were found predominately in kidney, spinal cord, testis, and brain suggesting that the invention potentially modulates leukocyte proliferation, differentiation, migration, and activation in these tissues. Therefore, the polynucleotide of the present invention has been tentatively named LTRPC3.
In the present invention, “isolated” refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term “isolated” does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.
In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
As used herein, a “polynucleotide” refers to a molecule having a nucleic acid sequence contained in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8. For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5′ and 3′ untranslated sequences, the coding region, with or without a signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a “polypeptide” refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.
In the present invention, the full length sequence identified as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11 was often generated by overlapping sequences contained in multiple clones (contig analysis). A representative clone containing all or most of the sequence for SEQ ID NO:1 was deposited with the American Type Culture Collection (“ATCC”). As shown in Table 1, each clone is identified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number. The ATCC is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for purposes of patent procedure. The deposited clone is inserted in the pBSII-SKΔSP-XB plasmid (Strategene) as described herein.
Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373, preferably a Model 3700, from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded bt the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
Using the information provided herein, such as the nucleotide sequence in FIGS. 1A-F (SEQ ID NO:1), a nucleic acid molecule of the present invention encoding the LTRPC3 polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in FIGS. 1A-F (SEQ ID NO:1) was discovered in a cDNA library derived from human kidney.
Using the information provided herein, such as the nucleotide sequence in FIGS. 2A-F (SEQ ID NO:3), a nucleic acid molecule of the present invention encoding the LTRPC3b polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in FIGS. 2A-F (SEQ ID NO:3) was discovered in a cDNA library derived from human kidney.
Using the information provided herein, such as the nucleotide sequence in FIGS. 3A-F (SEQ ID NO:5), a nucleic acid molecule of the present invention encoding the LTRPC3c polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in FIGS. 3A-F (SEQ ID NO:5) was discovered in a cDNA library derived from human kidney.
Using the information provided herein, such as the nucleotide sequence in FIGS. 5A-F (SEQ ID NO:9), a nucleic acid molecule of the present invention encoding the LTRPC3e polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in FIGS. 5A-F (SEQ ID NO:9) was discovered in a cDNA library derived human kidney.
A “polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, the complements thereof, to polynucleotide sequences encoding the sequences contained in SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:9, the complements thereof. “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65 degree C.
Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37 degree C. in a solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA; followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5×SSC).
Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of “polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).
The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, Proteins—Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)
As will be appreciated by the skilled practitioner, should the amino acid fragment comprise an antigenic epitope, for example, biological function per se need not be maintained. The terms LTRPC3, LTRPC3b, LTRPC3c, and/or LTRPC3e, polypeptide and LTRPC3, LTRPC3b, LTRPC3c, and/or LTRPC3e protein are used interchangeably herein to refer to the encoded product of the LTRPC3, LTRPC3b, LTRPC3c, and/or LTRPC3e nucleic acid sequence according to the present invention.
“SEQ ID NO:1”, “SEQ ID NO:3”, “SEQ ID NO:5”, and “SEQ ID NO:8”, refer to polynucleotide sequences, while “SEQ ID NO:2”, “SEQ ID NO:4”, “SEQ ID NO:6”, and “SEQ ID NO:9”, refer to polypeptide sequences, all sequences being identified by an integer specified in Table 1 herein.
“A polypeptide having biological activity” refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention).
The term “organism” as referred to herein is meant to encompass any organism referenced herein, though preferably to eukaryotic organisms, more preferably to mammals, and most preferably to humans.
As used herein the terms “modulate or modulates” refer to an increase or decrease in the amount, quality or effect of a particular activity, DNA, RNA, or protein.
It is another aspect of the present invention to provide modulators of the LTRPC3, LTRPC3b, LTRPC3c, and/or LTRPC3e protein and LTRPC3, LTRPC3b, LTRPC3c, and/or LTRPC3e peptide targets which can affect the function or activity of LTRPC3, LTRPC3b, LTRPC3c, and/or LTRPC3e in a cell in which LTRPC3, LTRPC3b, LTRPC3c, and/or LTRPC3e function or activity is to be modulated or affected. In addition, modulators of LTRPC3, LTRPC3b, LTRPC3c, and/or LTRPC3e can affect downstream systems and molecules that are regulated by, or which interact with, LTRPC3, LTRPC3b, LTRPC3c, and/or LTRPC3e in the cell. Modulators of LTRPC3, LTRPC3b, LTRPC3c, and/or LTRPC3e include compounds, materials, agents, drugs, and the like, that antagonize, inhibit, reduce, block, suppress, diminish, decrease, or eliminate LTRPC3, LTRPC3b, LTRPC3c, and/or LTRPC3e function and/or activity. Such compounds, materials, agents, drugs and the like can be collectively termed “antagonists”. Alternatively, modulators of LTRPC3, LTRPC3b, LTRPC3c, and/or LTRPC3e include compounds, materials, agents, drugs, and the like, that agonize, enhance, increase, augment, or amplify LTRPC3, LTRPC3b, LTRPC3c, and/or LTRPC3e function in a cell. Such compounds, materials, agents, drugs and the like can be collectively termed “agonists”.
The present invention encompasses the identification of proteins, nucleic acids, or other molecules, that bind to polypeptides and polynucleotides of the present invention (for example, in a receptor-ligand interaction). The polynucleotides of the present invention can also be used in interaction trap assays (such as, for example, that discribed by Ozenberger and Young (Mol. Endocrinol., 9(10):1321-9, (1995); and Ann. N.Y. Acad. Sci., 7; 766:279-81, (1995)).
The polynucleotide and polypeptides of the present invention are useful as probes for the identification and isolation of full-length cDNAs and/or genomic DNA which correspond to the polynucleotides of the present invention, as probes to hybridize and discover novel, related DNA sequences, as probes for positional cloning of this or a related sequence, as probe to “subtract-out” known sequences in the process of discovering other novel polynucleotides, as probes to quantify gene expression, and as probes for microarrays.
In addition, polynucleotides and polypeptides of the present invention may comprise one, two, three, four, five, six, seven, eight, or more membrane domains.
Also, in preferred embodiments the present invention provides methods for further refining the biological function of the polynucleotides and/or polypeptides of the present invention.
Specifically, the invention provides methods for using the polynucleotides and polypeptides of the invention to identify orthologs, homologs, paralogs, variants, and/or allelic variants of the invention. Also provided are methods of using the polynucleotides and polypeptides of the invention to identify the entire coding region of the invention, non-coding regions of the invention, regulatory sequences of the invention, and secreted, mature, pro-, prepro-, forms of the invention (as applicable).
In preferred embodiments, the invention provides methods for identifying the glycosylation sites inherent in the polynucleotides and polypeptides of the invention, and the subsequent alteration, deletion, and/or addition of said sites for a number of desirable characteristics which include, but are not limited to, augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.
The present invention further provides for other experimental methods and procedures currently available to derive functional assignments. These procedures include but are not limited to spotting of clones on arrays, micro-array technology, PCR based methods (e.g., quantitative PCR), anti-sense methodology, gene knockout experiments, and other procedures that could use sequence information from clones to build a primer or a hybrid partner.
Polynucleotides and Polypeptides of the Invention
Features of the Polypeptide Encoded by Gene No:1
The polypeptide of this gene provided as SEQ ID NO:2 (FIGS. 1A-F), encoded by the polynucleotide sequence according to SEQ ID NO:1 (FIGS. 1A-F), and/or encoded by the polynucleotide contained within the deposited clone, LTRPC3, has significant homology at the nucleotide and amino acid level to the human melastatin 1 protein (Melastatin1; Genbank Accession No. gi|3243075; SEQ ID NO:11). An alignment of the LTRPC3 polypeptide with this protein is provided in FIGS. 5A-D.
The LTRPC3 polypeptide was determined to share 65.7% identity and 73.5% similarity with the human melastatin 1 protein (Melastatin1; Genbank Accession No. gi|3243075; SEQ ID NO:11) as shown in FIG. 8.
The LTRPC3 protein is believed to represent a member of a new class of protein kinases referred to as alpha kinases (Curr. Biol. 9 (2), R43-R45 (1999)). These kinases represent a novel type of signaling molecule comprising both a catalytic protein kinase domain, in addition to, an ion channel domain. This family is commonly referred to as the transient receptor potential channel (TRP) family. Melastatin1 defines a separate subfamily of TRP channels referred to as TRPM (melastatin1). TRPM family members are characteristic of their unusually long cytoplasmic tails at both ends of the channel domain and some of the family members contain an enzyme domain at the C-terminal region. Thus, LTRPC3 represents a novel member of the TRPM subfamily.
The melastatin1 protein is believed to be negatively associated with the incidence of melanoma based upon its inverse correlative expression in highly aggressive melanomas (Genomics 54 (1), 116-123 (1998)). Thus, overexpression of melastatin1 could represent a novel therapeutic in the treatment of melanoma and potentially other cancers.
Based upon the observed homology, the polypeptide of the present invention is expected to share at least some biological activity with other transient receptor potential channel family members, more specifically with the melastatin1 protein, in addition to, other transient receptor potential channel family members referenced elsewhere herein or otherwise known in the art.
Most of the known transient receptor potential channel family members, possess one or more transmembrane domains. Likewise, the LTRPC3 polypeptide has been determined to comprise six transmembrane domains (TM1-TM6) as shown in FIGS. 1A-F. The transmembrane domains are located from about amino acid 740 to about amino acid 757 (TM1), from about amino acid 834 to about amino acid 851 (TM2), from about amino acid 908 to about amino acid 920 (TM3), from about amino acid 934 to about amino acid 951 (TM4), from about amino acid 968 to about amino acid 985 (TM5), and/or from about amino acid 1043 to about amino acid 1062 (TM6) of SEQ ID NO:2. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of the above referenced polypeptide.
In preferred embodiments, the following transmembrane domain polypeptides are encompassed by the present invention: LKVILGILLPPSILSLEF (SEQ ID NO:26), IVKFWFYTLAYIGYLMLF (SEQ ID NO:27), VTDLIAILLFSVGM (SEQ ID NO:28), RVIYCVNIIYWYIRLLDI (SEQ ID NO:29), MMIDMMYFVIIMLVVLMS (SEQ ID NO:30), and/or AIMACYLLVANILLVNLLIAV (SEQ ID NO:31). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the LTRPC3 transmembrane polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The present invention also encompasses polypeptides corresponding to the regions between each transmembrane domain of LTRPC3, referred to herein as inter-transmembrane domain polypeptides (inter TM1-2, inter TM2-3, inter TM3-4, inter TM4-5, and inter TM5-6). Such regions are typically solvent accessible (either extracellularly or intracellularly). Therefore, these regions are amenable to the development of agonists and/or antagonists to the LTRPC3 polypeptide, particularly for the development of antibodies specific to these regions.
In preferred embodiments, the following inter-transmembrane domain polypeptides are encompassed by the present invention: KNKDDMPYMSQAQEIHLQEKEAEEPEKPTKEKEEEDMELTAMLGRNNGESS RKKDEEEVQSKHRLIPLGRKIYEFYNAP (SEQ ID NO:50), NYIVLVKMERWPSTQEWIVISYIFTLGIEKMREILMSEPGKLLQKVKVWLQEY WN (SEQ ID NO:51), ILRLQDQPFRSDG (SEQ ID NO:52), FGVNKYLGPYVMMIGK (SEQ ID NO:53), and/or FGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFADQIDPPCGQNETRED GKIIQLPPCKTGAWIVP (SEQ ID NO:54). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the LTRPC3 inter-transmembrane polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3 inter TM1-2 domain deletion polypeptides are encompassed by the present invention: K1-P79, N2-P79, K3-P79, D4-P79, D5-P79, M6-P79, P7-P79, Y8-P79, M9-P79, S10-P79, Q11-P79, A12-P79, Q13-P79, E14-P79, I15-P79, H16-P79, L17-P79, Q18-P79, E19-P79, K20-P79, E21-P79, A22-P79, E23-P79, E24-P79, P25-P79, E26-P79, K27-P79, P28-P79, T29-P79, K30-P79, E31-P79, K32-P79, E33-P79, E34-P79, E35-P79, D36-P79, M37-P79, E38-P79, L39-P79, T40-P79, A41-P79, M42-P79, L43-P79, G44-P79, R45-P79, N46-P79, N47-P79, G48-P79, E49-P79, S50-P79, S51-P79, R52-P79, K53-P79, K54-P79, D55-P79, E56-P79, E57-P79, E58-P79, V59-P79, Q60-P79, S61-P79, K62-P79, H63-P79, R64-P79, L65-P79, I66-P79, P67-P79, L68-P79, G69-P79, R70-P79, K71-P79, I72-P79, and/or Y73-P79 of SEQ ID NO:50. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3 inter TM1-2 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3 inter TM1-2 domain deletion polypeptides are encompassed by the present invention: K1-P79, K1-A78, K1-N77, K1-Y76, K1-F75, K1-E74, K1-Y73, K1-I72, K1-K71, K1-R70, K1-G69, K1-L68, K1-P67, K1-I66, K1-L65, K1-R64, K1-H63, K1-K62, K1-S61, K1-Q60, K1-V59, K1-E58, K1-E57, K1-E56, K1-D55, K1-K54, K1-K53, K1-R52, K1-S51, K1-S50, K1-E49, K1-G48, K1-N47, K1-N46, K1-R45, K1-G44, K1-L43, K1-M42, K1-A41, K1-T40, K1-L39, K1-E38, K1-M37, K1-D36, K1-E35, K1-E34, K1-E33, K1-K32, K1-E31, K1-K30, K1-T29, K1-P28, K1-K27, K1-E26, K1-P25, K1-E24, K1-E23, K1-A22, K1-E21, K1-K20, K1-E19, K1-Q18, K1-L17, K1-H16, K1-I15, K1-E14, K1-Q13, K1-A12, K1-Q11, K1-S10, K1-M9, K1-Y8, and/or K1-P7 of SEQ ID NO:50. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3 inter TM1-2 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3 inter TM2-3 domain deletion polypeptides are encompassed by the present invention: F1-N56, N2-N56, Y3-N56, I4-N56, V5-N56, L6-N56, V7-N56, K8-N56, M9-N56, E10-N56, R11-N56, W12-N56, P13-N56, S14-N56, T15-N56, Q16-N56, E17-N56, W18-N56, I19-N56, V20-N56, I21-N56, S22-N56, Y23-N56, I24-N56, F25-N56, T26-N56, L27-N56, G28-N56, I29-N56, E30-N56, K31-N56, M32-N56, R33-N56, E34-N56, I35-N56, L36-N56, M37-N56, S38-N56, E39-N56, P40-N56, G41-N56, K42-N56, L43-N56, L44-N56, Q45-N56, K46-N56, V47-N56, K48-N56, V49-N56, and/or W50-N56 of SEQ ID NO:51. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3 inter TM2-3 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3 inter TM2-3 domain deletion polypeptides are encompassed by the present invention: F1-N56, F1-W55, F1-Y54, F1-E53, F1-Q52, F1-L51, F1-W50, F1-V49, F1-K48, F1-V47, F1-K46, F1-Q45, F1-L44, F1-L43, F1-K42, F1-G41, F1-P40, F1-E39, F1-S38, F1-M37, F1-L36, F1-I35, F1-E34, F1-R33, F1-M32, F1-K31, F1-E30, F1-I29, F1-G28, F1-L27, F1-T26, F1-F25, F1-I24, F1-Y23, F1-S22, F1-I21, F1-V20, F1-I19, F1-W18, F1-E17, F1-Q16, F1-T15, F1-S14, F1-P13, F1-W12, F1-R11, F1-E10, F1-M9, F1-K8, and/or F1-V7 of SEQ ID NO:51. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3 inter TM2-3 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3 inter TM3-4 domain deletion polypeptides are encompassed by the present invention: I1-G13, L2-G13, R3-G13, L4-G13, Q5-G13, D6-G13, and/or Q7-G13 of SEQ ID NO:52. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3 inter TM3-4 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3 inter TM3-4 domain deletion polypeptides are encompassed by the present invention: I1-G13, 1′-D12, I1-S11, I1-R10, I1-F9, I1-P8, and/or I1-Q7 of SEQ ID NO:52. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3 inter TM3-4 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3 inter TM4-5 domain deletion polypeptides are encompassed by the present invention: F1-K16, G2-K16, V3-K16, N4-K16, K5-K16, Y6-K16, L7-K16, G8-K16, P9-K16, and/or Y10-K16 of SEQ ID NO:53. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3 inter TM4-5 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3 inter TM4-5 domain deletion polypeptides are encompassed by the present invention: F1-K16, F1-G15, F1-I14, F1-M13, F1-M12, F1-V11, F1-Y10, F1-P9, F1-G8, and/or F1-L7 of SEQ ID NO:53. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3 inter TM4-5 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3 inter TM5-6 domain deletion polypeptides are encompassed by the present invention: F1-P69, G2-P69, V3-P69, A4-P69, R5-P69, Q6-P69, A7-P69, I8-P69, L9-P69, F10-P69, P11-P69, N12-P69, E13-P69, E14-P69, P15-P69, S16-P69, W17-P69, K18-P69, L19-P69, A20-P69, K21-P69, N22-P69, I23-P69, F24-P69, Y25-P69, M26-P69, P27-P69, Y28-P69, W29-P69, M30-P69, I31-P69, Y32-P69, G33-P69, E34-P69, V35-P69, F36-P69, A37-P69, D38-P69, Q39-P69, I40-P69, D41-P69, P42-P69, P43-P69, C44-P69, G45-P69, Q46-P69, N47-P69, E48-P69, T49-P69, R50-P69, E51-P69, D52-P69, G53-P69, K54-P69, I55-P69, I56-P69, Q57-P69, L58-P69, P59-P69, P60-P69, C61-P69, K62-P69, and/or T63-P69 of SEQ ID NO:54. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3 inter TM5-6 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3 inter TM5-6 domain deletion polypeptides are encompassed by the present invention: F1-P69, F1-V68, F1-I67, F1-W66, F1-A65, F1-G64, F1-T63, F1-K62, F1-C61, F1-P60, F1-P59, F1-L58, F1-Q57, F1-I56, F1-I55, F1-K54, F1-G53, F1-D52, F1-E51, F1-R50, F1-T49, F1-E48, F1-N47, F1-Q46, F1-G45, F1-C44, F1-P43, F1-P42, F1-D41, F1-I40, F1-Q39, F1-D38, F1-A37, F1-F36, F1-V35, F1-E34, F1-G33, F1-Y32, F1-I31, F1-M30, F1-W29, F1-Y28, F1-P27, F1-M26, F1-Y25, F1-F24, F1-I23, F1-N22, F1-K21, F1-A20, F1-L19, F1-K18, F1-W17, F1-S16, F1-P15, F1-E14, F1-E13, F1-N12, F1-P11, F1-F10, F1-L9, F1-I8, and/or F1-A7 of SEQ ID NO:54. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3 inter TM5-6 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The LTRPC3 polypeptide was determined to comprise several conserved cysteines, at amino acid 94, 209, 265, 419, 517, 582, 598, 821, 912, 929, 942, 1045, 1136, and 1402 of SEQ ID No: 2 (FIGS. 1A-F). Conservation of cysteines at key amino acid residues is indicative of conserved structural features, which may correlate with conservation of protein function and/or activity.
In confirmation of the LTRPC3 representing a member of the transient receptor channel family, the LTRPC3 polypeptide was determined to comprise a predicted TRP domain (EWKFAR) located from about amino acid 973 to about amino acid 978 of SEQ ID NO:2. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of the above referenced polypeptide.
In preferred embodiments, the following TRP domain polypeptide is encompassed by the present invention: VWKFQR (SEQ ID NO:55). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of this LTRPC3 TRP domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
In further confirmation of the LTRPC3 representing a member of the transient receptor channel family, the LTRPC3 polypeptide was determined to comprise a predicted ion transport signature domain located at about amino acid 748 to about amino acid 959 of SEQ ID NO:2. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of the above referenced polypeptide.
In preferred embodiments, the following ion transport signature domain polypeptide is encompassed by the present invention: TQEWIVISYIFTLGIEKMREILMSEPGKLLQKVKVWLQEYWNVTDLIAILLFSV GMILRLQDQPFRSDGRVIYCVNIIYWYIRLLDIFGVNKYLGPYVMMIGKMMID MMYFVIIMLVVLMSFGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFAD QIDPPCGQNETREDGKIIQLPPCKTGAWIVPAIMACYLLVANILLVNLLIAVF (SEQ ID NO:56). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of this LTRPC3 ion transport signature domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The LTRPC3 polypeptide was determined to comprise a predicted coiled-coil domain located at about amino acid 1063 to about amino acid 1117 of SEQ ID NO:2. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of the above referenced polypeptide.
In preferred embodiments, the following coiled-coil domain polypeptide is encompassed by the present invention: ERIRVTSERVENMSMRLEEVNEREHSMKASLQTVDIRLAQLEDLIGRMATAL ERLT (SEQ ID NO:57). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of this LTRPC3 coiled-coil domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
LTRPC3 polypeptides and polynucleotides are useful for diagnosing diseases related to the over and/or under expression of LTRPC3 by identifying mutations in the LTRPC3 gene using LTRPC3 sequences as probes or by determining LTRPC3 protein or mRNA expression levels. LTRPC3 polypeptides will be useful in screens for compounds that affect the activity of the protein. LTRPC3 peptides can also be used for the generation of specific antibodies and as bait in yeast two hybrid screens to find proteins the specifically interact with LTRPC3.
Consistent with LTRPC3 representing a transient receptor potential channel, immunohistochemical experiments have shown that LTRPC3 localizes to the cell membrane (see FIG. 17 and Example 6). Specifically, the complete open reading frame of LTRPC3 with a C-terminal HA tag was transiently transfected into HEK 293 cells to assess its cellular localization. The HA-tagged LTRPC3 was detected using a fluorescein-conjugated anti-HA antibody and a laser scanning confocal microscope which produces a green fluorescent signal. The green fluorescent signal was exclusively detected at the subplasma membrane region of the transfected cells (see FIG. 17), which is consistent with LTRPC3 being an integral membrane protein. Under these conditions approximately  ̃70% of cells are expressing LTRPC3. The expression of full-length protein was accessed with immunoblot using an anti-HA antibody and detected as the expected size of  ̃170 kD (data not shown).
Additionally, anti-HA staining was found to be associated with the membrane marker CM-DiI, indicating LTRPC3 protein in or near the plasmalemmal compartment of transfected cells (see FIG. 21). Plasmalemmal localization is consistent with the function of the TRP family as Ca2+-permeable cation channels. LTRPC3 was also observed in intracellular compartments, possibly resulting from overexpression in this heterologous expression system, as observed with other ion channels (Marshall, J., et al., (1995) Neuron 14, 211-215.).
Moreover, physiometric studies have shown that LTRPC3 is a functional Ca2+ permeable channel (see FIGS. 18 and 19, and Example 7). LTRPC3 function was assessed using a Fluorometric Imaging Plate Reader (FLIPRTM) that measures real-time intracellular fluorescence changes. Cells transiently transfected with vector or LTRPC3-HA were loaded with the cytoplasmic Ca2+ indicator Fluoro-4 in a 1 mM Ca2+ solution. Addition of Ca2+ to the media resulted in a concentration-dependent influx of Ca2+ into LTRPC3-expressing cells (FIG. 18; right panels), indicating that LTRPC3 is a functional Ca2+ channel. In contrast, vector-transfected cells showed minimal Ca2+ influx under the same experimental conditions (FIG. 18, left panels). The non-transfected cells were indistinguishable from the vector-transfected cells (data not shown). Therefore, LTRPC3 is a constitutively active channel capable of mediating Ca2+ influx.
To further address the mechanism of LTRPC3-mediated Ca2+ entry, similar Ca2+ addition experiments were performed on transfected cells incubated ( ̃30 min) in a nominally Ca2+-free solution. Previous studies have shown that lowering extracellular Ca2+ concentration below physiological levels can deplete intracellular Ca2+ stores in many cell types including HEK 293 (EMBO J. 17, 4274-4282, (1998)). Incubating vector-transfected HEK 293 cells in a nominally Ca2+-free solution gave rise to Ca2+ entry that was dependent on the concentration of Ca2+ added to the buffers, indicating Ca2+ influx was mediated through endogenous SOCs in HEK293 cells (FIG. 18, left panels). In LTRPC3 cells, the Ca2+ transients triggered by similar Ca2+ treatment were much larger (FIG. 18, right panels). This Ca2+ entry observed in LTRPC3 cells incubated in Ca2+-free media were greater than those observed in 1 mM Ca2+ media, indicating that LTRPC3-mediated Ca2+ entry can be potentiated by the store-depletion.
The store-operated mechanism of LTRPC3-mediated Ca2+ influx was tested further by passively depleting Ca2+ stores with thapsigargin (TG), an inhibitor of microsomal Ca2+ ATPases that pumps ions from the cytosol back into the stores. Addition of 2 μM thapsigargin equivalently depleted Ca2+ stores in LTRPC3-HA- and vector-transfected cells (FIG. 19A). Following store depletion with TG, addition of Ca2+ to the buffer induced a much larger Ca2+ entry in LTRPC3 cells compared to the vector control cells.
Receptor-mediated Ca2+ entry was also more pronounced in LTRPC3-HA-transfected cells. Carbachol (CCh) can activate an endogenous muscarinic receptor and trigger IP3 production, leading to store-depletion in HEK 293 cells. The addition of 50 μM of CCh caused a transient and rapid intracellular Ca2+ increase in both LTRPC3- and vector-transfected cells (FIG. 19B). After the store depletion with CCh, adding of Ca2+ to the buffer induced a much larger influx of Ca2+ into LTRPC3 cells, as compared to vector control cells. These results show that after store depletion with TG or CCh LTRPC3-transfected cells exhibit an increased Ca2+ influx when compared to control cells.
The lanthanides, gadolinium (Gd3+) and lanthanum (La3+), are noneselective Ca2+-permeable channel blockers, often used as part of the characterization of overexpressed TRP channels. Both lanthanides blocked LTRPC3 Ca2+ conductance, although La3+ was more potent (FIG. 19C). In the presence of 1 mM Ca2+ in which endogenous SOCs is minimally activated (FIG. 18A), pre-treatment with 100 μM of La3+ and Gd3+ blocked LTRPC3 Ca2+ currents, stimulated by adding 10 mM Ca2+, by 67 and 39%, respectively. These results indicated that LTRPC3 mediated currents are not non-specific leak currents resulting from protein overexpression.
Additional experiments were performed to further confirm the Ca2+ store-depleted activation of LTRPC3 using Gd3+. The effects of 100 μM Gd3+ on Ca2+ permeability were tested in vector- and LTRPC3-transfected cells. The minimal Ca2+ influx observed upon addition of 10 mM Ca2+ to the cells (cells were incubated in the presence of 1 mM Ca2+) in vector-transfected cells (see FIG. 22A) was strongly inhibited by 100 μM Gd3+. In contrast, 100 μM Gd3+ inhibited Ca2+ entry, induced by adding 10 mM Ca2+, by 53% in LTRPC3-transfected cells (see FIG. 22B). Gd3+ reduced fluorescence units in vector-transfected cells from 1470±140 to −58±8 and in LTRPC3-transfected cells from 6000±322 to 2080±199 (n=12). Fluorescence values were measured 150 seconds after adding 10 mM Ca2+ and percent blockade was calculated as 1 minus FLTRPC3−FVector in the presence of Gd3+ divided by FLTRPC3−FVector without blocker. The effects of Gd3+ on LTRPC3-mediated Ca2+ entry, induced by 10 mM Ca2+, in the presence of thapsigargin or carbachol was also examined. Cells were incubated in nominally Ca2+-free medium for thapsigargin and carbachol experiments. Gd3+ inhibited Ca2+ entry by 51% after depletion of intracellular stores with thapsigargin (see FIG. 22B). Gd3+ reduced peak fluorescence after 10 mM Ca2+ addition in vector-transfected cells from 26444±2410 to 1316±60 and in LTRPC3-transfected cells from 37676±2425 to 6783±250 (see FIGS. 22C and D, respectively; n=12). Gd3+ inhibited Ca2+ entry by 72% after depletion of intracellular stores with carbachol. Gd3+ reduced peak Ca2+ fluorescence in vector-transfected cells from 9327±466 to 453±15 and in LTRPC3-transfected cells from 14747±988 to 1975±79 (see FIGS. 22E and F, respectively; n=12). These results show that, under identical conditions, the endogenous Ca2+ entry pathway was strongly blocked by application of 100 μM Gd3+ whereas the LTRPC3-mediated pathway was partially blocked (53%, see FIGS. 22A and B). Stimulation of Ca2+ entry in LTRPC3-transfected cells in the presence of thapsigargin or carbachol was also partially blocked by 100 μM Gd3+. These results are consistent with the hypothesis that LTRPC3 mediates a Ca2+ entry pathway that apparently is distinct from the endogenous Ca2+ entry pathways present in HEK 293 cells.
LTRPC3 is constitutively active but can be potentiated by store-depletion and is partially sensitive to La3+ and Gd3+ blockade. LTRPC3 is believed to represent the first member of the TRPM subfamily that exhibits this store-operated mechanism, although some members of TRPC subfamily have been considered for this role. TRPM1 and TRPM4a are constitutive Ca2+ permeable channels but it is unclear whether they can be stimulated by store-depletion (Proc. Natl. Acad. Sci. U.S.A. 98, 10692-10697, (2001)). Distinct from TRPM4a, TRPM4b is directly activated by changes in intracellular Ca2+ without significant permeation of Ca2+ (Cell 109, 397-401, (2002)). TRPM2 is activated by ADP-ribose, NAD and changes in redox status (Nature 411, 595-599, (2001); Science 293, 1327-1330, (2001); and Mol. Cell. 9, 163-173, (2002)). TRPM7 is regulated by Mg2+-ATP and/or PIP2 (Science 291, 1043-1047, (2001); Nature 411, 690-695, (2001); and Nat. Cell Biol. 4, 329-36 (2002)). TRPM8 is activated by cold temperatures and cooling agents (Nature 416, 52-58, (2002); and Cell 108, 705-715, (2002)). Therefore, in conjunction with its fairly restricted tissue expression, which is not observed with any other family members, LTRPC3 may have a unique biological function in human.
Expression profiling designed to measure the steady state mRNA levels encoding the LTRPC3 polypeptide showed predominately high expression levels in kidney. The LTRPC3 polypeptide was also significantly expression in spinal cord, testis, and brain (as shown in FIG. 6).
Moreover, Northern hybridizations of the LTRPC3 mRNA confirmed the predominately high expression levels in kidney, and significant expression levels in testis, and brain (as shown in FIG. 7). The Northern hybridization was not performed on spinal cord tissue.
Expanded analysis of LTRPC3 expression levels by TaqManTM quantitative PCR (see FIG. 12) confirmed that the LTRPC3 polypeptide is expressed in kidney, brain, testis (FIGS. 6 and 7), although higher expression levels were observed in brain than previously appreciated. LTRPC3 mRNA was expressed predominately in the brain, specifically the cerebellum, choroid plexus, the locus coeruleus, the posterior hypothalamus and the substantia nigra. Expression of LTRPC3 was also significantly expressed in the kidney, with higher levels observed in the cortex than in the medulla or pelvis. LTRPC3 was also significantly expressed in the spinal cord, testis, and to a lesser extent in other tissues as shown.
Therefore, LTRPC3 polynucleotides and polypeptides, including modulators or fragments thereof, may be useful in treating, diagnosing, prognosing, and/or preventing a variety of neurological conditions, in particular various choroid plexus neoplasms, choroid plexus papilloma, Alzheimer's disease, prion disorders and multiple sclerosis and movement disorders that involve the cerebellum. Based upon the expression pattern of LTRPC3 in kidney, this novel TRP family member may also be the cause solitary metastasis in the choroid plexus, a rare type of carcinoma. For example, it has been shown that out of 15 cases of solitary metastasis of the choroid plexus, five originated from renal cell carcinoma (Neurol. Med. Chir. (Tokyo) 1997 December; 37(12):916-9). Additionally, given the rather selective expression of LTRPC3 in the choroid plexus and renal tissues, it may be possible that altered function of LTRPC3 may be responsible for solitary metastasis and renal carcinoma. LTRPC3 polynucleotides and polypeptides, including modulators or fragments thereof, may be useful in treating, diagnosing, prognosing, and/or preventing a variety of renal disorders, particularly solitary metastasis in the choroid plexus, and renal carcinoma.
Moreover, an additional analysis of LTRPC3 expression levels by TaqManTM quantitative PCR (see FIG. 13) in disease cells and tissues indicated that the LTRPC3 polypeptide is differentially expressed in renal, testicular, and ovarian tumor tissues. In the renal tumor tissue results, an average of 2 out of 3 matched samples, which represent 3 out of 4 samples total, showed a significant decrease in LTRPC3 steady state RNA levels in tumor compared to control samples. In the testicular tumor tissue results, differential expression of LTRPC3 in testicular cancers was observed with all 5 tumor samples showing a significant reduction in steady-state RNA levels compared to two control samples. In the ovarian tumor tissue results, differential expression of LTRPC3 in ovarian cancers was observed with 3 tumor samples showing a significant reduction in steady-state RNA levels compared to five control samples.
The differential expression of LTRPC3 in tumors relative to normal tissues suggests that loss of LTRPC3 expression during tumor progression might contribute to the metastatic process by altering internal calcium stores in a manner that reflects a loss of cellular control on apoptosis. Restoring LTRPC3 function might provide a novel therapeutic approach to treating certain cancers. Therefore, LTRPC3 polynucleotides and polypeptides, including modulators or fragments thereof, particularly agonists of LTRPC3 activity or expression, may be useful in treating, diagnosing, prognosing, ameloriating, and/or preventing a variety of cancers and proliferative conditions, particularly of the kidney, testis, and ovaries.
An additional analysis of LTRPC3 localized expression was assessed using In situ hybridization techniques (ISH). LTRPC3 expression was localized to the cytoplasm of collecting tubular epithelium in the medulla, medullary rays, and periglomerular regions (see FIG. 20, Plates i, ii, iv and v). Tubules in the medulla exhibited the most intense expression. Other tubular epithelia, e.g., proximal convoluted tubular epithelium, exhibited minimal expression. Expression patterns were compared to LTRPC3 sense mRNA-labeled human kidney sections as negative controls (FIG. 20, Plates iii and vi) and to human lysozyme antisense mRNA labeled human kidney sections as positive controls (data not shown).
The observed LTRPC3 mRNA expression in human kidney, suggests that LTRPC3 may play a role in renal handling of calcium and other cations. The kidney plays a major role in Ca2+ homeostasis. LTRPC3 could be involved in Ca2+ absorption directly due to its Ca2+ permeability. Indeed, the in situ hybridization analysis described herein demonstrates that LTRPC3 is predominantly expressed in the connecting tubule, which has frequently been implicated in active transcellular Ca2+ reabsorption (see Hoenderop, J. G., et al., (2002) Annu. Rev. Physiol. 64, 529-549; which is hereby incorporated by reference herein in its entirety). Alternatively, LTRPC3 may function as an SOC that regulates Ca2+ absorption. In the kidney, Ca2+ absorption is regulated by agonists such as calcitonin, parathyroid hormone, and parathyroid hormone-related peptide through their respective G protein-coupled receptors, and downstream SOCs (Friedman, P. A., et al., (1995) Physiol. Rev. 75, 429-471; which is hereby incorporated by reference herein in its entirety).
Potentially, LTRPC3 may involved in the pathogenesis of calcium homeostasis-related disorders such as hypercaliuric nephrolithiasis, certain forms of osteoporosis, Gitelman's disease and Bartter's syndrom.
In preferred embodiments, LTRPC3 polynucleotides and polypeptides, including fragments and modulators thereof, are useful for treating, diagnosing, and/or ameliorating Ca2+ reabsorption disorders, disorders associated with aberrant luminal Ca2+ entry via the epithelial Ca2+ channel (ECaC), disorders associated with aberrant cytosolic diffusion of Ca2+ bound to calbindin-D28K, disorders associated with aberrant basolateral extrusion of Ca2+ through the Na+/Ca2+ exchanger (NCX), disorders associated with aberrant plasma membrane Ca2+-ATPase (PMCA) activity and/or activation, disorders associated with the hypocalciuric effect of diuretics, disorders associated with the hypocalciuric effect of thiazide diuretics, disorders associated with hormone insufficiencies that affect the function of the kidney, disorders associated with hormone insufficiencies that affect renal Ca2+ homeostasis, disorders associated with aberrant renal Ca2+ homeostasis as a result of vitamin D deficiency, disorders associated with aberrant renal Ca2+ homeostasis as a result of PTH deficiency, disorders associated with aberrant renal Ca2+ homeostasis as a result of vasopressin deficiency, disorders associated with aberrant renal Ca2+ homeostasis as a result of prostaglandines deficiency, disorders associated with aberrant renal Ca2+ homeostasis as a result of estrogen deficiency, disorders associated with aberrant renal Ca2+ homeostasis as a result of calcitonin deficiency, disorders associated with aberrant renal Ca2+ homeostasis as a result of parathyroid hormone deficiency, disorders associated with aberrant renal Ca2+ homeostasis as a result of atrial natriuretic peptide deficiency, calcium homeostasis-related disorders, hypercaliuric nephrolithiasis, certain forms of osteoporosis, Gitelman's disease, Bartter's syndrom, disorders associated with aberrant function of kidney tubular epithelium, disorders associated with aberrant function of kidney tubular epithelium in the medulla, disorders associated with aberrant function of kidney tubular epithelium in the medullary rays, disorders associated with aberrant function of kidney tubular epithelium in the periglomerular.
In preferred embodiments, LTRPC3 polynucleotides and polypeptides, including fragments and modulators thereof, are useful for treating, diagnosing, and/or ameliorating acute kidney failure, chronic kidney failure, cystic kidney disease, horseshoe kidney, hypertensive kidney disease, kidney atrophy, kidney cancer, kidney disease, kidney failure, kidney infection, kidney metastasis, kidney stone, nephrolithiasis, multicystic dysplastic kidney disease, and polycystic kidney disease.
Characterization of the LTRPC3 polypeptide of the present invention using antisense oligonucleotides led to the determination that LTRPC3 is involved in the negative modulation of the FEN1 DNA base-excision repair/proliferation modulating protein as described in Example 5 herein.
Specifically, antisense inhibition of LTRPC3 resulted in upregulation of Fen1, a marker of proliferation. As such, LTRPC3 behaves in a manner similar to a tumor suppressor, in that loss results in increases in proliferation. While some increases in the proliferation markers p21 and IkB were also observed, they were less significant, and could be a response to the pro-proliferative effect of LTRPC3 antisense inhibition. Interestingly, expression of LTRPC3 was also observed to be significantly reduced in tumor tissues, relative to normal tissues (see FIG. 13). Specifically, renal, testicular, and ovarian tumor samples showed as much as 12 to 30 fold down regulation of LTRPC3 message levels compared to normal tissues. This reduction of LTRPC3 correlating with the tumor state is consistent with the negative FEN1 modulatory activity and suggests this gene may have antiproliferative activities. Therefore, for modulators of LTRPC3 to be useful for treating cancer, it would be necessary to increase the activity of the gene or gene product in order to have the reverse effect on cancerous cells. Thus, an agonist of LTRPC3 activity or a compound that increased LTRPC3 message levels would be a desired invention for cancer therapy.
In preferred embodiments, LTRPC3 polynucleotides and polypeptides, including fragments and modulators thereof, are useful for treating, diagnosing, and/or ameliorating DNA-repair deficiencies, particularly base-excision repair deficiencies, Xeroderma pigmentosum, skin cancer, melanoma, UV senstivity, alkylation sensivity, gamma irradiation sensitivity, pyrimidine dimer sensitivity, chemical mutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone, Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, blood vessel dilation aberrations in the skin, blood vessel dilation aberrations in the eye, conditions involving increased levels of apurinic sites, conditions involving increased levels of apyrimidinic sites, conditions involving increased levels of abasic sites, disorders related to aberrant signal transduction, proliferating disorders, and/or cancers.
Moreover, LTRPC3 polynucleotides and polypeptides, including fragments and modulators thereof, are useful for increasing mammalian base excision repair activity, increasing mammalian single-nucleotide base excision repair activity, and/or increasing mammalian long patch base excision repair activity.
In preferred embodiments, antagonists directed against LTRPC3 are useful for treating, diagnosing, and/or ameliorating DNA-repair deficiencies, particularly base-excision repair deficiencies, Xeroderma pigmentosum, skin cancer, melanoma, UV senstivity, alkylation sensivity, gamma irradiation sensitivity, pyrimidine dimer sensitivity, chemical mutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone, Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, blood vessel dilation aberrations in the skin, blood vessel dilation aberrations in the eye, conditions involving increased levels of apurinic sites, conditions involving increased levels of apyrimidinic sites, conditions involving increased levels of abasic sites, disorders related to aberrant signal transduction, proliferating disorders, and/or cancers.
Moreover, antagonists directed against LTRPC3 are useful for increasing mammalian base excision repair activity, increasing mammalian single-nucleotide base excision repair activity, and/or increasing mammalian long patch base excision repair activity.
In preferred embodiments, agonists directed against LTRPC3 are useful for treating, diagnosing, and/or ameliorating, disorders related to aberrant signal transduction, proliferating disorders, and/or cancers, particularly renal cell carcinomas, testicular cancers, and/or ovarian cancers.
Moreover, agonists directed against LTRPC3 are useful for decreasing mammalian base excision repair activity, decreasing mammalian single-nucleotide base excision repair activity, and/or decreasing mammalian long patch base excision repair activity.
The strong homology to human transient receptor potential channels (TRP), combined with the predominate localized expression in kidney tissue suggests the LTRPC3 polynucleotides and polypeptides, including modulators or fragments thereof, may be useful in treating, diagnosing, prognosing, and/or preventing renal diseases and/or disorders, which include, but are not limited to: nephritis, renal failure, nephrotic syndrome, urinary tract infection, hematuria, proteinuria, oliguria, polyuria, nocturia, edema, hypertension, electrolyte disorders, sterile pyuria, renal osteodystrophy, large kidneys, renal transport defects, nephrolithiasis, azotemia, anuria, urinary retention, slowing of urinary stream, large prostate, flank tenderness, full bladder sensation after voiding, enuresis, dysuria, bacteriuria, kidney stones, glomerulonephritis, vasculitis, hemolytic uremic syndromes, thrombotic thrombocytopenic purpura, malignant hypertension, casts, tubulointerstitial kidney diseases, renal tubular acidosis, pyelonephritis, hydronephritis, nephrotic syndrome, crush syndrome, and/or renal colic, in addition to Wilm's Tumor Disease, and congenital kidney abnormalities such as horseshoe kidney, polycystic kidney, and Falconi's syndrome.for example.
Several known TRP family members have been identified that are expressed significantly in kidney tissue. These TRP family members include, for example, Trp12 (Wissenbach, U., Bodding, M., Freichel, M., Flockerzi, V, Lett., 485(2-3):127-34, (2000)); OTRPC4 (Strotmann, R., Harteneck, C., Nunnenmacher, K., Schultz, G., Plant, T, D, Nat, Cell, Biol., 2(10):695-702, (2000)); polycystin-L2 (Guo, L., Schreiber, T, H., Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics., 64(3):241-51, (2000)); and EcaC (Hoenderop, J. G., van, der, Kemp, A, W., Hartog, A., van, de, Graaf, S, F., van, Os, C, H., Willems, P, H., Bindels, R, J. J. Biol, Chem., 274(13):8375-8, (1999)).
Thus, the LTRPC3 polynucleotides and polypeptides are expected to share at least some biological activity with TRP family members expressed in kidney cells and tissues, particularly those specifically referenced herein.
The strong homology to human transient receptor potential channels (TRP) proteins, combined with the localized expression in spinal cord and brain suggests the LTRPC3 polynucleotides and polypeptides, including modulators or fragments thereof, may be useful in treating, diagnosing, prognosing, and/or preventing neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in the Examples, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
The strong homology to human transient receptor potential channels (TRP), combined with the localized expression in testis tissue emphasizes the potential utility for LTRPC3 polynucleotides and polypeptides in treating, diagnosing, prognosing, and/or preventing testicular, in addition to reproductive disorders.
In preferred embodiments, LTRPC3 polynucleotides and polypeptides including agonists and fragments thereof, have uses which include treating, diagnosing, prognosing, and/or preventing the following, non-limiting, diseases or disorders of the testis: spermatogenesis, infertility, Klinefelter's syndrome, XX male, epididymitis, genital warts, germinal cell aplasia, cryptorchidism, varicocele, immotile cilia syndrome, and viral orchitis. The LTRPC3 polynucleotides and polypeptides including agonists and fragments thereof, may also have uses related to modulating testicular development, embryogenesis, reproduction, and in ameliorating, treating, and/or preventing testicular proliferative disorders (e.g., cancers, which include, for example, choriocarcinoma, Nonseminoma, seminona, and testicular germ cell tumors).
Likewise, the localized expression in testis tissue also emphasizes the potential utility for LTRPC3 polynucleotides and polypeptides in treating, diagnosing, prognosing, and/or preventing metabolic diseases and disorders which include the following, not limiting examples: premature puberty, incomplete puberty, Kallman syndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis, congenital adrenal hyperplasia, FSH deficiency, and granulomatous disease, for example.
This gene product may also be useful in assays designed to identify binding agents, as such agents (antagonists) are useful as male contraceptive agents. The testes are also a site of active gene expression of transcripts that is expressed, particularly at low levels, in other tissues of the body. Therefore, this gene product may be expressed in other specific tissues or organs where it may play related functional roles in other processes, such as hematopoiesis, inflammation, bone formation, and kidney function, to name a few possible target indications.
Several known TRP family members have been identified that are expressed significantly in testis tissue. These TRP family members include, for example, polycystin-L2 (Guo, L., Schreiber, T, H., Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics., 64(3):241-51, (2000)); TRP7 (Okada, T., Inoue, R., Yamazaki, K., Maeda, A., Kurosaki, T., Yamakuni, T., Tanaka, I., Shimizu, S., Ikenaka, K., Imoto, K., Mori, Y, J. Biol, Chem., 274(39):27359-70, (1999)); btrp2 (Wissenbach, U., Schroth, G., Philipp, S., Flockerzi, V, Lett., 429(1):61-6, (1998)); Htrp-1 (Zhu, X., Chu, P, B., Peyton, M., Bimbaumer, L, Lett., 373(3):193-8, (1995)); and TRPC1 (Wes, P, D., Chevesich, J., Jeromin, A., Rosenberg, C., Stetten, G., Montell, C, Proc, Natl, Acad, Sci, U,S,A., 92(21):9652-6, (1995)).
Thus, the LTRPC3 polynucleotides and polypeptides are expected to share at least some biological activity with TRP family members expressed in testis cells and tissues, particularly those specifically referenced herein.
As described elsewhere herein, transient receptor potential channel family members have been implicated in modulating cell proliferation, differentiation, migration, activation, exocytosis, muscle contraction, gene expression, apoptosis. signalling, pheromone sensory signaling, smooth muscle tone, pain perception, heat perception, osmosenstivity, and mechanosensitivity. Moreover, transient receptor potential channel family members have been implicated in disorders of the skin, skeletal-muscle, nervous, cardiac, and vascular systems, in addition to the following, non-limiting diseases and disorders, which include, for example, arteriosclerosis, neointimal hypoerplasia, metastatic melanomas, bipolar disorder, nonsyndromic hereditary deafness, Knobloch syndrome, holosencephaly, and various maligancies including prostate cancer.
In preferred embodiments, LTRPC3 polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include, modulating cell proliferation, differentiation, migration, activation, exocytosis, muscle contraction, gene expression, apoptosis. signalling, pheromone sensory signaling, smooth muscle tone, pain perception, heat perception, osmosenstivity, and mechanosensitivity.
In more preferred embodiments, LTRPC3 polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include, treating, ameliorating, preventing, detecting, and/or prognosing various diseases and disorders, particularly the following, non-limiting examples, disorders of the skin, skeletal-muscle, nervous, cardiac, and vascular systems, in addition to the following, non-limiting diseases and disorders, which include, for example, arteriosclerosis, neointimal hypoerplasia, metastatic melanomas, bipolar disorder, nonsyndromic hereditary deafness, Knobloch syndrome, holosencephaly, and various maligancies including prostate cancer.
LTRPC3 polynucleotides and polypeptides of the present invention, including agonists and/or fragments may be involved in intracellular Ca2+ homeostasis which affects various aspects of biological functions including mechano-regulation, pain transduction, vasorelaxation, gene expression, cell cycle and proliferation/apoptosis. Since LTRPC3 is dominantly expressed in kidney, it may particularly play an important role in regulating cytosolic Ca2+ in the renal system, potentially Ca2+ absorption.
The LTRPC3 gene maps to chromosome 9q21.11-21.31 between the two markers D9S1874 and D9S1807. This region is linked to amyotrophic lateral sclerosis with frontotemporal dementia, early-onset pulverulent cataract, infantile nephronophthisis, hypomagnesemia with secondary hypocalcemia (HSH), and familial hemophagocytic lymphohistiocytosis. Therefore, agonists and/or antagonists of the novel LTRPC3 can be used to treat diseases including various forms of neuronal degeneration, neurogenic inflammation, allergy, immunodeficiency/excessive immune activation, visual defects, hearing disorder, pain, cancer, hypertension and other cardiovascular diseases. In addition, the therapeutics may be useful in the treatment of diseases associated with disturbances in Ca2+ homeostasis including osteoporosis, hypercalciuric stone disease, and chronic renal failure.
Moreover, given the selective expression in kidney, LTRPC3 could be considered a candidate gene for HSH, since the phenotype is a renal insufficiency. LTRPC3 chromosomal location is  ̃600 kb downstream of a X;9 translocation breakpoint interval described for one patient with HSH (Hum. Mol. Genet. 6, 1491-1497, (1997)). Recently, two groups reported that a new member of the TRPM subfamily, TRPM6, expressed in both intestinal tissues and kidney, is associated with HSH (Nat. Genet. 31, 166-170, (2002); and Nat. Genet. 31, 171-174 (2002)). Indeed, LTRPC3 is  ̃4 mb 5′ to TRPM6 on 9q21. Although TRPM6 is the first component identified for HSH, this should not preclude involvement of LTRPC3 in the disease. Therefore, it is possible that LTRPC3 may co-localize with TRPM6 and/or forms a heteromultimer with TRPM6 in the kidney.
The kidney plays a major role in Ca2+ homeostasis (reviewed in Physiol. Rev. 75, 429-471, (1995)). LTRPC3 may play a direct role in Ca2+ absorption due to its constitutive Ca2+ permeability. Alternatively, LTRPC3 may be the SOC that regulates Ca2+ absorption. In kidney, Ca2+ absorption is regulated by agonists such as calcitonin, parathyroid hormone and parathyroid hormone-related peptide through their respective G protein-coupled receptors, and downstream SOCs. Additionally, in kidney a SOC has been described (J. Biol. Chem. 276, 25759-25765, (2001)) for the action of angiotensin II, a vasoactive peptide that plays a major role in regulating blood pressure.
In addition, LTRPC3 polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include modulating intracellular Ca++ ion concentrations, Ca++ ion flux, stored intracellular Ca++ ion concentrations, Ca++ ion pump activity, Ca++ ion flow into cell, Ca++ ion flow out of cells, the activation of Ca++ sensitive proteins, the activation of Ca++ sensitive signaling pathways, the activation of kinase-activatible proteins, and the activation of kinase-dependent signaling pathways.
The LTRPC3 polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include modulating proliferation, differentiation, migration, and activation in various cells, tissues, and organisms, and particularly in mammalian kidney, spinal cord, testis, and brain, preferably human. LTRPC3 polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, may be useful in diagnosing, treating, prognosing, and/or preventing renal, neural, and/or proliferative diseases or disorders, particularly of the renal system.
In addition, antagonists of the LTRPC3 polynucleotides and polypeptides, including modulators or fragments thereof, may have uses that include diagnosing, treating, prognosing, and/or preventing diseases or disorders related to transient receptor potential channel activity, which may include renal, neural, reproductive, and/or proliferative diseases or disorders.
Although it is believed the encoded polypeptide may share at least some biological activities with transient receptor potential channel family members, particularly those melastatin1, a number of methods of determining the exact biological function of this clone are either known in the art or are described elsewhere herein. Briefly, the function of this clone may be determined by applying microarray methodology. Nucleic acids corresponding to the LTRPC3 polynucleotides, in addition to, other clones of the present invention, may be arrayed on microchips for expression profiling. Depending on which polynucleotide probe is used to hybridize to the slides, a change in expression of a specific gene may provide additional insight into the function of this gene based upon the conditions being studied. For example, an observed increase or decrease in expression levels when the polynucleotide probe used comes from tissue that has been treated with known transient potential receptor inhibitors, which include, but are not limited to the drugs listed herein or otherwise known in the art, might indicate a function in modulating transient potential receptor function, for example. In the case of LTRPC3, kidney, spinal cord, testis, and brain, should be used to extract RNA to prepare the probe.
In addition, the function of the protein may be assessed by applying quantitative PCR methodology, for example. Real time quantitative PCR would provide the capability of following the expression of the LTRPC3 gene throughout development, for example. Quantitative PCR methodology requires only a nominal amount of tissue from each developmentally important step is needed to perform such experiments. Therefore, the application of quantitative PCR methodology to refining the biological function of this polypeptide is encompassed by the present invention. Also encompassed by the present invention are quantitative PCR probes corresponding to the polynucleotide sequence provided as SEQ ID NO:1 (FIGS. 1A-F).
The function of the protein may also be assessed through complementation assays in yeast. For example, in the case of the LTRPC3, transforming yeast deficient in transient receptor potential channel activity with LTRPC3 and assessing their ability to grow would provide convincing evidence the LTRPC3 polypeptide has transient receptor potential channel activity. Additional assay conditions and methods that may be used in assessing the function of the polynucleotides and polypeptides of the present invention are known in the art, some of which are disclosed elsewhere herein.
Alternatively, the biological function of the encoded polypeptide may be determined by disrupting a homologue of this polypeptide in Mice and/or rats and observing the resulting phenotype.
Moreover, the biological function of this polypeptide may be determined by the application of antisense and/or sense methodology and the resulting generation of transgenic mice and/or rats. Expressing a particular gene in either sense or antisense orientation in a transgenic mouse or rat could lead to respectively higher or lower expression levels of that particular gene. Altering the endogenous expression levels of a gene can lead to the observation of a particular phenotype that can then be used to derive indications on the function of the gene. The gene can be either over-expressed or under expressed in every cell of the organism at all times using a strong ubiquitous promoter, or it could be expressed in one or more discrete parts of the organism using a well characterized tissue-specific promoter (e.g., a kidney, spinal cord, testis, and/or brain-specific promoter), or it can be expressed at a specified time of development using an inducible and/or a developmentally regulated promoter.
In the case of LTRPC3 transgenic mice or rats, if no phenotype is apparent in normal growth conditions, observing the organism under diseased conditions (renal, reproductive, neural, or proliferative disorders, etc.) may lead to understanding the function of the gene. Therefore, the application of antisense and/or sense methodology to the creation of transgenic mice or rats to refine the biological function of the polypeptide is encompassed by the present invention.
In preferred embodiments, the following N-terminal LTRPC3 deletion polypeptides are encompassed by the present invention: M1-T1554, Y2-T1554, V3-T1554, R4-T1554, V5-T1554, S6-T1554, F7-T1554, D8-T1554, T9-T1554, K10-T1554, P11-T1554, D12-T1554, L13-T1554, L14-T1554, L15-T1554, H16-T1554, L17-T1554, M18-T1554, T19-T1554, K20-T1554, E21-T1554, W22-T1554, Q23-T1554, L24-T1554, E25-T1554, L26-T1554, P27-T1554, K28-T1554, L29-T1554, L30-T1554, I31-T1554, S32-T1554, V33-T1554, H34-T1554, G35-T1554, G36-T1554, L37-T1554, Q38-T1554, N39-T1554, F40-T1554, E41-T1554, L42-T1554, Q43-T1554, P44-T1554, K45-T1554, L46-T1554, K47-T1554, Q48-T1554, V49-T1554, F50-T1554, G51-T1554, K52-T1554, G53-T1554, L54-T1554, I55-T1554, K56-T1554, A57-T1554, A58-T1554, M59-T1554, T60-T1554, T61-T1554, G62-T1554, A63-T1554, W64-T1554, I65-T1554, F66-T1554, T67-T1554, G68-T1554, G69-T1554, V70-T1554, N71-T1554, T72-T1554, G73-T1554, V74-T1554, I75-T1554, R76-T1554, H77-T1554, V78-T1554, G79-T1554, D80-T1554, A81-T1554, L82-T1554, K83-T1554, D84-T1554, H85-T1554, A86-T1554, S87-T1554, K88-T1554, S89-T1554, R90-T1554, G91-T1554, K92-T1554, I93-T1554, C94-T1554, T95-T1554, I96-T1554, G97-T1554, I98-T1554, A99-T1554, P100-T1554, W101-T1554, G102-T1554, I103-T1554, V104-T1554, E105-T1554, N106-T1554, Q107-T1554, E108-T1554, D109-T1554, L110-T1554, I111-T1554, G112-T1554, R113-T1554, D114-T1554, V115-T1554, V116-T1554, R117-T1554, P118-T1554, Y119-T1554, Q120-T1554, T121-T1554, M122-T1554, S123-T1554, N124-T1554, P125-T1554, M126-T1554, S127-T1554, K128-T1554, L129-T1554, T130-T1554, V131-T1554, L132-T1554, N133-T1554, S134-T1554, M135-T1554, H136-T1554, S137-T1554, H138-T1554, F139-T1554, I140-T1554, L141-T1554, A142-T1554, D143-T1554, N144-T1554, G145-T1554, T146-T1554, T147-T1554, G148-T1554, K149-T1554, Y150-T1554, G151-T1554, A152-T1554, E153-T1554, V154-T1554, K155-T1554, L156-T1554, R157-T1554, R158-T1554, Q159-T1554, L160-T1554, E161-T1554, K162-T1554, H163-T1554, I164-T1554, S165-T1554, L166-T1554, Q167-T1554, K168-T1554, I169-T1554, N170-T1554, T171-T1554, R172-T1554, I173-T1554, G174-T1554, Q175-T1554, G176-T1554, V177-T1554, P178-T1554, V179-T1554, V180-T1554, A181-T1554, L182-T1554, I183-T1554, V184-T1554, E185-T1554, G186-T1554, G187-T1554, P188-T1554, N189-T1554, V190-T1554, I191-T1554, S192-T1554, I193-T1554, V194-T1554, L195-T1554, E196-T1554, Y197-T1554, L198-T1554, R199-T1554, D200-T1554, T201-T1554, P202-T1554, P203-T1554, V204-T1554, P205-T1554, V206-T1554, V207-T1554, V208-T1554, C209-T1554, D210-T1554, G211-T1554, S212-T1554, G213-T1554, R214-T1554, A215-T1554, S216-T1554, D217-T1554, I218-T1554, L219-T1554, A220-T1554, F221-T1554, G222-T1554, H223-T1554, K224-T1554, Y225-T1554, S226-T1554, E227-T1554, E228-T1554, G229-T1554, G230-T1554, L231-T1554, I232-T1554, N233-T1554, E234-T1554, S235-T1554, L236-T1554, R237-T1554, D238-T1554, Q239-T1554, L240-T1554, L241-T1554, V242-T1554, T243-T1554, I244-T1554, Q245-T1554, K246-T1554, T247-T1554, F248-T1554, T249-T1554, Y250-T1554, T251-T1554, R252-T1554, T253-T1554, Q254-T1554, A255-T1554, Q256-T1554, H257-T1554, L258-T1554, F259-T1554, I260-T1554, I261-T1554, L262-T1554, M263-T1554, E264-T1554, C265-T1554, M266-T1554, K267-T1554, K268-T1554, K269-T1554, E270-T1554, L271-T1554, I272-T1554, T273-T1554, V274-T1554, F275-T1554, R276-T1554, M277-T1554, G278-T1554, S279-T1554, E280-T1554, G281-T1554, H282-T1554, Q283-T1554, D284-T1554, I285-T1554, D286-T1554, L287-T1554, A288-T1554, I289-T1554, L290-T1554, T291-T1554, A292-T1554, L293-T1554, L294-T1554, K295-T1554, G296-T1554, A297-T1554, N298-T1554, A299-T1554, S300-T1554, A301-T1554, P302-T1554, D303-T1554, Q304-T1554, L305-T1554, S306-T1554, L307-T1554, A308-T1554, L309-T1554, A310-T1554, W311-T1554, N312-T1554, R313-T1554, V314-T1554, D315-T1554, I316-T1554, A317-T1554, R318-T1554, S319-T1554, Q320-T1554, I321-T1554, F322-T1554, I323-T1554, Y324-T1554, G325-T1554, Q326-T1554, Q327-T1554, W328-T1554, P329-T1554, V330-T1554, G331-T1554, 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E1073-T1554, N1074-T1554, M1075-T1554, S1076-T1554, M1077-T1554, R1078-T1554, L1079-T1554, E1080-T1554, E1081-T1554, V1082-T1554, N1083-T1554, E1084-T1554, R1085-T1554, E1086-T1554, H1087-T1554, S1088-T1554, M1089-T1554, K1090-T1554, A1091-T1554, S1092-T1554, L1093-T1554, Q1094-T1554, T1095-T1554, V1096-T1554, D1097-T1554, I1098-T1554, R1099-T1554, L1100-T1554, A110-T1554, Q1102-T1554, L1103-T1554, E1104-T1554, D1105-T1554, L1106-T1554, I1107-T1554, G1108-T1554, R1109-T1554, M1110-T1554, A1111-T1554, T1112-T1554, A1113-T1554, L1114-T1554, E1115-T1554, R1116-T1554, L1117-T1554, T1118-T1554, G1119-T1554, L1120-T1554, E1121-T1554, R1122-T1554, A1123-T1554, E1124-T1554, S1125-T1554, N1126-T1554, K1127-T1554, I1128-T1554, R1129-T1554, S1130-T1554, R1131-T1554, T1132-T1554, S1133-T1554, S1134-T1554, D1135-T1554, C1136-T1554, T1137-T1554, D1138-T1554, A1139-T1554, A1140-T1554, Y1141-T1554, I1142-T1554, V1143-T1554, R1144-T1554, Q1145-T1554, S1146-T1554, S1147-T1554, F1148-T1554, N1149-T1554, S1150-T1554, Q1151-T1554, E1152-T1554, G1153-T1554, N1154-T1554, T1155-T1554, F1156-T1554, K1157-T1554, L1158-T1554, Q1159-T1554, E1160-T1554, S1161-T1554, I1162-T1554, D1163-T1554, P1164-T1554, A1165-T1554, G1166-T1554, E1167-T1554, E1168-T1554, T1169-T1554, M1170-T1554, S1171-T1554, P1172-T1554, T1173-T1554, S1174-T1554, P1175-T1554, T1176-T1554, L1177-T1554, M1178-T1554, P1179-T1554, R1180-T1554, M1181-T1554, R1182-T1554, S1183-T1554, H1184-T1554, S1185-T1554, F1186-T1554, Y1187-T1554, S1188-T1554, V1189-T1554, N1190-T1554, M1191-T1554, K1192-T1554, D1193-T1554, K1194-T1554, G1195-T1554, G1196-T1554, I1197-T1554, E1198-T1554, K1199-T1554, L1200-T1554, E1201-T1554, S1202-T1554, I1203-T1554, F1204-T1554, K1205-T1554, E1206-T1554, R1207-T1554, S1208-T1554, L1209-T1554, S1210-T1554, L1211-T1554, H1212-T1554, R1213-T1554, A1214-T1554, T1215-T1554, S1216-T1554, S1217-T1554, H1218-T1554, S1219-T1554, V1220-T1554, A1221-T1554, K1222-T1554, E1223-T1554, P1224-T1554, K1225-T1554, A1226-T1554, P1227-T1554, A1228-T1554, A1229-T1554, P1230-T1554, A1231-T1554, N1232-T1554, T1233-T1554, L1234-T1554, A1235-T1554, I1236-T1554, V1237-T1554, P1238-T1554, D1239-T1554, S1240-T1554, R1241-T1554, R1242-T1554, P1243-T1554, S1244-T1554, S1245-T1554, C1246-T1554, I1247-T1554, D1248-T1554, I1249-T1554, Y1250-T1554, V1251-T1554, S1252-T1554, A1253-T1554, M1254-T1554, D1255-T1554, E1256-T1554, L1257-T1554, H1258-T1554, C1259-T1554, D1260-T1554, I1261-T1554, D1262-T1554, P1263-T1554, L1264-T1554, D1265-T1554, N1266-T1554, S1267-T1554, V1268-T1554, N1269-T1554, I1270-T1554, L1271-T1554, G1272-T1554, L1273-T1554, G1274-T1554, E1275-T1554, P1276-T1554, S1277-T1554, F1278-T1554, S1279-T1554, T1280-T1554, P1281-T1554, V1282-T1554, P1283-T1554, S1284-T1554, T1285-T1554, A1286-T1554, P1287-T1554, S1288-T1554, S1289-T1554, S1290-T1554, A1291-T1554, Y1292-T1554, A1293-T1554, T1294-T1554, L1295-T1554, A1296-T1554, P1297-T1554, T1298-T1554, D1299-T1554, R1300-T1554, P1301-T1554, P1302-T1554, S1303-T1554, R1304-T1554, S1305-T1554, I1306-T1554, D1307-T1554, F1308-T1554, E1309-T1554, D1310-T1554, I1311-T1554, T1312-T1554, S1313-T1554, M1314-T1554, D1315-T1554, T1316-T1554, R1317-T1554, S1318-T1554, F1319-T1554, S1320-T1554, S1321-T1554, D1322-T1554, Y1323-T1554, T1324-T1554, H1325-T1554, L1326-T1554, P1327-T1554, E1328-T1554, C1329-T1554, Q1330-T1554, N1331-T1554, P1332-T1554, W1333-T1554, D1334-T1554, S1335-T1554, E1336-T1554, P1337-T1554, P1338-T1554, M1339-T1554, Y1340-T1554, H1341-T1554, T1342-T1554, I1343-T1554, E1344-T1554, R1345-T1554, S1346-T1554, K1347-T1554, S1348-T1554, S1349-T1554, R1350-T1554, Y1351-T1554, L1352-T1554, A1353-T1554, T1354-T1554, T1355-T1554, P1356-T1554, F1357-T1554, L1358-T1554, L1359-T1554, E1360-T1554, E1361-T1554, A1362-T1554, P1363-T1554, I1364-T1554, V1365-T1554, K1366-T1554, S1367-T1554, H1368-T1554, S1369-T1554, F1370-T1554, M1371-T1554, F1372-T1554, S1373-T1554, P1374-T1554, S1375-T1554, R1376-T1554, S1377-T1554, Y1378-T1554, Y1379-T1554, A1380-T1554, N1381-T1554, F1382-T1554, G1383-T1554, V1384-T1554, P1385-T1554, V1386-T1554, K1387-T1554, T1388-T1554, A1389-T1554, E1390-T1554, Y1391-T1554, T1392-T1554, S1393-T1554, I1394-T1554, T1395-T1554, D1396-T1554, C1397-T1554, I1398-T1554, D1399-T1554, T1400-T1554, R1401-T1554, C1402-T1554, V1403-T1554, N1404-T1554, A1405-T1554, P1406-T1554, Q1407-T1554, A1408-T1554, I1409-T1554, A1410-T1554, D1411-T1554, R1412-T1554, A1413-T1554, A1414-T1554, F1415-T1554, P1416-T1554, G1417-T1554, G1418-T1554, L1419-T1554, G1420-T1554, D1421-T1554, K1422-T1554, V1423-T1554, E1424-T1554, D1425-T1554, L1426-T1554, T1427-T1554, C1428-T1554, C1429-T1554, H1430-T1554, P1431-T1554, E1432-T1554, R1433-T1554, E1434-T1554, A1435-T1554, E1436-T1554, L1437-T1554, S1438-T1554, H1439-T1554, P1440-T1554, S1441-T1554, S1442-T1554, D1443-T1554, S1444-T1554, E1445-T1554, E1446-T1554, N1447-T1554, E1448-T1554, A1449-T1554, K1450-T1554, G1451-T1554, R1452-T1554, R1453-T1554, A1454-T1554, T1455-T1554, I1456-T1554, A1457-T1554, I1458-T1554, S1459-T1554, S1460-T1554, Q1461-T1554, E1462-T1554, G1463-T1554, D1464-T1554, N1465-T1554, S1466-T1554, E1467-T1554, R1468-T1554, T1469-T1554, L1470-T1554, S1471-T1554, N1472-T1554, N1473-T1554, I1474-T1554, T1475-T1554, V1476-T1554, P1477-T1554, K1478-T1554, I1479-T1554, E1480-T1554, R1481-T1554, A1482-T1554, N1483-T1554, S1484-T1554, Y1485-T1554, S1486-T1554, A1487-T1554, E1488-T1554, E1489-T1554, P1490-T1554, S1491-T1554, A1492-T1554, P1493-T1554, Y1494-T1554, A1495-T1554, H1496-T1554, T1497-T1554, R1498-T1554, K1499-T1554, S1500-T1554, F1501-T1554, S1502-T1554, I1503-T1554, S1504-T1554, D1505-T1554, K1506-T1554, L1507-T1554, D1508-T1554, R1509-T1554, Q1510-T1554, R1511-T1554, N1512-T1554, T1513-T1554, A1514-T1554, S1515-T1554, L1516-T1554, Q1517-T1554, N1518-T1554, P1519-T1554, F1520-T1554, Q1521-T1554, R1522-T1554, S1523-T1554, K1524-T1554, S1525-T1554, S1526-T1554, K1527-T1554, P1528-T1554, E1529-T1554, G1530-T1554, R1531-T1554, G1532-T1554, D1533-T1554, S1534-T1554, L1535-T1554, S1536-T1554, M1537-T1554, R1538-T1554, R1539-T1554, L1540-T1554, S1541-T1554, R1542-T1554, T1543-T1554, S1544-T1554, A1545-T1554, F1546-T1554, Q1547-T1554, and/or S1548-T1554 of SEQ ID NO:2. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3 deletion polypeptides are encompassed by the present invention: M1-T1554, M1-H1553, M1-K1552, M1-S1551, M1-E1550, M1-F1549, M1-S1548, M1-Q1547, M1-F1546, M1-A1545, M1-S1544, M1-T1543, M1-R1542, M1-S1541, M1-L1540, M1-R1539, M1-R1538, M1-M1537, M1-S1536, M1-L1535, M1-S1534, M1-D1533, M1-G1532, M1-R1531, M1-G1530, M1-E1529, M1-P1528, M1-K1527, M1-S1526, M1-S1525, M1-K1524, M1-S1523, M1-R1522, M1-Q1521, M1-F1520, M1-P1519, M1-N1518, M1-Q1517, M1-L1516, M1-S1515, M1-A1514, M1-T1513, M1-N1512, M1-R1511, M1-Q1510, M1-R1509, M1-D1508, M1-L1507, M1-K1506, M1-D1505, M1-S1504, M1-I1503, M1-S1502, M1-F1501, M1-S1500, M1-K1499, M1-R1498, M1-T1497, M1-H1496, M1-A1495, M1-Y1494, M1-P1493, M1-A1492, M1-S1491, M1-P1490, M1-E1489, M1-E1488, M1-A1487, M1-S1486, M1-Y1485, M1-S1484, M1-N1483, M1-A1482, M1-R1481, M1-E1480, M1-I1479, M1-K1478, M1-P1477, M1-V1476, M1-T1475, M1-I1474, M1-N1473, M1-N1472, M1-S1471, M1-L1470, M1-T1469, M1-R1468, M1-E1467, M1-S1466, M1-N1465, M1-D1464, M1-G1463, M1-E1462, M1-Q1461, M1-S1460, M1-S1459, M1-I1458, M1-A1457, M1-I1456, M1-T1455, M1-A1454, M1-R1453, M1-R1452, M1-G1451, M1-K1450, M1-A1449, M1-E1448, M1-N1447, M1-E1446, M1-E1445, M1-S1444, M1-D1443, M1-S1442, M1-S1441, M1-P1440, M1-H1439, M1-S1438, M1-L1437, M1-E1436, M1-A1435, M1-E1434, M1-R1433, M1-E1432, M1-P1431, M1-H1430, M1-C1429, M1-C1428, M1-T1427, M1-L1426, M1-D1425, M1-E1424, M1-V1423, M1-K1422, M1-D1421, M1-G1420, M1-L1419, M1-G1418, M1-G1417, M1-P1416, M1-F1415, M1-A1414, M1-A1413, M1-R1412, M1-D1411, M1-A1410, M1-I1409, M1-A1408, M1-Q1407, M1-P1406, M1-A1405, M1-N1404, M1-V1403, M1-C1402, M1-R1401, M1-T1400, M1-D1399, M1-I1398, M1-C1397, M1-D1396, M1-T1395, M1-I1394, M1-S1393, M1-T1392, M1-Y1391, M1-E1390, M1-A1389, M1-T1388, M1-K1387, M1-V1386, M1-P1385, M1-V1384, M1-G1383, M1-F1382, M1-N1381, M1-A1380, M1-Y1379, M1-Y1378, M1-S1377, M1-R1376, M1-S1375, M1-P1374, M1-S1373, M1-F1372, M1-M1371, M1-F1370, M1-S1369, M1-H1368, M1-S1367, M1-K1366, M1-V1365, M1-I1364, M1-P1363, M1-A1362, M1-E1361, M1-E1360, M1-L1359, M1-L1358, M1-F1357, M1-P1356, M1-T1355, M1-T1354, M1-A1353, M1-L1352, M1-Y1351, M1-R1350, M1-S1349, M1-S1348, M1-K1347, M1-S1346, M1-R1345, M1-E1344, M1-I1343, M1-T1342, M1-H1341, M1-Y1340, M1-M1339, M1-P1338, M1-P1337, M1-E1336, M1-S1335, M1-D1334, M1-W1333, M1-P1332, M1-N1331, M1-Q1330, M1-C1329, M1-E1328, M1-P1327, M1-L1326, M1-H1325, M1-T1324, M1-Y1323, M1-D1322, M1-S1321, M1-S1320, M1-F1319, M1-S1318, M1-R1317, M1-T1316, M1-D1315, M1-M1314, M1-S1313, M1-T1312, M1-I1311, M1-D1310, M1-E1309, M1-F1308, M1-D1307, M1-I1306, M1-S1305, M1-R1304, M1-S1303, M1-P1302, M1-P1301, M1-R1300, M1-D1299, M1-T1298, M1-P1297, M1-A1296, M1-L1295, M1-T1294, M1-A1293, M1-Y1292, M1-A1291, M1-S1290, M1-S1289, M1-S1288, M1-P1287, M1-A1286, M1-T1285, M1-S1284, M1-P1283, M1-V1282, M1-P1281, M1-T1280, M1-S1279, M1-F1278, M1-S1277, M1-P1276, M1-E1275, M1-G1274, M1-L1273, M1-G1272, M1-L1271, M1-I1270, M1-N1269, M1-V1268, M1-S1267, M1-N1266, M1-D1265, M1-L1264, M1-P1263, M1-D1262, M1-I1261, M1-D1260, M1-C1259, M1-H1258, M1-L1257, M1-E1256, M1-D1255, M1-M1254, M1-A1253, M1-S1252, M1-V1251, M1-Y1250, M1-I1249, M1-D1248, M1-I1247, M1-C1246, M1-S1245, M1-S1244, M1-P1243, M1-R1242, M1-R1241, M1-S1240, M1-D1239, M1-P1238, M1-V1237, M1-I1236, M1-A1235, M1-L1234, M1-T1233, M1-N1232, M1-A1231, M1-P1230, M1-A1229, M1-A1228, M1-P1227, M1-A1226, M1-K1225, M1-P1224, M1-E1223, M1-K1222, M1-A1221, M1-V1220, M1-S1219, M1-H1218, M1-S1217, M1-S1216, M1-T1215, M1-A1214, M1-R1213, M1-H1212, M1-L1211, M1-S1210, M1-L1209, M1-S1208, M1-R1207, M1-E1206, M1-K1205, M1-F1204, M1-I1203, M1-S1202, M1-E1201, M1-L1200, M1-K1199, M1-E1198, M1-I1197, M1-G1196, M1-G1195, M1-K1194, M1-D1193, M1-K1192, M1-M1191, M1-N1190, M1-V1189, M1-S1188, M1-Y1187, M1-F1186, M1-S1185, M1-H1184, M1-S1183, M1-R1182, M1-M1181, M1-R1180, M1-P1179, M1-M1178, M1-L1177, M1-T1176, M1-P1175, M1-S1174, M1-T1173, M1-P1172, M1-S1171, M1-M1170, M1-T1169, M1-E1168, M1-E1167, M1-G1166, M1-A1165, M1-P1164, M1-D1163, M1-I1162, M1-S1161, M1-E1160, M1-Q1159, M1-L1158, M1-K1157, M1-F1156, M1-T1155, M1-N1154, M1-G1153, M1-E1152, M1-Q1151, M1-S1150, M1-N1149, M1-F1148, M1-S1147, M1-S1146, M1-Q1145, M1-R1144, M1-V1143, M1-I1142, M1-Y1141, M1-A1140, M1-A1139, M1-D1138, M1-T1137, M1-C1136, M1-D1135, M1-S1134, M1-S1133, M1-T1132, M1-R1131, M1-S1130, M1-R1129, M1-I1128, M1-K1127, M1-N1126, M1-S1125, M1-E1124, M1-A1123, M1-R1122, M1-E1121, M1-L1120, M1-G1119, M1-T1118, M1-L1117, M1-R1116, M1-E1115, M1-L1114, M1-A1113, M1-T1112, M1-A1111, M1-M1110, M1-R1109, M1-G1108, M1-I1107, M1-L1106, M1-D1105, M1-E1104, M1-L1103, M1-Q1102, M1-A1101, M1-L1100, M1-R1099, M1-I1098, M1-D1097, M1-V1096, M1-T1095, M1-Q1094, M1-L1093, M1-S1092, M1-A1091, M1-K1090, M1-M1089, M1-S1088, M1-H1087, M1-E1086, M1-R1085, M1-E1084, M1-N1083, M1-V1082, M1-E1081, M1-E1080, M1-L1079, M1-R1078, M1-M1077, M1-S1076, M1-M1075, M1-N1074, M1-E1073, M1-V1072, M1-R1071, M1-E1070, M1-S1069, M1-T1068, M1-V1067, M1-R1066, M1-I1065, M1-R1064, M1-E1063, M1-D1062, M1-N1061, M1-S1060, M1-S1059, M1-N1058, M1-F1057, M1-R1056, M1-D1055, M1-D1054, M1-K1053, M1-E1052, M1-R1051, M1-F1050, M1-Y1049, M1-E1048, M1-E1047, M1-I1046, M1-C1045, M1-Q1044, M1-E1043, M1-E1042, M1-F1041, M1-D1040, M1-H1039, M1-V1038, M1-K1037, M1-K1036, M1-L1035, M1-E1034, M1-D1033, M1-D1032, M1-T1031, M1-I1030, M1-F1029, M1-L1028, M1-K1027, M1-L1026, M1-G1025, M1-Y1024, M1-D1023, M1-R1022, M1-E1021, M1-D1020, M1-P1019, M1-D1018, M1-S1017, M1-E1016, M1-H1015, M1-K1014, M1-R1013, M1-W1012, M1-R1011, M1-C1010, M1-C1009, M1-L1008, M1-H1007, M1-Q1006, M1-F1005, M1-I1004, M1-M1003, M1-T1002, M1-M1001, M1-H1000, M1-S999, M1-F998, M1-I997, M1-I996, M1-L995, M1-P994, M1-P993, M1-P992, M1-L991, M1-V990, M1-P989, M1-R988, M1-E987, M1-H986, M1-F985, M1-T984, M1-M983, M1-I982, M1-L981, M1-Q980, M1-Y979, M1-R978, M1-Q977, M1-F976, M1-K975, M1-W974, M1-V973, M1-Q972, M1-N971, M1-S970, M1-I969, M1-S968, M1-K967, M1-V966, M1-E965, M1-F964, M1-F963, M1-T962, M1-N961, M1-N960, M1-F959, M1-V958, M1-A957, M1-I956, M1-L955, M1-L954, M1-N953, M1-V952, M1-L951, M1-L950, M1-I949, M1-N948, M1-A947, M1-V946, M1-L945, M1-L944, M1-Y943, M1-C942, M1-A941, M1-M940, M1-I939, M1-A938, M1-P937, M1-V936, M1-I935, M1-W934, M1-A933, M1-G932, M1-T931, M1-K930, M1-C929, M1-P928, M1-P927, M1-L926, M1-Q925, M1-I924, M1-I923, M1-K922, M1-G921, M1-D920, M1-E919, M1-R918, M1-T917, M1-E916, M1-N915, M1-Q914, M1-G913, M1-C912, M1-P911, M1-P910, M1-D909, M1-I908, M1-Q907, M1-D906, M1-A905, M1-F904, M1-V903, M1-E902, M1-G901, M1-Y900, M1-I899, M1-M898, M1-W897, M1-Y896, M1-P895, M1-M894, M1-Y893, M1-F892, M1-I891, M1-N890, M1-K889, M1-A888, M1-L887, M1-K886, M1-W885, M1-S884, M1-P883, M1-E882, M1-E881, M1-N880, M1-P879, M1-F878, M1-L877, M1-I876, M1-A875, M1-Q874, M1-R873, M1-A872, M1-V871, M1-G870, M1-F869, M1-S868, M1-M867, M1-L866, M1-V865, M1-V864, M1-L863, M1-M862, M1-I861, M1-I860, M1-V859, M1-F858, M1-Y857, M1-M856, M1-M855, M1-D854, M1-I853, M1-M852, M1-M851, M1-K850, M1-G849, M1-I848, M1-M847, M1-M846, M1-V845, M1-Y844, M1-P843, M1-G842, M1-L841, M1-Y840, M1-K839, M1-N838, M1-V837, M1-G836, M1-F835, M1-I834, M1-D833, M1-L832, M1-L831, M1-R830, M1-I829, M1-Y828, M1-W827, M1-Y826, M1-I825, M1-I824, M1-N823, M1-V822, M1-C821, M1-Y820, M1-I819, M1-V818, M1-R817, M1-G816, M1-D815, M1-S814, M1-R813, M1-F812, M1-P811, M1-Q810, M1-D809, M1-Q808, M1-L807, M1-R806, M1-L805, M1-I804, M1-M803, M1-G802, M1-V801, M1-S800, M1-F799, M1-L798, M1-L797, M1-I796, M1-A795, M1-I794, M1-L793, M1-D792, M1-T791, M1-V790, M1-N789, M1-W788, M1-Y787, M1-E786, M1-Q785, M1-L784, M1-W783, M1-V782, M1-K781, M1-V780, M1-K779, M1-Q778, M1-L777, M1-L776, M1-K775, M1-G774, M1-P773, M1-E772, M1-S771, M1-M770, M1-L769, M1-I768, M1-E767, M1-R766, M1-M765, M1-K764, M1-E763, M1-I762, M1-G761, M1-L760, M1-T759, M1-F758, M1-I757, M1-Y756, M1-S755, M1-I754, M1-V753, M1-I752, M1-W751, M1-E750, M1-Q749, M1-T748, M1-S747, M1-P746, M1-W745, M1-R744, M1-E743, M1-M742, M1-K741, M1-V740, M1-L739, M1-V738, M1-I737, M1-Y736, M1-N735, M1-F734, M1-L733, M1-M732, M1-L731, M1-Y730, M1-G729, M1-I728, M1-Y727, M1-A726, M1-L725, M1-T724, M1-Y723, M1-F722, M1-W721, M1-F720, M1-K719, M1-V718, M1-I717, M1-P716, M1-A715, M1-N714, M1-Y713, M1-F712, M1-E711, M1-Y710, M1-I709, M1-K708, M1-R707, M1-G706, M1-L705, M1-P704, M1-I703, M1-L702, M1-R701, M1-H700, M1-K699, M1-S698, M1-Q697, M1-V696, M1-E695, M1-E694, M1-E693, M1-D692, M1-K691, M1-K690, M1-R689, M1-S688, M1-S687, M1-E686, M1-G685, M1-N684, M1-N683, M1-R682, M1-G681, M1-L680, M1-M679, M1-A678, M1-T677, M1-L676, M1-E675, M1-M674, M1-D673, M1-E672, M1-E671, M1-E670, M1-K669, M1-E668, M1-K667, M1-T666, M1-P665, M1-K664, M1-E663, M1-P662, M1-E661, M1-E660, M1-A659, M1-E658, M1-K657, M1-E656, M1-Q655, M1-L654, M1-H653, M1-I652, M1-E651, M1-Q650, M1-A649, M1-Q648, M1-S647, M1-M646, M1-Y645, M1-P644, M1-M643, M1-D642, M1-D641, M1-K640, M1-N639, M1-K638, M1-F637, M1-E636, M1-L635, M1-S634, M1-L633, M1-I632, M1-S631, M1-P630, M1-P629, M1-L628, M1-L627, M1-I626, M1-G625, M1-L624, M1-I623, M1-V622, M1-K621, M1-L620, M1-G619, M1-S618, M1-N617, M1-K616, M1-R615, M1-M614, M1-R613, M1-L612, M1-R611, M1-G610, M1-M609, M1-W608, M1-M607, M1-D606, M1-T605, M1-L604, M1-L603, M1-M602, M1-Q601, M1-S600, M1-C599, M1-T598, M1-H597, M1-A596, M1-I595, M1-F594, M1-D593, M1-R592, M1-H591, M1-K590, M1-A589, M1-A588, M1-V587, M1-A586, M1-L585, M1-Q584, M1-L583, M1-C582, M1-T581, M1-A580, M1-N579, M1-S578, M1-W577, M1-N576, M1-K575, M1-L574, M1-E573, M1-Y572, M1-T571, M1-L570, M1-L569, M1-K568, M1-M567, M1-A566, M1-L565, M1-Q564, M1-E563, M1-D562, M1-Q561, M1-K560, M1-Y559, M1-S558, M1-Q557, M1-D556, M1-L555, M1-L554, M1-E553, M1-V552, M1-A551, M1-L550, M1-Q549, M1-G548, M1-F547, M1-D546, M1-R545, M1-S544, M1-N543, M1-H542, M1-N541, M1-L540, M1-E539, M1-Q538, M1-S537, M1-I536, M1-D535, M1-D534, M1-V533, M1-M532, M1-D531, M1-N530, M1-E529, M1-S528, M1-A527, M1-E526, M1-H525, M1-A524, M1-M523, M1-A522, M1-K521, M1-C520, M1-L519, M1-K518, M1-C517, M1-A516, M1-V515, M1-L514, M1-A513, M1-K512, M1-A511, M1-M510, M1-A509, M1-E508, M1-E507, M1-G506, M1-H505, M1-Q504, M1-W503, M1-F502, M1-F501, M1-L500, M1-A499, M1-M498, M1-K497, M1-Q496, M1-R495, M1-K494, M1-M493, M1-L492, M1-V491, M1-A490, M1-W489, M1-V488, M1-M487, M1-L486, M1-E485, M1-H484, M1-F483, M1-P482, M1-F481, M1-P480, M1-F479, M1-H478, M1-N477, M1-I476, M1-E475, M1-P474, M1-D473, M1-D472, M1-L471, M1-D470, M1-I469, M1-D468, M1-V467, M1-E466, M1-E465, M1-E464, M1-R463, M1-K462, M1-K461, M1-T460, M1-T459, M1-K458, M1-R457, M1-G456, M1-R455, M1-R454, M1-L453, M1-P452, M1-I451, M1-D450, M1-D449, M1-E448, M1-M447, M1-G446, M1-L445, M1-L444, M1-K443, M1-L442, M1-A441, M1-K440, M1-P439, M1-R438, M1-K437, M1-P436, M1-G435, M1-F434, M1-L433, M1-N432, M1-H431, M1-Y430, M1-L429, M1-T428, M1-R427, M1-F426, M1-R425, M1-K424, M1-R423, M1-T422, M1-Y421, M1-N420, M1-C419, M1-R418, M1-Y417, M1-A416, M1-G415, M1-G414, M1-M413, M1-L412, M1-Y411, M1-E410, M1-I409, M1-V408, M1-L407, M1-G406, M1-I405, M1-D404, M1-I403, M1-L402, M1-S401, M1-I400, M1-R399, M1-Y398, M1-D397, M1-P396, M1-P395, M1-L394, M1-N393, M1-G392, M1-K391, M1-K390, M1-V389, M1-D388, M1-R387, M1-V386, M1-L385, M1-H384, M1-Y383, M1-L382, M1-T381, M1-N380, M1-S379, M1-P378, M1-G377, M1-H376, M1-R375, M1-T374, M1-N373, M1-Y372, M1-L371, M1-E370, M1-E369, M1-L368, M1-R367, M1-S366, M1-I365, M1-T364, M1-L363, M1-F362, M1-R361, M1-H360, M1-M359, M1-S358, M1-V357, M1-G356, M1-N355, M1-E354, M1-I353, M1-L352, M1-L351, M1-K350, M1-V349, M1-F348, M1-D347, M1-V346, M1-R345, M1-D344, M1-L343, M1-V342, M1-L341, M1-A340, M1-D339, M1-L338, M1-M337, M1-A336, M1-Q335, M1-E334, M1-L333, M1-S332, M1-G331, M1-V330, M1-P329, M1-W328, M1-Q327, M1-Q326, M1-G325, M1-Y324, M1-I323, M1-F322, M1-I321, M1-Q320, M1-S319, M1-R318, M1-A317, M1-I316, M1-D315, M1-V314, M1-R313, M1-N312, M1-W311, M1-A310, M1-L309, M1-A308, M1-L307, M1-S306, M1-L305, M1-Q304, M1-D303, M1-P302, M1-A301, M1-S300, M1-A299, M1-N298, M1-A297, M1-G296, M1-K295, M1-L294, M1-L293, M1-A292, M1-T291, M1-L290, M1-I289, M1-A288, M1-L287, M1-D286, M1-I285, M1-D284, M1-Q283, M1-H282, M1-G281, M1-E280, M1-S279, M1-G278, M1-M277, M1-R276, M1-F275, M1-V274, M1-T273, M1-I272, M1-L271, M1-E270, M1-K269, M1-K268, M1-K267, M1-M266, M1-C265, M1-E264, M1-M263, M1-L262, M1-I261, M1-I260, M1-F259, M1-L258, M1-H257, M1-Q256, M1-A255, M1-Q254, M1-T253, M1-R252, M1-T251, M1-Y250, M1-T249, M1-F248, M1-T247, M1-K246, M1-Q245, M1-I244, M1-T243, M1-V242, M1-L241, M1-L240, M1-Q239, M1-D238, M1-R237, M1-L236, M1-S235, M1-E234, M1-N233, M1-I232, M1-L231, M1-G230, M1-G229, M1-E228, M1-E227, M1-S226, M1-Y225, M1-K224, M1-H223, M1-G222, M1-F221, M1-A220, M1-L219, M1-I218, M1-D217, M1-S216, M1-A215, M1-R214, M1-G213, M1-S212, M1-G211, M1-D210, M1-C209, M1-V208, M1-V207, M1-V206, M1-P205, M1-V204, M1-P203, M1-P202, M1-T201, M1-D200, M1-R199, M1-L198, M1-Y197, M1-E196, M1-L195, M1-V194, M1-I193, M1-S192, M1-I191, M1-V190, M1-N189, M1-P188, M1-G187, M1-G186, M1-E185, M1-V184, M1-I183, M1-L182, M1-A181, M1-V180, M1-V179, M1-P178, M1-V177, M1-G176, M1-Q175, M1-G174, M1-I173, M1-R172, M1-T171, M1-N170, M1-I169, M1-K168, M1-Q167, M1-L166, M1-S165, M1-I164, M1-H163, M1-K162, M1-E161, M1-L160, M1-Q159, M1-R158, M1-R157, M1-L156, M1-K155, M1-V154, M1-E153, M1-A152, M1-G151, M1-Y150, M1-K149, M1-G148, M1-T147, M1-T146, M1-G145, M1-N144, M1-D143, M1-A142, M1-L141, M1-I140, M1-F139, M1-H138, M1-S137, M1-H136, M1-M135, M1-S134, M1-N133, M1-L132, M1-V131, M1-T130, M1-L129, M1-K128, M1-S127, M1-M126, M1-P125, M1-N124, M1-S123, M1-M122, M1-T121, M1-Q120, M1-Y119, M1-P118, M1-R117, M1-V116, M1-V115, M1-D114, M1-R113, M1-G112, M1-I111, M1-L110, M1-D109, M1-E108, M1-Q107, M1-N106, M1-E105, M1-V104, M1-I103, M1-G102, M1-W10, M1-P100, M1-A99, M1-I98, M1-G97, M1-I96, M1-T95, M1-C94, M1-I93, M1-K92, M1-G91, M1-R90, M1-S89, M1-K88, M1-S87, M1-A86, M1-H85, M1-D84, M1-K83, M1-L82, M1-A81, M1-D80, M1-G79, M1-V78, M1-H77, M1-R76, M1-I75, M1-V74, M1-G73, M1-T72, M1-N71, M1-V70, M1-G69, M1-G68, M1-T67, M1-F66, M1-I65, M1-W64, M1-A63, M1-G62, M1-T61, M1-T60, M1-M59, M1-A58, M1-A57, M1-K56, M1-I55, M1-L54, M1-G53, M1-K52, M1-G51, M1-F50, M1-V49, M1-Q48, M1-K47, M1-L46, M1-K45, M1-P44, M1-Q43, M1-L42, M1-E41, M1-F40, M1-N39, M1-Q38, M1-L37, M1-G36, M1-G35, M1-H34, M1-V33, M1-S32, M1-I31, M1-L30, M1-L29, M1-K28, M1-P27, M1-L26, M1-E25, M1-L24, M1-Q23, M1-W22, M1-E21, M1-K20, M1-T19, M1-M18, M1-L17, M1-H16, M1-L15, M1-L14, M1-L13, M1-D12, M1-P11, M1-K10, M1-T9, M1-D8, and/or M1-F7 of SEQ ID NO:2. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3 deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
Alternatively, preferred polypeptides of the present invention may comprise polypeptide sequences corresponding to, for example, internal regions of the LTRPC3 polypeptide (e.g., any combination of both N- and C-terminal LTRPC3 polypeptide deletions) of SEQ ID NO:2. For example, internal regions could be defined by the equation: amino acid NX to amino acid CX, wherein NX refers to any N-terminal deletion polypeptide amino acid of LTRPC3 (SEQ ID NO:2), and where CX refers to any C-terminal deletion polypeptide amino acid of LTRPC3 (SEQ ID NO:2). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.
The LTRPC3 polypeptides of the present invention were determined to comprise several phosphorylation sites based upon the Motif algorithm (Genetics Computer Group, Inc.). The phosphorylation of such sites may regulate some biological activity of the LTRPC3 polypeptide. For example, phosphorylation at specific sites may be involved in regulating the proteins ability to associate or bind to other molecules (e.g., proteins, ligands, substrates, DNA, etc.). In the present case, phosphorylation may modulate the ability of the LTRPC3 polypeptide to associate with other potassium channel alpha subunits, beta subunits, or its ability to modulate potassium channel function.
Specifically, the LTRPC3 polypeptide was predicted to comprise two tyrosine phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). Such sites are phosphorylated at the tyrosine amino acid residue. The consensus pattern for tyrosine phosphorylation sites are as follows: [RK]-x(2)-[DE]-x(3)-Y, or [RK]-x(3)-[DE]-x(2)-Y, where Y represents the phosphorylation site and ‘x’ represents an intervening amino acid residue. Additional information specific to tyrosine phosphorylation sites can be found in Patschinsky T., Hunter T., Esch F. S., Cooper J. A., Sefton B. M., Proc. Natl. Acad. Sci. U.S.A. 79:973-977 (1982); Hunter T., J. Biol. Chem. 257:4843-4848 (1982), and Cooper J. A., Esch F. S., Taylor S. S., Hunter T., J. Biol. Chem. 259:7835-7841(1984), which are hereby incorporated herein by reference.
In preferred embodiments, the following tyrosine phosphorylation site polypeptides are encompassed by the present invention: LSLEFKNKDDMPYMSQAQ (SEQ ID NO:58), and/or VMMIGKMMIDMMYFVIIM (SEQ ID NO:59). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these LTRPC3 tyrosine phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The LTRPC3 polypeptide was predicted to comprise twenty three PKC phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). In vivo, protein kinase C exhibits a preference for the phosphorylation of serine or threonine residues. The PKC phosphorylation sites have the following consensus pattern: [ST]-x-[RK], where S or T represents the site of phosphorylation and ‘x’ an intervening amino acid residue. Additional information regarding PKC phosphorylation sites can be found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem. 161:177-184 (1986), and Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem. 260:12492-12499 (1985); which are hereby incorporated by reference herein.
In preferred embodiments, the following PKC phosphorylation site polypeptides are encompassed by the present invention: ADNGTTGKYGAEV (SEQ ID NO:60), VVCDGSGRASDIL (SEQ ID NO:61), GLINESLRDQLLV (SEQ ID NO:62), YRCNYTRKRFRTL (SEQ ID NO:63), RRGRKTTKKREEE (SEQ ID NO:64), RGRKTTKKREEEV (SEQ ID NO:65), ELLDQSYKQDEQL (SEQ ID NO:66), RNNGESSRKKDEE (SEQ ID NO:67), NNGESSRKKDEEE (SEQ ID NO:68), PNEEPSWKLAKNI (SEQ ID NO:69), RIRVTSERVENMS (SEQ ID NO:70), RVENMSMRLEEVN (SEQ ID NO:71), NEREHSMKASLQT (SEQ ID NO:72), LERAESNKIRSRT (SEQ ID NO:73), SQEGNTFKLQESI (SEQ ID NO:74), AIVPDSRRPSSCI (SEQ ID NO:75), ATLAPTDRPPSRS (SEQ ID NO:76), IERSKSSRYLATT (SEQ ID NO:77), QEGDNSERTLSNN (SEQ ID NO:78), APYAHTRKSFSIS (SEQ ID NO:79), KSFSISDKLDRQR (SEQ ID NO:80), FQRSKSSKPEGRG (SEQ ID NO:81), and/or RGDSLSMRRLSRT (SEQ ID NO:82). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these LTRPC3 PKC phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The present invention also encompasses immunogenic and/or antigenic epitopes of the LTRPC3 polypeptide.
The LTRPC3 polypeptide has been shown to comprise twelve glycosylation sites according to the Motif algorithm (Genetics Computer Group, Inc.). As discussed more specifically herein, protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.
Asparagine phosphorylation sites have the following consensus pattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation site. However, it is well known that that potential N-glycosylation sites are specific to the consensus sequence Asn-Xaa-Ser/Thr. However, the presence of the consensus tripeptide is not sufficient to conclude that an asparagine residue is glycosylated, due to the fact that the folding of the protein plays an important role in the regulation of N-glycosylation. It has been shown that the presence of proline between Asn and Ser/Thr will inhibit N-glycosylation; this has been confirmed by a recent statistical analysis of glycosylation sites, which also shows that about 50% of the sites that have a proline C-terminal to Ser/Thr are not glycosylated. Additional information relating to asparagine glycosylation may be found in reference to the following publications, which are hereby incorporated by reference herein: Marshall R. D., Annu. Rev. Biochem. 41:673-702 (1972); Pless D. D., Lennarz W. J., Proc. Natl. Acad. Sci. U.S.A. 74:134-138 (1977); Bause E., Biochem. J. 209:331-336 (1983); Gavel Y., von Heijne G., Protein Eng. 3:433-442 (1990); and Miletich J. P., Broze G. J. Jr., J. Biol. Chem. 265:11397-11404 (1990).
In preferred embodiments, the following asparagine glycosylation site polypeptides are encompassed by the present invention: FILADNGTTGKYGA (SEQ ID NO:83), EGGLINESLRDQLL (SEQ ID NO:84), LLKGANASAPDQLS (SEQ ID NO:85), GAYRCNYTRKRFRT (SEQ ID NO:86), TYELKNWSNATCLQ (SEQ ID NO:87), LKNWSNATCLQLAV (SEQ ID NO:88), LQEYWNVTDLIAIL (SEQ ID NO:89), PPCGQNETREDGKI (SEQ ID NO:90), LIAVFNNTFFEVKS (SEQ ID NO:91), KDDRFNSSNDERIR (SEQ ID NO:92), SERVENMSMRLEEV (SEQ ID NO:93), and/or RTLSNNITVPKIER (SEQ ID NO:94). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these LTRPC3 asparagine glycosylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The LTRPC3 polypeptide has been shown to comprise one RGD cell attachment site domain according to the Motif algorithm (Genetics Computer Group, Inc.). The sequence Arg-Gly-Asp, found in fibronectin, is crucial for its interaction with its cell surface receptor, an integrin. What has been called the ‘RGD’ tripeptide is also found in the sequences of a number of other proteins, where it has been shown to play a role in cell adhesion. Non-limiting examples of these proteins are the following: some forms of collagens, fibrinogen, vitronectin, von Willebrand factor (VWF), snake disintegrins, and slime mold discoidins. The ‘RGD’ tripeptide is also found in other proteins where it may serve the same purpose. A consensus pattern for RGD cell attachment sites is the following: R-G-D. Additional information relating to RGD cell attachment site domains may be found in reference to the following publications, which are hereby incorporated by reference herein: Ruoslahti E., Pierschbacher M. D., Cell 44:517-518 (1986); and d'Souza S. E., Ginsberg M. H., Plow E. F., Trends Biochem. Sci. 16:246-250 (1991).
In preferred embodiments, the following RGD cell attachment site domain polypeptide is encompassed by the present invention: SKPEGRGDSLSMR (SEQ ID NO:95). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this RGD cell attachment site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The LTRPC3 polypeptide has been shown to comprise one aminoacyl-transfer RNA synthetases class-II domain according to the Motif algorithm (Genetics Computer Group, Inc.). Aminoacyl-tRNA synthetases (EC 6.1.1.-) are a group of enzymes which activate amino acids and transfer them to specific tRNA molecules as the first step in protein biosynthesis. In prokaryotic organisms there are at least twenty different types of aminoacyl-tRNA synthetases, one for each different amino acid. In eukaryotes there are generally two aminoacyl-tRNA synthetases for each different amino acid: one cytosolic form and a mitochondrial form. While all these enzymes have a common function, they are widely diverse in terms of subunit size and of quaternary structure.
The synthetases specific for alanine, asparagine, aspartic acid, glycine, histidine, lysine, phenylalanine, proline, serine, and threonine are referred to as class-II synthetases and probably have a common folding pattern in their catalytic domain for the binding of ATP and amino acid which is different to the Rossmann fold observed for the class I synthetases.
Class-II tRNA synthetases do not share a high degree of similarity, however at least three conserved regions are present.
The consensus pattern for aminoacyl-transfer RNA synthetases class-II domains are as follows: [FYH]-R-x-[DE]-x(4,12)-[RH]-x(3)-F-x(3)-[DE]; and [GSTALVF]-{DENQHRKP}-[GSTA]-[LIVMF]-[DE]-R-[LIVMF]-x-[LIVMSTAG]-[LIVMFY], where ‘x’ represents an intervening amino acid residue.
Additional information specific to aminoacyl-transfer RNA synthetases class-II domains may be found in reference to the following publications, Schimmel P., Annu. Rev. Biochem. 56:125-158 (1987); Delarue M., Moras D., BioEssays 15:675-687 (1993); Schimmel P., Trends Biochem. Sci. 16:1-3 (1991); Nagel G. M., Doolittle R. F., Proc. Natl. Acad. Sci. U.S.A. 88:8121-8125 (1991); Cusack S., Haertlein M., Leberman R., Nucleic Acids Res. 19:3489-3498 (1991); Cusack S., Biochimie 75:1077-1081 (1993); Cusack S., Berthet-Colominas C., Haertlein M., Nassar N., Leberman R., Nature 347:249-255 (1990); and Leveque F., Plateau P., Dessen P., Blanquet S., Nucleic Acids Res. 18:305-312 (1990); which are hereby incorporated herein by reference in their entirety.
In preferred embodiments, the following aminoacyl-transfer RNA synthetases class-II domain polypeptide is encompassed by the present invention: LIGRMATALERLTGLERAES (SEQ ID NO:96). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this RGD cell attachment site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The LTRPC3 polypeptide has been shown to comprise three amidation sites according to the Motif algorithm (Genetics Computer Group, Inc.). The precursor of hormones and other active peptides which are C-terminally amidated is always directly followed by a glycine residue which provides the amide group, and most often by at least two consecutive basic residues (Arg or Lys) which generally function as an active peptide precursor cleavage site. Although all amino acids can be amidated, neutral hydrophobic residues such as Val or Phe are good substrates, while charged residues such as Asp or Arg are much less reactive. A consensus pattern for amidation sites is the following: x-G-[RK]-[RK], wherein “X” represents the amidation site. Additional information relating to asparagine glycosylation may be found in reference to the following publications, which are hereby incorporated by reference herein: Kreil G., Meth. Enzymol. 106:218-223(1984); and Bradbury A. F., Smyth D. G., Biosci. Rep. 7:907-916 (1987).
In preferred embodiments, the following amidation site polypeptides are encompassed by the present invention: DIPLRRGRKTTKKR (SEQ ID NO:97), HRLIPLGRKIYEFY (SEQ ID NO:98), and/or EENEAKGRRATIAI (SEQ ID NO:99). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these LTRPC3 amidation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:1 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 4651 of SEQ ID NO:1, b is an integer between 15 to 4665, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:1, and where b is greater than or equal to a+14.
Features of the Polypeptide Encoded by Gene No:2
The polypeptide of this gene provided as SEQ ID NO:4 (FIGS. 2A-F), encoded by the polynucleotide sequence according to SEQ ID NO:3 (FIGS. 2A-F), and/or encoded by the polynucleotide contained within the deposited clone, LTRPC3b, has significant homology at the nucleotide and amino acid level to the human melastatin 1 protein (Melastatin1; Genbank Accession No. gi|3243075; SEQ ID NO:11). An alignment of the LTRPC3b polypeptide with this protein is provided in FIGS. 5A-D.
The LTRPC3b polypeptide was determined to share 65.7% identity and 73.5% similarity with the human melastatin 1 protein (Melastatin1; Genbank Accession No. gi|3243075; SEQ ID NO:11) as shown in FIG. 8.
The LTRPC3b protein is believed to represent a member of a new class of protein kinases referred to as alpha kinases (Curr. Biol. 9 (2), R43-R45 (1999)). These kinases represent a novel type of signaling molecule comprising both a catalytic protein kinase domain, in addition to, an ion channel domain. This family is commonly referred to as the transient receptor potential channel (TRP) family. Melastatin1 defines a separate subfamily of TRP channels referred to as TRPM (melastatin1). TRPM family members are characteristic of their unusually long cytoplasmic tails at both ends of the channel domain and some of the family members contain an enzyme domain at the C-terminal region. Thus, LTRPC3 represents a novel member of the TRPM subfamily.
The melastatin1 protein is believed to be negatively associated with the incidence of melanoma based upon its inverse correlative expression in highly aggressive melanomas (Genomics 54 (1), 116-123 (1998)). Thus, overexpression of melastatin1 could represent a novel therapeutic in the treatment of melanoma and potentially other cancers.
Based upon the observed homology, the polypeptide of the present invention is expected to share at least some biological activity with other transient receptor potential channel family members, more specifically with the melastatin1 protein, in addition to, other transient receptor potential channel family members referenced elsewhere herein or otherwise known in the art.
Most of the known transient receptor potential channel family members, possess one or more transmembrane domains. Likewise, the LTRPC3b polypeptide has been determined to comprise six transmembrane domains (TM1-TM6) as shown in FIGS. 2A-F. The transmembrane domains are located from about amino acid 632 to about amino acid 649 (TM1), from about amino acid 729 to about amino acid 746 (TM2), from about amino acid 802 to about amino acid 815 (TM3), from about amino acid 829 to about amino acid 846 (TM4), from about amino acid 863 to about amino acid 880 (TM5), and/or from about amino acid 950 to about amino acid 970 (TM6) of SEQ ID NO:4. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of the above referenced polypeptide.
In preferred embodiments, the following transmembrane domain polypeptides are encompassed by the present invention: LKVILGILLPPSILSLEF (SEQ ID NO:32), IVKFWFYTLAYIGYLMLF (SEQ ID NO:33), VTDLIAILLFSVGM (SEQ ID NO:34), RVIYCVNIIYWYIRLLDI (SEQ ID NO:35), MMIDMMYFVIIMLVVLMS (SEQ ID NO:36), and/or AIMACYLLVANILLVNLLIAV (SEQ ID NO:37). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the LTRPC3b transmembrane polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The present invention also encompasses polypeptides corresponding to the regions between each transmembrane domain of LTRPC3b, referred to herein as inter-transmembrane domain polypeptides (inter TM1-2, inter TM2-3, inter TM3-4, inter TM4-5, and inter TM5-6). Such regions are typically solvent accessible (either extracellularly or intracellularly). Therefore, these regions are amenable to the development of agonists and/or antagonists to the LTRPC3b polypeptide, particularly for the development of antibodies specific to these regions.
In preferred embodiments, the following inter-transmembrane domain polypeptides are encompassed by the present invention: KNKDDMPYMSQAQEIHLQEKEAEEPEKPTKEKEEEDMELTAMLGRNNGESS RKKDEEEVQSKHRLIPLGRKIYEFYNAP (SEQ ID NO:100), NYIVLVKMERWPSTQEWIVISYIFTLGIEKMREILMSEPGKLLQKVKVWLQEY WN (SEQ ID NO:101), ILRLQDQPFRSDG (SEQ ID NO:102), FGVNKYLGPYVMMIGK (SEQ ID NO:103), and/or FGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFADQIDPPCGQNETRED GKIIQLPPCKTGAWIVP (SEQ ID NO:104). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the LTRPC3b inter-transmembrane polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3b inter TM1-2 domain deletion polypeptides are encompassed by the present invention: K1-P79, N2-P79, K3-P79, D4-P79, D5-P79, M6-P79, P7-P79, Y8-P79, M9-P79, S10-P79, Q11-P79, A12-P79, Q13-P79, E14-P79, I15-P79, H16-P79, L17-P79, Q18-P79, E19-P79, K20-P79, E21-P79, A22-P79, E23-P79, E24-P79, P25-P79, E26-P79, K27-P79, P28-P79, T29-P79, K30-P79, E31-P79, K32-P79, E33-P79, E34-P79, E35-P79, D36-P79, M37-P79, E38-P79, L39-P79, T40-P79, A41-P79, M42-P79, L43-P79, G44-P79, R45-P79, N46-P79, N47-P79, G48-P79, E49-P79, S50-P79, S51-P79, R52-P79, K53-P79, K54-P79, D55-P79, E56-P79, E57-P79, E58-P79, V59-P79, Q60-P79, S61-P79, K62-P79, H63-P79, R64-P79, L65-P79, I66-P79, P67-P79, L68-P79, G69-P79, R70-P79, K71-P79, I72-P79, and/or Y73-P79 of SEQ ID NO:100. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3b inter TM1-2 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3b inter TM1-2 domain deletion polypeptides are encompassed by the present invention: K1-P79, K1-A78, K1-N77, K1-Y76, K1-F75, K1-E74, K1-Y73, K1-I72, K1-K71, K1-R70, K1-G69, K1-L68, K1-P67, K1-I66, K1-L65, K1-R64, K1-H63, K1-K62, K1-S61, K1-Q60, K1-V59, K1-E58, K1-E57, K1-E56, K1-D55, K1-K54, K1-K53, K1-R52, K1-S51, K1-S50, K1-E49, K1-G48, K1-N47, K1-N46, K1-R45, K1-G44, K1-L43, K1-M42, K1-A41, K1-T40, K1-L39, K1-E38, K1-M37, K1-D36, K1-E35, K1-E34, K1-E33, K1-K32, K1-E31, K1-K30, K1-T29, K1-P28, K1-K27, K1-E26, K1-P25, K1-E24, K1-E23, K1-A22, K1-E21, K1-K20, K1-E19, K1-Q18, K1-L17, K1-H16, K1-I15, K1-E14, K1-Q13, K1-A12, K1-Q11, K1-S10, K1-M9, K1-Y8, and/or K1-P7 of SEQ ID NO:100. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3b inter TM1-2 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3b inter TM2-3 domain deletion polypeptides are encompassed by the present invention: F1-N56, N2-N56, Y3-N56, I4-N56, V5-N56, L6-N56, V7-N56, K8-N56, M9-N56, E10-N56, R1′-N56, W12-N56, P13-N56, S14-N56, T15-N56, Q16-N56, E17-N56, W18-N56, I19-N56, V20-N56, I21-N56, S22-N56, Y23-N56, I24-N56, F25-N56, T26-N56, L27-N56, G28-N56, I29-N56, E30-N56, K31-N56, M32-N56, R33-N56, E34-N56, I35-N56, L36-N56, M37-N56, S38-N56, E39-N56, P40-N56, G41-N56, K42-N56, L43-N56, L44-N56, Q45-N56, K46-N56, V47-N56, K48-N56, V49-N56, and/or W50-N56 of SEQ ID NO:101. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3b inter TM2-3 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3b inter TM2-3 domain deletion polypeptides are encompassed by the present invention: F1-N56, F1-W55, F1-Y54, F1-E53, F1-Q52, F1-L51, F1-W50, F1-V49, F1-K48, F1-V47, F1-K46, F1-Q45, F1-L44, F1-L43, F1-K42, F1-G41, F1-P40, F1-E39, F1-S38, F1-M37, F1-L36, F1-I35, F1-E34, F1-R33, F1-M32, F1-K31, F1-E30, F1-I29, F1-G28, F1-L27, F1-T26, F1-F25, F1-I24, F1-Y23, F1-S22, F1-I21, F1-V20, F1-I19, F1-W18, F1-E17, F1-Q16, F1-T15, F1-S14, F1-P13, F1-W12, F1-R11, F1-E10, F1-M9, F1-K8, and/or F1-V7 of SEQ ID NO:101. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3b inter TM2-3 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3b inter TM3-4 domain deletion polypeptides are encompassed by the present invention: I1-G13, L2-G13, R3-G13, L4-G13, Q5-G13, D6-G13, and/or Q7-G13 of SEQ ID NO:102. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3b inter TM3-4 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3b inter TM3-4 domain deletion polypeptides are encompassed by the present invention: I1-G13, I1-D12, I1-S11, I1-R10, I1-F9, I1-P8, and/or I1-Q7 of SEQ ID NO:102. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3b inter TM3-4 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3b inter TM4-5 domain deletion polypeptides are encompassed by the present invention: F1-K16, G2-K16, V3-K16, N4-K16, K5-K16, Y6-K16, L7-K16, G8-K16, P9-K16, and/or Y10-K16 of SEQ ID NO:103. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3b inter TM4-5 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3b inter TM4-5 domain deletion polypeptides are encompassed by the present invention: F1-K16, F1-G15, F1-I14, F1-M13, F1-M12, F1-V11, F1-Y10, F1-P9, F1-G8, and/or F1-L7 of SEQ ID NO:103. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3b inter TM4-5 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3b inter TM5-6 domain deletion polypeptides are encompassed by the present invention: F1-P69, G2-P69, V3-P69, A4-P69, R5-P69, Q6-P69, A7-P69, I8-P69, L9-P69, F10-P69, P11-P69, N12-P69, E13-P69, E14-P69, P15-P69, S16-P69, W17-P69, K18-P69, L19-P69, A20-P69, K21-P69, N22-P69, I23-P69, F24-P69, Y25-P69, M26-P69, P27-P69, Y28-P69, W29-P69, M30-P69, I31-P69, Y32-P69, G33-P69, E34-P69, V35-P69, F36-P69, A37-P69, D38-P69, Q39-P69, I40-P69, D41-P69, P42-P69, P43-P69, C44-P69, G45-P69, Q46-P69, N47-P69, E48-P69, T49-P69, R50-P69, E51-P69, D52-P69, G53-P69, K54-P69, I55-P69, I56-P69, Q57-P69, L58-P69, P59-P69, P60-P69, C61-P69, K62-P69, and/or T63-P69 of SEQ ID NO:104. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3b inter TM5-6 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3b inter TM5-6 domain deletion polypeptides are encompassed by the present invention: F1-P69, F1-V68, F1-I67, F1-W66, F1-A65, F1-G64, F1-T63, F1-K62, F1-C61, F1-P60, F1-P59, F1-L58, F1-Q57, F1-I56, F1-I55, F1-K54, F1-G53, F1-D52, F1-E51, F1-R50, F1-T49, F1-E48, F1-N47, F1-Q46, F1-G45, F1-C44, F1-P43, F1-P42, F1-D41, F1-I40, F1-Q39, F1-D38, F1-A37, F1-F36, F1-V35, F1-E34, F1-G33, F1-Y32, F1-I31, F1-M30, F1-W29, F1-Y28, F1-P27, F1-M26, F1-Y25, F1-F24, F1-I23, F1-N22, F1-K21, F1-A20, F1-L19, F1-K18, F1-W17, F1-S16, F1-P15, F1-E14, F1-E13, F1-N12, F1-P11, F1-F10, F1-L9, F1-I8, and/or F1-A7 of SEQ ID NO:104. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3b inter TM5-6 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The LTRPC3b polypeptide was determined to comprise several conserved cysteines, at amino acid 94, 209, 265, 431, 529, 594, 611, 833, 924, 941, 954, 1057, 1148, and 1114 of SEQ ID No: 2 (FIGS. 1A-F). Conservation of cysteines at key amino acid residues is indicative of conserved structural features, which may correlate with conservation of protein function and/or activity.
In confirmation of the LTRPC3b representing a member of the transient receptor channel family, the LTRPC3b polypeptide was determined to comprise a predicted TRP domain (EWKFAR) located from about amino acid 985 to about amino acid 990 of SEQ ID NO:4. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of the above referenced polypeptide.
In preferred embodiments, the following TRP domain polypeptide is encompassed by the present invention: VWKFQR (SEQ ID NO:105). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of this LTRPC3b TRP domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
In further confirmation of the LTRPC3b representing a member of the transient receptor channel family, the LTRPC3b polypeptide was determined to comprise a predicted ion transport signature domain located at about amino acid 748 to about amino acid 959 of SEQ ID NO:4. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of the above referenced polypeptide.
In preferred embodiments, the following ion transport signature domain polypeptide is encompassed by the present invention: TQEWIVISYIFTLGIEKMREILMSEPGKLLQKVKVWLQEYWNVTDLIAILLFSV GMILRLQDQPFRSDGRVIYCVNIIYWYIRLLDIFGVNKYLGPYVMMIGKMMID MMYFVIIMLVVLMSFGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFAD QIDPPCGQNETREDGKIIQLPPCKTGAWIVPAIMACYLLVANILLVNLLIAVF (SEQ ID NO:109). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of this LTRPC3b ion transport signature domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The LTRPC3b polypeptide was determined to comprise a predicted coiled-coil domain located at about amino acid 1075 to about amino acid 1129 of SEQ ID NO:4. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of the above referenced polypeptide.
In preferred embodiments, the following coiled-coil domain polypeptide is encompassed by the present invention: ERIRVTSERVENMSMRLEEVNEREHSMKASLQTVDIRLAQLEDLIGRMATAL ERL (SEQ ID NO:106). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of this LTRPC3b coiled-coil domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The present invention is also directed to polynucleotides comprising at least about 45 nt of the polynucleotide sequence provided as SEQ ID NO:3. Specifically, the present invention is directed to a polynucleotide sequence comprising nucleotides from about nucleotide 1174 to about nucleotide 1212; nucleotides from about nucleotide 1174 to about nucleotide 1215; nucleotides from about nucleotide 1168 to about nucleotide 1209; nucleotides from about nucleotide 1165 to about nucleotide 1209; of SEQ ID NO:3; and/or nucleotides from about nucleotide 1162 to about nucleotide 1209. In this context “about” includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus, or at both termini.
The present invention is also directed to polypeptides comprising at least about 15 amino acids of the polypeptides sequence provided as SEQ ID NO:4. Specifically, the present invention is directed to a polypeptides sequence comprising amino acids from about amino acid 392 to about amino acid 404; amino acids from about amino acid 392 to about amino acid 405; amino acids from about amino acid 392 to about amino acid 406; amino acids from about amino acid 390 to about amino acid 403; amino acids from about amino acid 389 to about amino acid 403; and/or amino acids from about amino acid 388 to about amino acid 403 of SEQ ID NO:4. In this context “about” includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus, or at both termini.
LTRPC3b polypeptides and polynucleotides are useful for diagnosing diseases related to the over and/or under expression of LTRPC3b by identifying mutations in the LTRPC3b gene using LTRPC3b sequences as probes or by determining LTRPC3b protein or mRNA expression levels. LTRPC3b polypeptides will be useful in screens for compounds that affect the activity of the protein. LTRPC3b peptides can also be used for the generation of specific antibodies and as bait in yeast two hybrid screens to find proteins the specifically interact with LTRPC3b.
Consistent with LTRPC3 representing a transient receptor potential channel, immunohistochemical experiments have shown that LTRPC3 localizes to the cell membrane (see FIG. 17 and Example 6). Specifically, the complete open reading frame of LTRPC3 with a C-terminal HA tag was transiently transfected into HEK 293 cells to assess its cellular localization. The HA-tagged LTRPC3 was detected using a fluorescein-conjugated anti-HA antibody and a laser scanning confocal microscope which produces a green fluorescent signal. The green fluorescent signal was exclusively detected at the subplasma membrane region of the transfected cells (see FIG. 17), which is consistent with LTRPC3 being an integral membrane protein. Under these conditions approximately  ̃70% of cells are expressing LTRPC3. The expression of full-length protein was accessed with immunoblot using an anti-HA antibody and detected as the expected size of  ̃170 kD (data not shown). Cellular localization of the LTRPC3b splice variant is expected to be similarly localized to the subplasma membrane region.
Moreover, physiometric studies have shown that LTRPC3 is a functional Ca2+ permeable channel (see FIGS. 18 and 19, and Example 7). LTRPC3 function was assessed using a Fluorometric Imaging Plate Reader (FLIPRTM) that measures real-time intracellular fluorescence changes. Cells transiently transfected with vector or LTRPC3-HA were loaded with the cytoplasmic Ca2+ indicator Fluoro-4 in a 1 mM Ca2+ solution. Addition of Ca2+ to the media resulted in a concentration-dependent influx of Ca2+ into LTRPC3-expressing cells (FIG. 18; right panels), indicating that LTRPC3 is a functional Ca2+ channel. In contrast, vector-transfected cells showed minimal Ca2+ influx under the same experimental conditions (FIG. 18, left panels). The non-transfected cells were indistinguishable from the vector-transfected cells (data not shown). Therefore, LTRPC3 is a constitutively active channel capable of mediating Ca2+ influx. The Ca2+ influx activity of the LTRPC3b splice variants is expected to be similar to LTRPC3.
To further address the mechanism of LTRPC3-mediated Ca2+ entry, similar Ca2+ addition experiments were performed on transfected cells incubated ( ̃30 min) in a nominally Ca2+-free solution. Previous studies have shown that lowering extracellular Ca2+ concentration below physiological levels can deplete intracellular Ca2+ stores in many cell types including HEK 293 (EMBO J. 17, 4274-4282, (1998)). Incubating vector-transfected HEK 293 cells in a nominally Ca2+-free solution gave rise to Ca2+ entry that was dependent on the concentration of Ca2+ added to the buffers, indicating Ca2+ influx was mediated through endogenous SOCs in HEK293 cells (FIG. 18, left panels). In LTRPC3 cells, the Ca2+ transients triggered by similar Ca2+ treatment were much larger (FIG. 18, right panels). This Ca2+ entry observed in LTRPC3 cells incubated in Ca2+-free media were greater than those observed in 1 mM Ca2+ media, indicating that LTRPC3-mediated Ca2+ entry can be potentiated by the store-depletion. The store-depletion potentiation of LTRPC3-mediated Ca2+ entry is expected to be similar for the LTRPC3b splice variant.
The store-operated mechanism of LTRPC3-mediated Ca2+ influx was tested further by passively depleting Ca2+ stores with thapsigargin (TG), an inhibitor of microsomal Ca2+ ATPases that pumps ions from the cytosol back into the stores. Addition of 2 μM thapsigargin equivalently depleted Ca2+ stores in LTRPC3-HA- and vector-transfected cells (FIG. 19A). Following store depletion with TG, addition of Ca2+ to the buffer induced a much larger Ca2+ entry in LTRPC3 cells compared to the vector control cells. The increased Ca2+ entry of LTRPC3 cells, relative to non-LTRPC3 transfected cells, post store depletion with TG is expected to be similar for the LTRPC3b splice variant.
Receptor-mediated Ca2+ entry was also more pronounced in LTRPC3-HA-transfected cells. Carbachol (CCh) can activate an endogenous muscarinic receptor and trigger IP3 production, leading to store-depletion in HEK 293 cells. The addition of 50 μM of CCh caused a transient and rapid intracellular Ca2+ increase in both LTRPC3- and vector-transfected cells (FIG. 19B). After the store depletion with CCh, adding of Ca2+ to the buffer induced a much larger influx of Ca2+ into LTRPC3 cells, as compared to vector control cells. These results show that after store depletion with TG or CCh LTRPC3-transfected cells exhibit an increased Ca2+ influx when compared to control cells. The increased Ca2+ entry of LTRPC3 cells, relative to non-LTRPC3 transfected cells, post store depletion with TG or CCh is expected to be similar for the LTRPC3b splice variant.
The lanthanides, gadolinium (Gd3+) and lanthanum (La3+), are noneselective Ca2+-permeable channel blockers, often used as part of the characterization of overexpressed TRP channels. Both lanthanides blocked LTRPC3 Ca2+ conductance, although La3+ was more potent (FIG. 19C). In the presence of 1 mM Ca2+ in which endogenous SOCs is minimally activated (FIG. 18A), pre-treatment with 100 μM of La3+ and Gd3+ blocked LTRPC3 Ca2+ currents, stimulated by adding 10 mM Ca2+, by 67 and 39%, respectively. These results indicated that LTRPC3 mediated currents are not non-specific leak currents resulting from protein overexpression.
LTRPC3 is constitutively active but can be potentiated by store-depletion and is partially sensitive to La3+ and Gd3+ blockade. LTRPC3 is believed to represent the first member of the TRPM subfamily that exhibits this store-operated mechanism, although some members of TRPC subfamily have been considered for this role. TRPM1 and TRPM4a are constitutive Ca2+ permeable channels but it is unclear whether they can be stimulated by store-depletion (Proc. Natl. Acad. Sci. U.S.A. 98, 10692-10697, (2001)). Distinct from TRPM4a, TRPM4b is directly activated by changes in intracellular Ca2+ without significant permeation of Ca2+ (Cell 109, 397-401, (2002)). TRPM2 is activated by ADP-ribose, NAD and changes in redox status (Nature 411, 595-599, (2001); Science 293, 1327-1330, (2001); and Mol. Cell 9, 163-173, (2002)). TRPM7 is regulated by Mg2+-ATP and/or PIP2 (Science 291, 1043-1047, (2001); Nature 411, 690-695, (2001); and Nat. Cell Biol. 4, 329-36 (2002)). TRPM8 is activated by cold temperatures and cooling agents (Nature 416, 52-58, (2002); and Cell 108, 705-715, (2002)). Therefore, in conjunction with its fairly restricted tissue expression, which is not observed with any other family members, LTRPC3 may have a unique biological function in human.
Expression profiling designed to measure the steady state mRNA levels encoding the LTRPC3 polypeptide showed predominately high expression levels in kidney. The LTRPC3 polypeptide was also significantly expression in spinal cord, testis, and brain (as shown in FIG. 6). The expression profile of the LTRPC3b splice variant is expected to be similar to LTRPC3.
Moreover, Northern hybridizations of the LTRPC3 mRNA confirmed the predominately high expression levels in kidney, and significant expression levels in testis, and brain (as shown in FIG. 7). The Northern hybridization was not performed on spinal cord tissue.
Expanded analysis of LTRPC3 expression levels by TaqManTM quantitative PCR (see FIG. 12) confirmed that the LTRPC3 polypeptide is expressed in kidney, brain, testis (FIGS. 6 and 7), although higher expression levels were observed in brain than previously appreciated. LTRPC3 mRNA was expressed predominately in the brain, specifically the cerebellum, choroid plexus, the locus coeruleus, the posterior hypothalamus and the substantia nigra. Expression of LTRPC3 was also significantly expressed in the kidney, with higher levels observed in the cortex than in the medulla or pelvis. LTRPC3 was also significantly expressed in the spinal cord, testis, and to a lesser extent in other tissues as shown.
Therefore, LTRPC3b polynucleotides and polypeptides, including modulators or fragments thereof, may be useful in treating, diagnosing, prognosing, and/or preventing a variety of neurological conditions, in particular various choroid plexus neoplasms, choroid plexus papilloma, Alzheimer's disease, prion disorders and multiple sclerosis and movement disorders that involve the cerebellum. Based upon the expression pattern of LTRPC3 in kidney, this novel TRP family member, or a splice variant or polymorphism thereof, may also be the cause solitary metastasis in the choroid plexus, a rare type of carcinoma. For example, it has been shown that out of 15 cases of solitary metastasis of the choroid plexus, five originated from renal cell carcinoma (Neurol. Med. Chir. (Tokyo) 1997 December; 37(12):916-9). Additionally, given the rather selective expression of LTRPC3 in the choroid plexus and renal tissues, it may be possible that altered function of LTRPC3 or a splice variant or polymorphism thereof, may be responsible for solitary metastasis and renal carcinoma. LTRPC3 polynucleotides and polypeptides, including modulators or fragments thereof, may be useful in treating, diagnosing, prognosing, and/or preventing a variety of renal disorders, particularly solitary metastasis in the choroid plexus, and renal carcinoma.
Moreover, an additional analysis of LTRPC3 expression levels by TaqManTM quantitative PCR (see FIG. 13) in disease cells and tissues indicated that the LTRPC3 polypeptide is differentially expressed in renal, testicular, and ovarian tumor tissues. In the renal tumor tissue results, an average of 2 out of 3 matched samples, which represent 3 out of 4 samples total, showed a significant decrease in LTRPC3 steady state RNA levels in tumor compared to control samples. In the testicular tumor tissue results, differential expression of LTRPC3 in testicular cancers was observed with all 5 tumor samples showing a significant reduction in steady-state RNA levels compared to two control samples. In the ovarian tumor tissue results, differential expression of LTRPC3 in ovarian cancers was observed with 3 tumor samples showing a significant reduction in steady-state RNA levels compared to five control samples.
The differential expression of LTRPC3 in tumors relative to normal tissues suggests that loss of LTRPC3 expression during tumor progression might contribute to the metastatic process by altering internal calcium stores in a manner that reflects a loss of cellular control on apoptosis. Restoring LTRPC3b function might provide a novel therapeutic approach to treating certain cancers. Therefore, LTRPC3b polynucleotides and polypeptides, including modulators or fragments thereof, particularly agonists of LTRPC3b activity or expression, may be useful in treating, diagnosing, prognosing, ameloriating, and/or preventing a variety of cancers and proliferative conditions, particularly of the kidney, testis, and ovaries.
Characterization of the LTRPC3 polypeptide of the present invention using antisense oligonucleotides led to the determination that LTRPC3 is involved in the negative modulation of the FEN1 DNA base-excision repair/proliferation modulating protein as described in Example 5 herein.
Specifically, antisense inhibition of LTRPC3 resulted in upregulation of Fen1, a marker of proliferation. As such, LTRPC3 behaves in a manner similar to a tumor suppressor, in that loss results in increases in proliferation. While some increases in the proliferation markers p21 and IkB were also observed, they were less significant, and could be a response to the pro-proliferative effect of LTRPC3 antisense inhibition. Interestingly, expression of LTRPC3 was also observed to be significantly reduced in tumor tissues, relative to normal tissues (see FIG. 13). Specifically, renal, testicular, and ovarian tumor samples showed as much as 12 to 30 fold down regulation of LTRPC3 message levels compared to normal tissues. This reduction of LTRPC3 correlating with the tumor state is consistent with the negative FEN1 modulatory activity and suggests this gene may have antiproliferative activities. Therefore, for modulators of LTRPC3 to be useful for treating cancer, it would be necessary to increase the activity of the gene or gene product in order to have the reverse effect on cancerous cells. Thus, an agonist of LTRPC3 activity or a compound that increased LTRPC3 message levels would be a desired invention for cancer therapy. The same regimen may also be applicable to LTRPC3 splice variants and/or polymorphisms, such as LTRPC3b.
In preferred embodiments, LTRPC3b polynucleotides and polypeptides, including fragments and modulators thereof, are useful for treating, diagnosing, and/or ameliorating DNA-repair deficiencies, particularly base-excision repair deficiencies, Xeroderma pigmentosum, skin cancer, melanoma, UV senstivity, alkylation sensivity, gamma irradiation sensitivity, pyrimidine dimer sensitivity, chemical mutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone, Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, blood vessel dilation aberrations in the skin, blood vessel dilation aberrations in the eye, conditions involving increased levels of apurinic sites, conditions involving increased levels of apyrimidinic sites, conditions involving increased levels of abasic sites, disorders related to aberrant signal transduction, proliferating disorders, and/or cancers.
Moreover, LTRPC3b polynucleotides and polypeptides, including fragments and modulators thereof, are useful for increasing mammalian base excision repair activity, increasing mammalian single-nucleotide base excision repair activity, and/or increasing mammalian long patch base excision repair activity.
In preferred embodiments, antagonists directed against LTRPC3b are useful for treating, diagnosing, and/or ameliorating DNA-repair deficiencies, particularly base-excision repair deficiencies, Xeroderma pigmentosum, skin cancer, melanoma, UV senstivity, alkylation sensivity, gamma irradiation sensitivity, pyrimidine dimer sensitivity, chemical mutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone, Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, blood vessel dilation aberrations in the skin, blood vessel dilation aberrations in the eye, conditions involving increased levels of apurinic sites, conditions involving increased levels of apyrimidinic sites, conditions involving increased levels of abasic sites, disorders related to aberrant signal transduction, proliferating disorders, and/or cancers.
Moreover, antagonists directed against LTRPC3b are useful for increasing mammalian base excision repair activity, increasing mammalian single-nucleotide base excision repair activity, and/or increasing mammalian long patch base excision repair activity.
In preferred embodiments, agonists directed against LTRPC3b are useful for treating, diagnosing, and/or ameliorating, disorders related to aberrant signal transduction, proliferating disorders, and/or cancers, particularly renal cell carcinomas, testicular cancers, and/or ovarian cancers.
The strong homology to human transient receptor potential channels (TRP), combined with the predominate localized expression in kidney tissue of LTRPC3 suggests the LTRPC3b polynucleotides and polypeptides, including modulators or fragments thereof, may be useful in treating, diagnosing, prognosing, and/or preventing renal diseases and/or disorders, which include, but are not limited to: nephritis, renal failure, nephrotic syndrome, urinary tract infection, hematuria, proteinuria, oliguria, polyuria, nocturia, edema, hypertension, electrolyte disorders, sterile pyuria, renal osteodystrophy, large kidneys, renal transport defects, nephrolithiasis, azotemia, anuria, urinary retention, slowing of urinary stream, large prostate, flank tenderness, full bladder sensation after voiding, enuresis, dysuria, bacteriuria, kidney stones, glomerulonephritis, vasculitis, hemolytic uremic syndromes, thrombotic thrombocytopenic purpura, malignant hypertension, casts, tubulointerstitial kidney diseases, renal tubular acidosis, pyelonephritis, hydronephritis, nephrotic syndrome, crush syndrome, and/or renal colic, in addition to Wilm's Tumor Disease, and congenital kidney abnormalities such as horseshoe kidney, polycystic kidney, and Falconi's syndrome.for example.
Several known TRP family members have been identified that are expressed significantly in kidney tissue. These TRP family members include, for example, Trp12 (Wissenbach, U., Bodding, M., Freichel, M., Flockerzi, V, Lett., 485(2-3):127-34, (2000)); OTRPC4 (Strotmann, R., Harteneck, C., Nunnenmacher, K., Schultz, G., Plant, T, D, Nat, Cell, Biol., 2(10):695-702, (2000)); polycystin-L2 (Guo, L., Schreiber, T, H., Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics., 64(3):241-51, (2000)); and EcaC (Hoenderop, J. G., van, der, Kemp, A, W., Hartog, A., van, de, Graaf, S, F., van, Os, C, H., Willems, P, H., Bindels, R, J. J. Biol, Chem., 274(13):8375-8, (1999)).
Thus, the LTRPC3b polynucleotides and polypeptides are expected to share at least some biological activity with TRP family members expressed in kidney cells and tissues, particularly those specifically referenced herein.
The strong homology to human transient receptor potential channels (TRP) proteins, combined with the localized expression in spinal cord and brain of LTRPC3 suggests the LTRPC3b polynucleotides and polypeptides, including modulators or fragments thereof, may be useful in treating, diagnosing, prognosing, and/or preventing neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in the Examples, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
The strong homology to human transient receptor potential channels (TRP), combined with the localized expression in testis tissue of LTRPC3 emphasizes the potential utility for LTRPC3b polynucleotides and polypeptides in treating, diagnosing, prognosing, and/or preventing testicular, in addition to reproductive disorders.
In preferred embodiments, LTRPC3b polynucleotides and polypeptides including agonists and fragments thereof, have uses which include treating, diagnosing, prognosing, and/or preventing the following, non-limiting, diseases or disorders of the testis: spermatogenesis, infertility, Klinefelter's syndrome, XX male, epididymitis, genital warts, germinal cell aplasia, cryptorchidism, varicocele, immotile cilia syndrome, and viral orchitis. The LTRPC3b polynucleotides and polypeptides including agonists and fragments thereof, may also have uses related to modulating testicular development, embryogenesis, reproduction, and in ameliorating, treating, and/or preventing testicular proliferative disorders (e.g., cancers, which include, for example, choriocarcinoma, Nonseminoma, seminona, and testicular germ cell tumors).
Likewise, the localized expression in testis tissue also emphasizes the potential utility for LTRPC3b polynucleotides and polypeptides in treating, diagnosing, prognosing, and/or preventing metabolic diseases and disorders which include the following, not limiting examples: premature puberty, incomplete puberty, Kallman syndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis, congenital adrenal hyperplasia, FSH deficiency, and granulomatous disease, for example.
This gene product may also be useful in assays designed to identify binding agents, as such agents (antagonists) are useful as male contraceptive agents. The testes are also a site of active gene expression of transcripts that is expressed, particularly at low levels, in other tissues of the body. Therefore, this gene product may be expressed in other specific tissues or organs where it may play related functional roles in other processes, such as hematopoiesis, inflammation, bone formation, and kidney function, to name a few possible target indications.
Several known TRP family members have been identified that are expressed significantly in testis tissue. These TRP family members include, for example, polycystin-L2 (Guo, L., Schreiber, T, H., Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics., 64(3):241-51, (2000)); TRP7 (Okada, T., Inoue, R., Yamazaki, K., Maeda, A., Kurosaki, T., Yamakuni, T., Tanaka, I., Shimizu, S., Ikenaka, K., Imoto, K., Mori, Y, J. Biol, Chem., 274(39):27359-70, (1999)); btrp2 (Wissenbach, U., Schroth, G., Philipp, S., Flockerzi, V, Lett., 429(1):61-6, (1998)); Htrp-1 (Zhu, X., Chu, P, B., Peyton, M., Bimbaumer, L, Lett., 373(3):193-8, (1995)); and TRPC1 (Wes, P, D., Chevesich, J., Jeromin, A., Rosenberg, C., Stetten, G., Montell, C, Proc, Natl, Acad, Sci, U,S,A., 92(21):9652-6, (1995)).
Thus, the LTRPC3b polynucleotides and polypeptides are expected to share at least some biological activity with TRP family members expressed in testis cells and tissues, particularly those specifically referenced herein.
As described elsewhere herein, transient receptor potential channel family members have been implicated in modulating cell proliferation, differentiation, migration, activation, exocytosis, muscle contraction, gene expression, apoptosis. signalling, pheromone sensory signaling, smooth muscle tone, pain perception, heat perception, osmosenstivity, and mechanosensitivity. Moreover, transient receptor potential channel family members have been implicated in disorders of the skin, skeletal-muscle, nervous, cardiac, and vascular systems, in addition to the following, non-limiting diseases and disorders, which include, for example, arteriosclerosis, neointimal hypoerplasia, metastatic melanomas, bipolar disorder, nonsyndromic hereditary deafness, Knobloch syndrome, holosencephaly, and various maligancies including prostate cancer.
In preferred embodiments, LTRPC3b polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include, modulating cell proliferation, differentiation, migration, activation, exocytosis, muscle contraction, gene expression, apoptosis. signalling, pheromone sensory signaling, smooth muscle tone, pain perception, heat perception, osmosenstivity, and mechanosensitivity.
In more preferred embodiments, LTRPC3b polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include, treating, ameliorating, preventing, detecting, and/or prognosing various diseases and disorders, particularly the following, non-limiting examples, disorders of the skin, skeletal-muscle, nervous, cardiac, and vascular systems, in addition to the following, non-limiting diseases and disorders, which include, for example, arteriosclerosis, neointimal hypoerplasia, metastatic melanomas, bipolar disorder, nonsyndromic hereditary deafness, Knobloch syndrome, holosencephaly, and various maligancies including prostate cancer.
LTRPC3b polynucleotides and polypeptides of the present invention, including agonists and/or fragments may be involved in intracellular Ca2+ homeostasis which affects various aspects of biological functions including mechano-regulation, pain transduction, vasorelaxation, gene expression, cell cycle and proliferation/apoptosis. Since LTRPC3b is dominantly expressed in kidney, it may particularly play an important role in regulating cytosolic Ca2+ in the renal system, potentially Ca2+ absorption.
The LTRPC3b gene maps to chromosome 9q21.11-21.31 between the two markers D9S1874 and D9S1807. This region is linked to amyotrophic lateral sclerosis with frontotemporal dementia, early-onset pulverulent cataract, infantile nephronophthisis, hypomagnesemia with secondary hypocalcemia (HSH), and familial hemophagocytic lymphohistiocytosis. Therefore, agonists and/or antagonists of the novel LTRPC3b can be used to treat diseases including various forms of neuronal degeneration, neurogenic inflammation, allergy, immunodeficiency/excessive immune activation, visual defects, hearing disorder, pain, cancer, hypertension and other cardiovascular diseases. In addition, the therapeutics may be useful in the treatment of diseases associated with disturbances in Ca2+ homeostasis including osteoporosis, hypercalciuric stone disease, and chronic renal failure.
Moreover, given the selective expression in kidney, LTRPC3b could be considered a candidate gene for HSH, since the phenotype is a renal insufficiency. LTRPC3b chromosomal location is  ̃600 kb downstream of a X;9 translocation breakpoint interval described for one patient with HSH (Hum. Mol. Genet. 6, 1491-1497, (1997)). Recently, two groups reported that a new member of the TRPM subfamily, TRPM6, expressed in both intestinal tissues and kidney, is associated with HSH (Nat. Genet. 31, 166-170, (2002); and Nat. Genet. 31, 171-174 (2002)). Indeed, LTRPC3 is  ̃4 mb 5′ to TRPM6 on 9q21. Although TRPM6 is the first component identified for HSH, this should not preclude involvement of LTRPC3b in the disease. Therefore, it is possible that LTRPC3b may co-localize with TRPM6 and/or forms a heteromultimer with TRPM6 in the kidney.
The kidney plays a major role in Ca2+ homeostasis (reviewed in Physiol. Rev. 75, 429-471, (1995)). LTRPC3b may play a direct role in Ca2+ absorption due to its constitutive Ca2+ permeability. Alternatively, LTRPC3b may be the SOC that regulates Ca2+ absorption. In kidney, Ca2+ absorption is regulated by agonists such as calcitonin, parathyroid hormone and parathyroid hormone-related peptide through their respective G protein-coupled receptors, and downstream SOCs. Additionally, in kidney a SOC has been described (J. Biol. Chem. 276, 25759-25765, (2001)) for the action of angiotensin II, a vasoactive peptide that plays a major role in regulating blood pressure.
In addition, LTRPC3b polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include modulating intracellular Ca++ ion concentrations, Ca++ ion flux, stored intracellular Ca++ ion concentrations, Ca++ ion pump activity, Ca++ ion flow into cell, Ca++ ion flow out of cells, the activation of Ca++ sensitive proteins, the activation of Ca++ sensitive signaling pathways, the activation of kinase-activatible proteins, and the activation of kinase-dependent signaling pathways.
The LTRPC3b polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include modulating proliferation, differentiation, migration, and activation in various cells, tissues, and organisms, and particularly in mammalian kidney, spinal cord, testis, and brain, preferably human. LTRPC3b polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, may be useful in diagnosing, treating, prognosing, and/or preventing renal, neural, and/or proliferative diseases or disorders, particularly of the renal system.
In addition, antagonists of the LTRPC3b polynucleotides and polypeptides, including modulators or fragments thereof, may have uses that include diagnosing, treating, prognosing, and/or preventing diseases or disorders related to transient receptor potential channel activity, which may include renal, neural, reproductive, and/or proliferative diseases or disorders.
Although it is believed the encoded polypeptide may share at least some biological activities with transient receptor potential channel family members, particularly those melastatin1, a number of methods of determining the exact biological function of this clone are either known in the art or are described elsewhere herein. Briefly, the function of this clone may be determined by applying microarray methodology. Nucleic acids corresponding to the LTRPC3b polynucleotides, in addition to, other clones of the present invention, may be arrayed on microchips for expression profiling. Depending on which polynucleotide probe is used to hybridize to the slides, a change in expression of a specific gene may provide additional insight into the function of this gene based upon the conditions being studied. For example, an observed increase or decrease in expression levels when the polynucleotide probe used comes from tissue that has been treated with known transient potential receptor inhibitors, which include, but are not limited to the drugs listed herein or otherwise known in the art, might indicate a function in modulating transient potential receptor function, for example. In the case of LTRPC3b, kidney, spinal cord, testis, and brain, should be used to extract RNA to prepare the probe.
In addition, the function of the protein may be assessed by applying quantitative PCR methodology, for example. Real time quantitative PCR would provide the capability of following the expression of the LTRPC3b gene throughout development, for example. Quantitative PCR methodology requires only a nominal amount of tissue from each developmentally important step is needed to perform such experiments. Therefore, the application of quantitative PCR methodology to refining the biological function of this polypeptide is encompassed by the present invention. Also encompassed by the present invention are quantitative PCR probes corresponding to the polynucleotide sequence provided as SEQ ID NO:3 (FIGS. 2A-F).
The function of the protein may also be assessed through complementation assays in yeast. For example, in the case of the LTRPC3b, transforming yeast deficient in transient receptor potential channel activity with LTRPC3b and assessing their ability to grow would provide convincing evidence the LTRPC3b polypeptide has transient receptor potential channel activity. Additional assay conditions and methods that may be used in assessing the function of the polynucleotides and polypeptides of the present invention are known in the art, some of which are disclosed elsewhere herein.
Alternatively, the biological function of the encoded polypeptide may be determined by disrupting a homologue of this polypeptide in Mice and/or rats and observing the resulting phenotype.
Moreover, the biological function of this polypeptide may be determined by the application of antisense and/or sense methodology and the resulting generation of transgenic mice and/or rats. Expressing a particular gene in either sense or antisense orientation in a transgenic mouse or rat could lead to respectively higher or lower expression levels of that particular gene. Altering the endogenous expression levels of a gene can lead to the observation of a particular phenotype that can then be used to derive indications on the function of the gene. The gene can be either over-expressed or under expressed in every cell of the organism at all times using a strong ubiquitous promoter, or it could be expressed in one or more discrete parts of the organism using a well characterized tissue-specific promoter (e.g., a kidney, spinal cord, testis, and/or brain-specific promoter), or it can be expressed at a specified time of development using an inducible and/or a developmentally regulated promoter.
In the case of LTRPC3b transgenic mice or rats, if no phenotype is apparent in normal growth conditions, observing the organism under diseased conditions (renal, reproductive, neural, or proliferative disorders, etc.) may lead to understanding the function of the gene. Therefore, the application of antisense and/or sense methodology to the creation of transgenic mice or rats to refine the biological function of the polypeptide is encompassed by the present invention.
In preferred embodiments, the following N-terminal LTRPC3b deletion polypeptides are encompassed by the present invention: M1-T1566, Y2-T1566, V3-T1566, R4-T1566, V5-T1566, S6-T1566, F7-T1566, D8-T1566, T9-T1566, K10-T1566, P11-T1566, D12-T1566, L13-T1566, L14-T1566, L15-T1566, H16-T1566, L17-T1566, M18-T1566, T19-T1566, K20-T1566, E21-T1566, W22-T1566, Q23-T1566, L24-T1566, E25-T1566, L26-T1566, P27-T1566, K28-T1566, L29-T1566, L30-T1566, I31-T1566, S32-T1566, V33-T1566, H34-T1566, G35-T1566, G36-T1566, L37-T1566, Q38-T1566, N39-T1566, F40-T1566, E41-T1566, L42-T1566, Q43-T1566, P44-T1566, K45-T1566, L46-T1566, K47-T1566, Q48-T1566, V49-T1566, F50-T1566, G51-T1566, K52-T1566, G53-T1566, L54-T1566, I55-T1566, K56-T1566, A57-T1566, A58-T1566, M59-T1566, T60-T1566, T61-T1566, G62-T1566, A63-T1566, W64-T1566, I65-T1566, F66-T1566, T67-T1566, G68-T1566, G69-T1566, V70-T1566, N71-T1566, T72-T1566, G73-T1566, V74-T1566, I75-T1566, R76-T1566, H77-T1566, V78-T1566, G79-T1566, D80-T1566, A81-T1566, L82-T1566, K83-T1566, D84-T1566, H85-T1566, A86-T1566, S87-T1566, K88-T1566, S89-T1566, R90-T1566, G91-T1566, K92-T1566, I93-T1566, C94-T1566, T95-T1566, I96-T1566, G97-T1566, I98-T1566, A99-T1566, P100-T1566, W101-T1566, G102-T1566, I103-T1566, V104-T1566, E105-T1566, N106-T1566, Q107-T1566, E108-T1566, D109-T1566, L110-T1566, I111-T1566, G112-T1566, R113-T1566, D114-T1566, V115-T1566, V116-T1566, R117-T1566, P118-T1566, Y119-T1566, Q120-T1566, T121-T1566, M122-T1566, S123-T1566, N124-T1566, P125-T1566, M126-T1566, S127-T1566, K128-T1566, L129-T1566, T130-T1566, V131-T1566, L132-T1566, N133-T1566, S134-T1566, M135-T1566, H136-T1566, S137-T1566, H138-T1566, F139-T1566, I140-T1566, L141-T1566, A142-T1566, D143-T1566, N144-T1566, G145-T1566, T146-T1566, T147-T1566, G148-T1566, K149-T1566, Y150-T1566, G151-T1566, A152-T1566, E153-T1566, V154-T1566, K155-T1566, L156-T1566, R157-T1566, R158-T1566, Q159-T1566, L160-T1566, E161-T1566, K162-T1566, H163-T1566, I164-T1566, S165-T1566, L166-T1566, Q167-T1566, K168-T1566, I169-T1566, N170-T1566, T171-T1566, R172-T1566, I173-T1566, G174-T1566, Q175-T1566, G176-T1566, V177-T1566, P178-T1566, V179-T1566, V180-T1566, A181-T1566, L182-T1566, I183-T1566, V184-T1566, E185-T1566, G186-T1566, G187-T1566, P188-T1566, N189-T1566, V190-T1566, I191-T1566, S192-T1566, I193-T1566, V194-T1566, L195-T1566, E196-T1566, Y197-T1566, L198-T1566, R199-T1566, D200-T1566, T201-T1566, P202-T1566, P203-T1566, V204-T1566, P205-T1566, V206-T1566, V207-T1566, V208-T1566, C209-T1566, D210-T1566, G211-T1566, S212-T1566, G213-T1566, R214-T1566, A215-T1566, S216-T1566, D217-T1566, I218-T1566, L219-T1566, A220-T1566, F221-T1566, G222-T1566, H223-T1566, K224-T1566, Y225-T1566, S226-T1566, E227-T1566, E228-T1566, G229-T1566, G230-T1566, L231-T1566, I232-T1566, N233-T1566, E234-T1566, S235-T1566, L236-T1566, R237-T1566, D238-T1566, Q239-T1566, L240-T1566, L241-T1566, V242-T1566, T243-T1566, I244-T1566, Q245-T1566, K246-T1566, T247-T1566, F248-T1566, T249-T1566, Y250-T1566, T251-T1566, R252-T1566, T253-T1566, Q254-T1566, A255-T1566, Q256-T1566, H257-T1566, L258-T1566, F259-T1566, I260-T1566, I261-T1566, L262-T1566, M263-T1566, E264-T1566, C265-T1566, M266-T1566, K267-T1566, K268-T1566, K269-T1566, E270-T1566, L271-T1566, I272-T1566, T273-T1566, V274-T1566, F275-T1566, R276-T1566, M277-T1566, G278-T1566, S279-T1566, E280-T1566, G281-T1566, H282-T1566, Q283-T1566, D284-T1566, I285-T1566, D286-T1566, L287-T1566, A288-T1566, I289-T1566, L290-T1566, T291-T1566, A292-T1566, L293-T1566, L294-T1566, K295-T1566, G296-T1566, A297-T1566, N298-T1566, A299-T1566, S300-T1566, A301-T1566, P302-T1566, D303-T1566, Q304-T1566, L305-T1566, S306-T1566, L307-T1566, A308-T1566, L309-T1566, A310-T1566, W311-T1566, N312-T1566, R313-T1566, V314-T1566, D315-T1566, I316-T1566, A317-T1566, R318-T1566, S319-T1566, Q320-T1566, I321-T1566, F322-T1566, I323-T1566, Y324-T1566, G325-T1566, Q326-T1566, Q327-T1566, W328-T1566, P329-T1566, V330-T1566, G331-T1566, S332-T1566, L333-T1566, E334-T1566, Q335-T1566, A336-T1566, M337-T1566, L338-T1566, D339-T1566, A340-T1566, L341-T1566, V342-T1566, L343-T1566, D344-T1566, R345-T1566, V346-T1566, D347-T1566, F348-T1566, V349-T1566, K350-T1566, L351-T1566, L352-T1566, I353-T1566, E354-T1566, N355-T1566, G356-T1566, V357-T1566, S358-T1566, M359-T1566, H360-T1566, R361-T1566, F362-T1566, L363-T1566, T364-T1566, I365-T1566, S366-T1566, R367-T1566, L368-T1566, E369-T1566, E370-T1566, L371-T1566, Y372-T1566, N373-T1566, T374-T1566, R375-T1566, H376-T1566, G377-T1566, P378-T1566, S379-T1566, N380-T1566, T381-T1566, L382-T1566, Y383-T1566, H384-T1566, L385-T1566, V386-T1566, R387-T1566, D388-T1566, V389-T1566, K390-T1566, K391-T1566, R392-T1566, E393-T1566, Y394-T1566, P395-T1566, G396-T1566, F397-T1566, G398-T1566, W399-T1566, I400-T1566, Y401-T1566, F402-T1566, and/or K403-T1566 of SEQ ID NO:4. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3b deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3b deletion polypeptides are encompassed by the present invention: M1-T1566, M1-H1565, M1-K1564, M1-S1563, M1-E1562, M1-F1561, M1-S1560, M1-Q1559, M1-F1558, M1-A1557, M1-S1556, M1-T1555, M1-R1554, M1-S1553, M1-L1552, M1-R1551, M1-R1550, M1-M1549, M1-S1548, M1-L1547, M1-S1546, M1-D1545, M1-G1544, M1-R1543, M1-G1542, M1-E1541, M1-P1540, M1-K1539, M1-S1538, M1-S1537, M1-K1536, M1-S1535, M1-R1534, M1-Q1533, M1-F1532, M1-P1531, M1-N1530, M1-Q1529, M1-L1528, M1-S1527, M1-A1526, M1-T1525, M1-N1524, M1-R1523, M1-Q1522, M1-R1521, M1-D1520, M1-L1519, M1-K1518, M1-D1517, M1-S1516, M1-I1515, M1-S1514, M1-F1513, M1-S1512, M1-K1511, M1-R1510, M1-T1509, M1-H1508, M1-A1507, M1-Y1506, M1-P1505, M1-A1504, M1-S1503, M1-P1502, M1-E1501, M1-E1500, M1-A1499, M1-S1498, M1-Y1497, M1-S1496, M1-N1495, M1-A1494, M1-R1493, M1-E1492, M1-I1491, M1-K1490, M1-P1489, M1-V1488, M1-T1487, M1-I1486, M1-N1485, M1-N1484, M1-S1483, M1-L1482, M1-T1481, M1-R1480, M1-E1479, M1-S1478, M1-N1477, M1-D1476, M1-G1475, M1-E1474, M1-Q1473, M1-S1472, M1-S1471, M1-I1470, M1-A1469, M1-I1468, M1-T1467, M1-A1466, M1-R1465, M1-R1464, M1-G1463, M1-K1462, M1-A1461, M1-E1460, M1-N1459, M1-E1458, M1-E1457, M1-S1456, M1-D1455, M1-S1454, M1-S1453, M1-P1452, M1-H1451, M1-S1450, M1-L1449, M1-E1448, M1-A1447, M1-E1446, M1-R1445, M1-E1444, M1-P1443, M1-H1442, M1-C1441, M1-C1440, M1-T1439, M1-L1438, M1-D1437, M1-E1436, M1-V1435, M1-K1434, M1-D1433, M1-G1432, M1-L1431, M1-G1430, M1-G1429, M1-P1428, M1-F1427, M1-A1426, M1-A1425, M1-R1424, M1-D1423, M1-A1422, M1-I1421, M1-A1420, M1-Q1419, M1-P1418, M1-A1417, M1-N1416, M1-V1415, M1-C1414, M1-R1413, M1-T1412, M1-D1411, M1-I1410, M1-C1409, M1-D1408, M1-T1407, M1-I1406, M1-S1405, M1-T1404, M1-Y1403, M1-E1402, M1-A1401, M1-T1400, M1-K1399, M1-V1398, M1-P1397, M1-V1396, M1-G1395, M1-F1394, M1-N1393, M1-A1392, M1-Y1391, M1-Y1390, M1-S1389, M1-R1388, M1-S1387, M1-P1386, M1-S1385, M1-F1384, M1-M1383, M1-F1382, M1-S1381, M1-H1380, M1-S1379, M1-K1378, M1-V1377, M1-I1376, M1-P1375, M1-A1374, M1-E1373, M1-E1372, M1-L1371, M1-L1370, M1-F1369, M1-P1368, M1-T1367, M1-T1366, M1-A1365, M1-L1364, M1-Y1363, M1-R1362, M1-S1361, M1-S1360, M1-K1359, M1-S1358, M1-R1357, M1-E1356, M1-I1355, M1-T1354, M1-H1353, M1-Y1352, M1-M1351, M1-P1350, M1-P1349, M1-E1348, M1-S1347, M1-D1346, M1-W1345, M1-P1344, M1-N1343, M1-Q1342, M1-C1341, M1-E1340, M1-P1339, M1-L1338, M1-H1337, M1-T1336, M1-Y1335, M1-D1334, M1-S1333, M1-S1332, M1-F1331, M1-S1330, M1-R1329, M1-T1328, M1-D1327, M1-M1326, M1-S1325, M1-T1324, M1-I1323, M1-D1322, M1-E1321, M1-F1320, M1-D1319, M1-I1318, M1-S1317, M1-R1316, M1-S1315, M1-P1314, M1-P1313, M1-R1312, M1-D1311, M1-T1310, M1-P1309, M1-A1308, M1-L1307, M1-T1306, M1-A1305, M1-Y1304, M1-A1303, M1-S1302, M1-S1301, M1-S1300, M1-P1299, M1-A1298, M1-T1297, M1-S1296, M1-P1295, M1-V1294, M1-P1293, M1-T1292, M1-S1291, M1-F1290, M1-S1289, M1-P1288, M1-E1287, M1-G1286, M1-L1285, M1-G1284, M1-L1283, M1-I1282, M1-N1281, M1-V1280, M1-S1279, M1-N1278, M1-D1277, M1-L1276, M1-P1275, M1-D1274, M1-I1273, M1-D1272, M1-C1271, M1-H1270, M1-L1269, M1-E1268, M1-D1267, M1-M1266, M1-A1265, M1-S1264, M1-V1263, M1-Y1262, M1-I1261, M1-D1260, M1-I1259, M1-C1258, M1-S1257, M1-S1256, M1-P1255, M1-R1254, M1-R1253, M1-S1252, M1-D1251, M1-P1250, M1-V1249, M1-I1248, M1-A1247, M1-L1246, M1-T1245, M1-N1244, M1-A1243, M1-P1242, M1-A1241, M1-A1240, M1-P1239, M1-A1238, M1-K1237, M1-P1236, M1-E1235, M1-K1234, M1-A1233, M1-V1232, M1-S1231, M1-H1230, M1-S1229, M1-S1228, M1-T1227, M1-A1226, M1-R1225, M1-H1224, M1-L1223, M1-S1222, M1-L1221, M1-S1220, M1-R1219, M1-E1218, M1-K1217, M1-F1216, M1-I1215, M1-S1214, M1-E1213, M1-L1212, M1-K1211, M1-E1210, M1-I1209, M1-G1208, M1-G1207, M1-K1206, M1-D1205, M1-K1204, M1-M1203, M1-N1202, M1-V1201, M1-S1200, M1-Y1199, M1-F1198, M1-S1197, M1-H1196, M1-S1195, M1-R1194, M1-M1193, M1-R1192, M1-P191, M1-M1190, M1-L1189, M1-T1188, M1-P1187, M1-S1186, M1-T1185, M1-P1184, M1-S1183, M1-M1182, M1-T1181, M1-E1180, M1-E1179, M1-G1178, M1-A1177, M1-P1176, M1-D1175, M1-I1174, M1-S1173, M1-E1172, M1-Q1171, M1-L1170, M1-K1169, M1-F1168, M1-T1167, M1-N1166, M1-G1165, M1-E1164, M1-Q1163, M1-S1162, M1-N1161, M1-F1160, M1-S1159, M1-S1158, M1-Q1157, M1-R1156, M1-V1155, M1-I1154, M1-Y1153, M1-A1152, M1-A1151, M1-D1150, M1-T1149, M1-C1148, M1-D1147, M1-S1146, M1-S1145, M1-T1144, M1-R1143, M1-S1142, M1-R1141, M1-I1140, M1-K1139, M1-N1138, M1-S1137, M1-E1136, M1-A1135, M1-R1134, M1-E1133, M1-L1132, M1-G1131, M1-T1130, M1-L1129, M1-R1128, M1-E1127, M1-L1126, M1-A1125, M1-T1124, M1-A1123, M1-M1122, M1-R1121, M1-G1120, M1-I1119, M1-L1118, M1-D1117, M1-E1116, M1-L1115, M1-Q1114, M1-A1113, M1-L1112, M1-R1111, M1-I1110, M1-D1109, M1-V1108, M1-T1107, M1-Q1106, M1-L1105, M1-S1104, M1-A1103, M1-K1102, M1-M111, M1-S110, M1-H1099, M1-E1098, M1-R1097, M1-E1096, M1-N1095, M1-V1094, M1-E1093, M1-E1092, M1-L1091, M1-R1090, M1-M1089, M1-S1088, M1-M1087, M1-N1086, M1-E1085, M1-V1084, M1-R1083, M1-E1082, M1-S1081, M1-T1080, M1-V1079, M1-R1078, M1-I1077, M1-R1076, M1-E1075, M1-D1074, M1-N1073, M1-S1072, M1-S1071, M1-N1070, M1-F1069, M1-R1068, M1-D1067, M1-D1066, M1-K1065, M1-E1064, M1-R1063, M1-F1062, M1-Y1061, M1-E1060, M1-E1059, M1-I1058, M1-C1057, M1-Q1056, M1-E1055, M1-E1054, M1-F1053, M1-D1052, M1-H1051, M1-V1050, M1-K1049, M1-K1048, M1-L1047, M1-E1046, M1-D1045, M1-D1044, M1-T1043, M1-I1042, M1-F1041, M1-L1040, M1-K1039, M1-L1038, M1-G1037, M1-Y1036, M1-D1035, M1-R1034, M1-E1033, M1-D1032, M1-P1031, M1-D1030, M1-S1029, M1-E1028, M1-H1027, M1-K1026, M1-R1025, M1-W1024, M1-R1023, M1-C1022, M1-C1021, M1-L1020, M1-H1019, M1-Q1008, M1-F1017, M1-I1016, M1-M1015, M1-T1014, M1-M1013, M1-H1012, M1-S1011, M1-F1010, M1-I1009, M1-I1008, M1-L1007, M1-P1006, M1-P1005, M1-P1004, M1-L1003, M1-V1002, M1-P1001, M1-R1000, M1-E999, M1-H998, M1-F997, M1-T996, M1-M995, M1-I994, M1-L993, M1-Q992, M1-Y991, M1-R990, M1-Q989, M1-F988, M1-K987, M1-W986, M1-V985, M1-Q984, M1-N983, M1-S982, M1-I981, M1-S980, M1-K979, M1-V978, M1-E977, M1-F976, M1-F975, M1-T974, M1-N973, M1-N972, M1-F971, M1-V970, M1-A969, M1-I968, M1-L967, M1-L966, M1-N965, M1-V964, M1-L963, M1-L962, M1-I961, M1-N960, M1-A959, M1-V958, M1-L957, M1-L956, M1-Y955, M1-C954, M1-A953, M1-M952, M1-I951, M1-A950, M1-P949, M1-V948, M1-I947, M1-W946, M1-A945, M1-G944, M1-T943, M1-K942, M1-C941, M1-P940, M1-P939, M1-L938, M1-Q937, M1-I936, M1-I935, M1-K934, M1-G933, M1-D932, M1-E931, M1-R930, M1-T929, M1-E928, M1-N927, M1-Q926, M1-G925, M1-C924, M1-P923, M1-P922, M1-D921, M1-I920, M1-Q919, M1-D918, M1-A917, M1-F916, M1-V915, M1-E914, M1-G913, M1-Y912, M1-I911, M1-M910, M1-W909, M1-Y908, M1-P907, M1-M906, M1-Y905, M1-F904, M1-I903, M1-N902, M1-K901, M1-A900, M1-L899, M1-K898, M1-W897, M1-S896, M1-P895, M1-E894, M1-E893, M1-N892, M1-P891, M1-F890, M1-L889, M1-I888, M1-A887, M1-Q886, M1-R885, M1-A884, M1-V883, M1-G882, M1-F881, M1-S880, M1-M879, M1-L878, M1-V877, M1-V876, M1-L875, M1-M874, M1-I873, M1-I872, M1-V871, M1-F870, M1-Y869, M1-M868, M1-M867, M1-D866, M1-I865, M1-M864, M1-M863, M1-K862, M1-G861, M1-I860, M1-M859, M1-M858, M1-V857, M1-Y856, M1-P855, M1-G854, M1-L853, M1-Y852, M1-K851, M1-N850, M1-V849, M1-G848, M1-F847, M1-I846, M1-D845, M1-L844, M1-L843, M1-R842, M1-I841, M1-Y840, M1-W839, M1-Y838, M1-I837, M1-I836, M1-N835, M1-V834, M1-C833, M1-Y832, M1-I831, M1-V830, M1-R829, M1-G828, M1-D827, M1-S826, M1-R825, M1-F824, M1-P823, M1-Q822, M1-D821, M1-Q820, M1-L819, M1-R818, M1-L817, M1-I816, M1-M815, M1-G814, M1-V813, M1-S812, M1-F811, M1-L810, M1-L809, M1-I808, M1-A807, M1-I806, M1-L805, M1-D804, M1-T803, M1-V802, M1-N801, M1-W800, M1-Y799, M1-E798, M1-Q797, M1-L796, M1-W795, M1-V794, M1-K793, M1-V792, M1-K791, M1-Q790, M1-L789, M1-L788, M1-K787, M1-G786, M1-P785, M1-E784, M1-S783, M1-M782, M1-L781, M1-I780, M1-E779, M1-R778, M1-M777, M1-K776, M1-E775, M1-I774, M1-G773, M1-L772, M1-T771, M1-F770, M1-I769, M1-Y768, M1-S767, M1-I766, M1-V765, M1-I764, M1-W763, M1-E762, M1-Q761, M1-T760, M1-S759, M1-P758, M1-W757, M1-R756, M1-E755, M1-M754, M1-K753, M1-V752, M1-L751, M1-V750, M1-I749, M1-Y748, M1-N747, M1-F746, M1-L745, M1-M744, M1-L743, M1-Y742, M1-G741, M1-I740, M1-Y739, M1-A738, M1-L737, M1-T736, M1-Y735, M1-F734, M1-W733, M1-F732, M1-K731, M1-V730, M1-I729, M1-P728, M1-A727, M1-N726, M1-Y725, M1-F724, M1-E723, M1-Y722, M1-I721, M1-K720, M1-R719, M1-G718, M1-L717, M1-P716, M1-I715, M1-L714, M1-R713, M1-H712, M1-K711, M1-S710, M1-Q709, M1-V708, M1-E707, M1-E706, M1-E705, M1-D704, M1-K703, M1-K702, M1-R701, M1-S700, M1-S699, M1-E698, M1-G697, M1-N696, M1-N695, M1-R694, M1-G693, M1-L692, M1-M691, M1-A690, M1-T689, M1-L688, M1-E687, M1-M686, M1-D685, M1-E684, M1-E683, M1-E682, M1-K681, M1-E680, M1-K679, M1-T678, M1-P677, M1-K676, M1-E675, M1-P674, M1-E673, M1-E672, M1-A671, M1-E670, M1-K669, M1-E668, M1-Q667, M1-L666, M1-H665, M1-I664, M1-E663, M1-Q662, M1-A661, M1-Q660, M1-S659, M1-M658, M1-Y657, M1-P656, M1-M655, M1-D654, M1-D653, M1-K652, M1-N651, M1-K650, M1-F649, M1-E648, M1-L647, M1-S646, M1-L645, M-I644, M1-S643, M1-P642, M1-P641, M1-L640, M1-L639, M1-I638, M1-G637, M1-L636, M1-I635, M1-V634, M1-K633, M1-L632, M1-G631, M1-S630, M1-N629, M1-K628, M1-R627, M1-M626, M1-R625, M1-L624, M1-R623, M1-G622, M1-M621, M1-W620, M1-M619, M1-D618, M1-T617, M1-L616, M1-L615, M1-M614, M1-Q613, M1-S612, M1-C611, M1-T610, M1-H609, M1-A608, M1-I607, M1-F606, M1-D605, M1-R604, M1-H603, M1-K602, M1-A601, M1-A600, M1-V599, M1-A598, M1-L597, M1-Q596, M1-L595, M1-C594, M1-T593, M1-A592, M1-N591, M1-S590, M1-W589, M1-N588, M1-K587, M1-L586, M1-E585, M1-Y584, M1-T583, M1-L582, M1-L581, M1-K580, M1-M579, M1-A578, M1-L577, M1-Q576, M1-E575, M1-D574, M1-Q573, M1-K572, M1-Y571, M1-S570, M1-Q569, M1-D568, M1-L567, M1-L566, M1-E565, M1-V564, M1-A563, M1-L562, M1-Q561, M1-G560, M1-F559, M1-D558, M1-R557, M1-S556, M1-N555, M1-H554, M1-N553, M1-L552, M1-E551, M1-Q550, M1-S549, M1-I548, M1-D547, M1-D546, M1-V545, M1-M544, M1-D543, M1-N542, M1-E541, M1-S540, M1-A539, M1-E538, M1-H537, M1-A536, M1-M535, M1-A534, M1-K533, M1-C532, M1-L531, M1-K530, M1-C529, M1-A528, M1-V527, M1-L526, M1-A525, M1-K524, M1-A523, M1-M522, M1-A521, M1-E520, M1-E519, M1-G518, M1-H517, M1-Q516, M1-W515, M1-F514, M1-F513, M1-L512, M1-A511, M1-M510, M1-K509, M1-Q508, M1-R507, M1-K506, M1-M505, M1-L504, M1-V503, M1-A502, M1-W501, M1-V500, M1-M499, M1-L498, M1-E497, M1-H496, M1-F495, M1-P494, M1-F493, M1-P492, M1-F491, M1-H490, M1-N489, M1-I488, M1-E487, M1-P486, M1-D485, M1-D484, M1-L483, M1-D482, M1-I481, M1-D480, M1-V479, M1-E478, M1-E477, M1-E476, M1-R475, M1-K474, M1-K473, M1-T472, M1-T471, M1-K470, M1-R469, M1-G468, M1-R467, M1-R466, M1-L465, M1-P464, M1-I463, M1-D462, M1-D461, M1-E460, M1-M459, M1-G458, M1-L457, M1-L456, M1-K455, M1-L454, M1-A453, M1-K452, M1-P451, M1-R450, M1-K449, M1-P448, M1-G447, M1-F446, M1-L445, M1-N444, M1-H443, M1-Y442, M1-L441, M1-T440, M1-R439, M1-F438, M1-R437, M1-K436, M1-R435, M1-T434, M1-Y433, M1-N432, M1-C431, M1-R430, M1-Y429, M1-A428, M1-G427, M1-G426, M1-M425, M1-L424, M1-Y423, M1-E422, M1-I421, M1-V420, M1-L419, M1-G418, M1-I417, M1-D416, M1-I415, M1-L414, M1-S413, M1-I412, M1-R411, M1-Y410, M1-D409, M1-P408, M1-P407, M1-L406, M1-N405, M1-G404, M1-K403, M1-F402, M1-Y401, M1-I400, M1-W399, M1-G398, M1-F397, M1-G396, M1-P395, M1-Y394, M1-E393, M1-R392, and/or M1-K391 of SEQ ID NO:4. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3b deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
Alternatively, preferred polypeptides of the present invention may comprise polypeptide sequences corresponding to, for example, internal regions of the LTRPC3b polypeptide (e.g., any combination of both N- and C-terminal LTRPC3b polypeptide deletions) of SEQ ID NO:4. For example, internal regions could be defined by the equation: amino acid NX to amino acid CX, wherein NX refers to any N-terminal deletion polypeptide amino acid of LTRPC3b (SEQ ID NO:4), and where CX refers to any C-terminal deletion polypeptide amino acid of LTRPC3b (SEQ ID NO:4). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.
The LTRPC3b polypeptides of the present invention were determined to comprise several phosphorylation sites based upon the Motif algorithm (Genetics Computer Group, Inc.). The phosphorylation of such sites may regulate some biological activity of the LTRPC3b polypeptide. For example, phosphorylation at specific sites may be involved in regulating the proteins ability to associate or bind to other molecules (e.g., proteins, ligands, substrates, DNA, etc.). In the present case, phosphorylation may modulate the ability of the LTRPC3b polypeptide to associate with other potassium channel alpha subunits, beta subunits, or its ability to modulate potassium channel function.
Specifically, the LTRPC3b polypeptide was predicted to comprise two tyrosine phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). Such sites are phosphorylated at the tyrosine amino acid residue. The consensus pattern for tyrosine phosphorylation sites are as follows: [RK]-x(2)-[DE]-x(3)-Y, or [RK]-x(3)-[DE]-x(2)-Y, where Y represents the phosphorylation site and ‘x’ represents an intervening amino acid residue. Additional information specific to tyrosine phosphorylation sites can be found in Patschinsky T., Hunter T., Esch F. S., Cooper J. A., Sefton B. M., Proc. Natl. Acad. Sci. U.S.A. 79:973-977 (1982); Hunter T., J. Biol. Chem. 257:4843-4848 (1982), and Cooper J. A., Esch F. S., Taylor S. S., Hunter T., J. Biol. Chem. 259:7835-7841(1984), which are hereby incorporated herein by reference.
In preferred embodiments, the following tyrosine phosphorylation site polypeptides are encompassed by the present invention: LSLEFKNKDDMPYMSQAQ (SEQ ID NO:110), and/or VMMIGKMMIDMMYFVIIM (SEQ ID NO:111). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these LTRPC3b tyrosine phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The LTRPC3b polypeptide was predicted to comprise twenty three PKC phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). In vivo, protein kinase C exhibits a preference for the phosphorylation of serine or threonine residues. The PKC phosphorylation sites have the following consensus pattern: [ST]-x-[RK], where S or T represents the site of phosphorylation and ‘x’ an intervening amino acid residue. Additional information regarding PKC phosphorylation sites can be found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem. 161:177-184 (1986), and Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem. 260:12492-12499 (1985); which are hereby incorporated by reference herein.
In preferred embodiments, the following PKC phosphorylation site polypeptides are encompassed by the present invention: ADNGTTGKYGAEV (SEQ ID NO:112), VVCDGSGRASDIL (SEQ ID NO:113), GLINESLRDQLLV (SEQ ID NO:114), YRCNYTRKRFRTL (SEQ ID NO:115), RRGRKTTKKREEE (SEQ ID NO:116), RGRKTTKKREEEV (SEQ ID NO:117), ELLDQSYKQDEQL (SEQ ID NO:118), RNNGESSRKKDEE (SEQ ID NO:119), NNGESSRKKDEEE (SEQ ID NO:120), PNEEPSWKLAKNI (SEQ ID NO:121), RIRVTSERVENMS (SEQ ID NO:122), RVENMSMRLEEVN (SEQ ID NO:123), NEREHSMKASLQT (SEQ ID NO:124), LERAESNKIRSRT (SEQ ID NO:125), SQEGNTFKLQESI (SEQ ID NO:126), AIVPDSRRPSSCI (SEQ ID NO:127), ATLAPTDRPPSRS (SEQ ID NO:128), IERSKSSRYLATT (SEQ ID NO:129), QEGDNSERTLSNN (SEQ ID NO:130), APYAHTRKSFSIS (SEQ ID NO:131), KSFSISDKLDRQR (SEQ ID NO:132), FQRSKSSKPEGRG (SEQ ID NO:133), and/or RGDSLSMRRLSRT (SEQ ID NO:134). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these LTRPC3b PKC phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The present invention also encompasses immunogenic and/or antigenic epitopes of the LTRPC3b polypeptide.
The LTRPC3b polypeptide has been shown to comprise twelve glycosylation sites according to the Motif algorithm (Genetics Computer Group, Inc.). As discussed more specifically herein, protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.
Asparagine phosphorylation sites have the following consensus pattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation site. However, it is well known that that potential N-glycosylation sites are specific to the consensus sequence Asn-Xaa-Ser/Thr. However, the presence of the consensus tripeptide is not sufficient to conclude that an asparagine residue is glycosylated, due to the fact that the folding of the protein plays an important role in the regulation of N-glycosylation. It has been shown that the presence of proline between Asn and Ser/Thr will inhibit N-glycosylation; this has been confirmed by a recent statistical analysis of glycosylation sites, which also shows that about 50% of the sites that have a proline C-terminal to Ser/Thr are not glycosylated. Additional information relating to asparagine glycosylation may be found in reference to the following publications, which are hereby incorporated by reference herein: Marshall R. D., Annu. Rev. Biochem. 41:673-702 (1972); Pless D. D., Lennarz W. J., Proc. Natl. Acad. Sci. U.S.A. 74:134-138 (1977); Bause E., Biochem. J. 209:331-336 (1983); Gavel Y., von Heijne G., Protein Eng. 3:433-442 (1990); and Miletich J. P., Broze G. J. Jr., J. Biol. Chem. 265:11397-11404 (1990).
In preferred embodiments, the following asparagine glycosylation site polypeptides are encompassed by the present invention: FILADNGTTGKYGA (SEQ ID NO:135), EGGLINESLRDQLL (SEQ ID NO:136), LLKGANASAPDQLS (SEQ ID NO:137), GAYRCNYTRKRFRT (SEQ ID NO:138), TYELKNWSNATCLQ (SEQ ID NO:139), LKNWSNATCLQLAV (SEQ ID NO:140), LQEYWNVTDLIAIL (SEQ ID NO:141), PPCGQNETREDGKI (SEQ ID NO:142), LIAVFNNTFFEVKS (SEQ ID NO:143), KDDRFNSSNDERIR (SEQ ID NO:144), SERVENMSMRLEEV (SEQ ID NO:145), and/or RTLSNNITVPKIER (SEQ ID NO:146). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these LTRPC3b asparagine glycosylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The LTRPC3b polypeptide has been shown to comprise one RGD cell attachment site domain according to the Motif algorithm (Genetics Computer Group, Inc.). The sequence Arg-Gly-Asp, found in fibronectin, is crucial for its interaction with its cell surface receptor, an integrin. What has been called the ‘RGD’ tripeptide is also found in the sequences of a number of other proteins, where it has been shown to play a role in cell adhesion. Non-limiting examples of these proteins are the following: some forms of collagens, fibrinogen, vitronectin, von Willebrand factor (VWF), snake disintegrins, and slime mold discoidins. The ‘RGD’ tripeptide is also found in other proteins where it may serve the same purpose. A consensus pattern for RGD cell attachment sites is the following: R-G-D. Additional information relating to RGD cell attachment site domains may be found in reference to the following publications, which are hereby incorporated by reference herein: Ruoslahti E., Pierschbacher M. D., Cell 44:517-518 (1986); and d'Souza S. E., Ginsberg M. H., Plow E. F., Trends Biochem. Sci. 16:246-250 (1991).
In preferred embodiments, the following RGD cell attachment site domain polypeptide is encompassed by the present invention: SKPEGRGDSLSMR (SEQ ID NO:108). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this RGD cell attachment site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The LTRPC3b polypeptide has been shown to comprise one aminoacyl-transfer RNA synthetases class-II domain according to the Motif algorithm (Genetics Computer Group, Inc.). Aminoacyl-tRNA synthetases (EC 6.1.1.-) are a group of enzymes which activate amino acids and transfer them to specific tRNA molecules as the first step in protein biosynthesis. In prokaryotic organisms there are at least twenty different types of aminoacyl-tRNA synthetases, one for each different amino acid. In eukaryotes there are generally two aminoacyl-tRNA synthetases for each different amino acid: one cytosolic form and a mitochondrial form. While all these enzymes have a common function, they are widely diverse in terms of subunit size and of quaternary structure.
The synthetases specific for alanine, asparagine, aspartic acid, glycine, histidine, lysine, phenylalanine, proline, serine, and threonine are referred to as class-II synthetases and probably have a common folding pattern in their catalytic domain for the binding of ATP and amino acid which is different to the Rossmann fold observed for the class I synthetases.
Class-II tRNA synthetases do not share a high degree of similarity, however at least three conserved regions are present.
The consensus pattern for aminoacyl-transfer RNA synthetases class-II domains are as follows: [FYH]-R-x-[DE]-x(4,12)-[RH]-x(3)-F-x(3)-[DE]; and [GSTALVF]-{DENQHRKP}-[GSTA]-[LIVMF]-[DE]-R-[LIVMF]-x-[LIVMSTAG]-[LIVMFY], where ‘x’ represents an intervening amino acid residue.
Additional information specific to aminoacyl-transfer RNA synthetases class-II domains may be found in reference to the following publications, Schimmel P., Annu. Rev. Biochem. 56:125-158 (1987); Delarue M., Moras D., BioEssays 15:675-687 (1993); Schimmel P., Trends Biochem. Sci. 16:1-3 (1991); Nagel G. M., Doolittle R. F., Proc. Natl. Acad. Sci. U.S.A. 88:8121-8125 (1991); Cusack S., Haertlein M., Leberman R., Nucleic Acids Res. 19:3489-3498 (1991); Cusack S., Biochimie 75:1077-1081 (1993); Cusack S., Berthet-Colominas C., Haertlein M., Nassar N., Leberman R., Nature 347:249-255 (1990); and Leveque F., Plateau P., Dessen P., Blanquet S., Nucleic Acids Res. 18:305-312 (1990); which are hereby incorporated herein by reference in their entirety.
In preferred embodiments, the following aminoacyl-transfer RNA synthetases class-II domain polypeptide is encompassed by the present invention: LIGRMATALERLTGLERAES (SEQ ID NO:107). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this RGD cell attachment site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The LTRPC3b polypeptide has been shown to comprise three amidation sites according to the Motif algorithm (Genetics Computer Group, Inc.). The precursor of hormones and other active peptides which are C-terminally amidated is always directly followed by a glycine residue which provides the amide group, and most often by at least two consecutive basic residues (Arg or Lys) which generally function as an active peptide precursor cleavage site. Although all amino acids can be amidated, neutral hydrophobic residues such as Val or Phe are good substrates, while charged residues such as Asp or Arg are much less reactive. A consensus pattern for amidation sites is the following: x-G-[RK]-[RK], wherein “X” represents the amidation site. Additional information relating to asparagine glycosylation may be found in reference to the following publications, which are hereby incorporated by reference herein: Kreil G., Meth. Enzymol. 106:218-223(1984); and Bradbury A. F., Smyth D. G., Biosci. Rep. 7:907-916 (1987).
In preferred embodiments, the following amidation site polypeptides are encompassed by the present invention: DIPLRRGRKTTKKR (SEQ ID NO:147), HRLIPLGRKIYEFY (SEQ ID NO:148), and/or EENEAKGRRATIAI (SEQ ID NO:149). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these LTRPC3b amidation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:3 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 4687 of SEQ ID NO:3, b is an integer between 15 to 4701, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:3, and where b is greater than or equal to a+14.
Features of the Polypeptide Encoded by Gene No:3
The polypeptide of this gene provided as SEQ ID NO:6 (FIGS. 3A-F), encoded by the polynucleotide sequence according to SEQ ID NO:5 (FIGS. 3A-F), and/or encoded by the polynucleotide contained within the deposited clone, LTRPC3c, has significant homology at the nucleotide and amino acid level to the human melastatin 1 protein (Melastatin1; Genbank Accession No. gi|3243075; SEQ ID NO:11). An alignment of the LTRPC3c polypeptide with this protein is provided in FIGS. 5A-D.
The LTRPC3c polypeptide was determined to share 65.4% identity and 73.1% similarity with the human melastatin 1 protein (Melastatin1; Genbank Accession No. gi|3243075; SEQ ID NO:11) as shown in FIG. 8.
The LTRPC3c protein is believed to represent a member of a new class of protein kinases referred to as alpha kinases (Curr. Biol. 9 (2), R43-R45 (1999)). These kinases represent a novel type of signaling molecule comprising both a catalytic protein kinase domain, in addition to, an ion channel domain. This family is commonly referred to as the transient receptor potential channel (TRP) family. Melastatin1 defines a separate subfamily of TRP channels referred to as TRPM (melastatin1). TRPM family members are characteristic of their unusually long cytoplasmic tails at both ends of the channel domain and some of the family members contain an enzyme domain at the C-terminal region. Thus, LTRPC3 represents a novel member of the TRPM subfamily.
The melastatin1 protein is believed to be negatively associated with the incidence of melanoma based upon its inverse correlative expression in highly aggressive melanomas (Genomics 54 (1), 116-123 (1998)). Thus, overexpression of melastatin1 could represent a novel therapeutic in the treatment of melanoma and potentially other cancers.
Based upon the observed homology, the polypeptide of the present invention is expected to share at least some biological activity with other transient receptor potential channel family members, more specifically with the melastatin1 protein, in addition to, other transient receptor potential channel family members referenced elsewhere herein or otherwise known in the art.
Most of the known transient receptor potential channel family members, possess one or more transmembrane domains. Likewise, the LTRPC3c polypeptide has been determined to comprise six transmembrane domains (TM1-TM6) as shown in FIGS. 3A-F. The transmembrane domains are located from about amino acid 620 to about amino acid 637 (TM1), from about amino acid 717 to about amino acid 734 (TM2), from about amino acid 790 to about amino acid 803 (TM3), from about amino acid 817 to about amino acid 834 (TM4), from about amino acid 851 to about amino acid 868 (TM5), and/or from about amino acid 950 to about amino acid 970 (TM6) of SEQ ID NO:6. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of the above referenced polypeptide.
In preferred embodiments, the following transmembrane domain polypeptides are encompassed by the present invention: LKVILGILLPPSILSLEF (SEQ ID NO:38), IVKFWFYTLAYIGYLMLF (SEQ ID NO:39), VTDLIAILLFSVGM (SEQ ID NO:40), RVIYCVNIIYWYIRLLDI (SEQ ID NO:41), MMIDMMYFVIIMLVVLMS (SEQ ID NO:42), and/or AIMACYLLVANILLVNLLIAV (SEQ ID NO:43). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the LTRPC3c transmembrane polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The present invention also encompasses polypeptides corresponding to the regions between each transmembrane domain of LTRPC3c, referred to herein as inter-transmembrane domain polypeptides (inter TM1-2, inter TM2-3, inter TM3-4, inter TM4-5, and inter TM5-6). Such regions are typically solvent accessible (either extracellularly or intracellularly). Therefore, these regions are amenable to the development of agonists and/or antagonists to the LTRPC3c polypeptide, particularly for the development of antibodies specific to these regions.
In preferred embodiments, the following inter-transmembrane domain polypeptides are encompassed by the present invention: KNKDDMPYMSQAQEIHLQEKEAEEPEKPTKEKEEEDMELTAMLGRNNGESS RKKDEEEVQSKHRLIPLGRKIYEFYNAP (SEQ ID NO:150), NYIVLVKMERWPSTQEWIVISYIFTLGIEKMREILMSEPGKLLQKVKVWLQEY WN (SEQ ID NO:151), ILRLQDQPFRSDG (SEQ ID NO:152), FGVNKYLGPYVMMIGK (SEQ ID NO:153), and/or FGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFADQIDRKQVYDSHTPK SAPCGQNETREDGKIIQLPPCKTGAWIVP (SEQ ID NO:154). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the LTRPC3c inter-transmembrane polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3c inter TM1-2 domain deletion polypeptides are encompassed by the present invention: K1-P79, N2-P79, K3-P79, D4-P79, D5-P79, M6-P79, P7-P79, Y8-P79, M9-P79, S10-P79, Q11-P79, A12-P79, Q13-P79, E14-P79, I15-P79, H16-P79, L17-P79, Q18-P79, E19-P79, K20-P79, E21-P79, A22-P79, E23-P79, E24-P79, P25-P79, E26-P79, K27-P79, P28-P79, T29-P79, K30-P79, E31-P79, K32-P79, E33-P79, E34-P79, E35-P79, D36-P79, M37-P79, E38-P79, L39-P79, T40-P79, A41-P79, M42-P79, L43-P79, G44-P79, R45-P79, N46-P79, N47-P79, G48-P79, E49-P79, S50-P79, S51-P79, R52-P79, K53-P79, K54-P79, D55-P79, E56-P79, E57-P79, E58-P79, V59-P79, Q60-P79, S61-P79, K62-P79, H63-P79, R64-P79, L65-P79, I66-P79, P67-P79, L68-P79, G69-P79, R70-P79, K71-P79, I72-P79, and/or Y73-P79 of SEQ ID NO:150. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3c inter TM1-2 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3c inter TM1-2 domain deletion polypeptides are encompassed by the present invention: K1-P79, K1-A78, K1-N77, K1-Y76, K1-F75, K1-E74, K1-Y73, K1-I72, K1-K71, K1-R70, K1-G69, K1-L68, K1-P67, K1-I66, K1-L65, K1-R64, K1-H63, K1-K62, K1-S61, K1-Q60, K1-V59, K1-E58, K1-E57, K1-E56, K1-D55, K1-K54, K1-K53, K1-R52, K1-S51, K1-S50, K1-E49, K1-G48, K1-N47, K1-N46, K1-R45, K1-G44, K1-L43, K1-M42, K1-A41, K1-T40, K1-L39, K1-E38, K1-M37, K1-D36, K1-E35, K1-E34, K1-E33, K1-K32, K1-E31, K1-K30, K1-T29, K1-P28, K1-K27, K1-E26, K1-P25, K1-E24, K1-E23, K1-A22, K1-E21, K1-K20, K1-E19, K1-Q18, K1-L17, K1-H16, K1-I15, K1-E14, K1-Q13, K1-A12, K1-Q11, K1-S10, K1-M9, K1-Y8, and/or K1-P7 of SEQ ID NO:150. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3c inter TM1-2 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3c inter TM2-3 domain deletion polypeptides are encompassed by the present invention: F1-N56, N2-N56, Y3-N56, I4-N56, V5-N56, L6-N56, V7-N56, K8-N56, M9-N56, E10-N56, R11-N56, W12-N56, P13-N56, S14-N56, T15-N56, Q16-N56, E17-N56, W18-N56, I19-N56, V20-N56, I21-N56, S22-N56, Y23-N56, I24-N56, F25-N56, T26-N56, L27-N56, G28-N56, I29-N56, E30-N56, K31-N56, M32-N56, R33-N56, E34-N56, I35-N56, L36-N56, M37-N56, S38-N56, E39-N56, P40-N56, G41-N56, K42-N56, L43-N56, L44-N56, Q45-N56, K46-N56, V47-N56, K48-N56, V49-N56, and/or W50-N56 of SEQ ID NO:151. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3c inter TM2-3 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3c inter TM2-3 domain deletion polypeptides are encompassed by the present invention: F1-N56, F1-W55, F1-Y54, F1-E53, F1-Q52, F1-L51, F1-W50, F1-V49, F1-K48, F1-V47, F1-K46, F1-Q45, F1-L44, F1-L43, F1-K42, F1-G41, F1-P40, F1-E39, F1-S38, F1-M37, F1-L36, F1-I35, F1-E34, F1-R33, F1-M32, F1-K31, F1-E30, F1-I29, F1-G28, F1-L27, F1-T26, F1-F25, F1-I24, F1-Y23, F1-S22, F1-I21, F1-V20, F1-I19, F1-W18, F1-E17, F1-Q16, F1-T15, F1-S14, F1-P13, F1-W12, F1-R11, F1-E10, F1-M9, F1-K8, and/or F1-V7 of SEQ ID NO:151. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3c inter TM2-3 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3c inter TM3-4 domain deletion polypeptides are encompassed by the present invention: I1-G13, L2-G13, R3-G13, L4-G13, Q5-G13, D6-G13, and/or Q7-G13 of SEQ ID NO:152. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3c inter TM3-4 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3c inter TM3-4 domain deletion polypeptides are encompassed by the present invention: I1-G13, I1-D12, I1-S11, I1-R10, I1-F9, I1-P8, and/or I1-Q7 of SEQ ID NO:152. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3c inter TM3-4 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3c inter TM4-5 domain deletion polypeptides are encompassed by the present invention: F1-K16, G2-K16, V3-K16, N4-K16, K5-K16, Y6-K16, L7-K16, G8-K16, P9-K16, and/or Y10-K16 of SEQ ID NO:153. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3c inter TM4-5 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3c inter TM4-5 domain deletion polypeptides are encompassed by the present invention: F1-K16, F1-G15, F1-I14, F1-M13, F1-M12, F1-V11, F1-Y10, F1-P9, F1-G8, and/or F1-L7 of SEQ ID NO:153. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3c inter TM4-5 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3c inter TM5-6 domain deletion polypeptides are encompassed by the present invention: F1-P81, G2-P81, V3-P81, A4-P81, R5-P81, Q6-P81, A7-P81, I8-P81, L9-P81, F10-P81, P11-P81, N12-P81, E13-P81, E14-P81, P15-P81, S16-P81, W17-P81, K18-P81, L19-P81, A20-P81, K21-P81, N22-P81, I23-P81, F24-P81, Y25-P81, M26-P81, P27-P81, Y28-P81, W29-P81, M30-P81, I31-P81, Y32-P81, G33-P81, E34-P81, V35-P81, F36-P81, A37-P81, D38-P81, Q39-P81, I40-P81, D41-P81, R42-P81, K43-P81, Q44-P81, V45-P81, Y46-P81, D47-P81, S48-P81, H49-P81, T50-P81, P51-P81, K52-P81, S53-P81, A54-P81, P55-P81, C56-P81, G57-P81, Q58-P81, N59-P81, E60-P81, T61-P81, R62-P81, E63-P81, D64-P81, G65-P81, K66-P81, I67-P81, I68-P81, Q69-P81, L70-P81, P71-P81, P72-P81, C73-P81, K74-P81, and/or T75-P81 of SEQ ID NO:154. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3c inter TM5-6 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3c inter TM5-6 domain deletion polypeptides are encompassed by the present invention: F1-P81, F1-V80, F1-I79, F1-W78, F1-A77, F1-G76, F1-T75, F1-K74, F1-C73, F1-P72, F1-P71, F1-L70, F1-Q69, F1-I68, F1-I67, F1-K66, F1-G65, F1-D64, F1-E63, F1-R62, F1-T61, F1-E60, F1-N59, F1-Q58, F1-G57, F1-C56, F1-P55, F1-A54, F1-S53, F1-K52, F1-P51, F1-T50, F1-H49, F1-S48, F1-D47, F1-Y46, F1-V45, F1-Q44, F1-K43, F1-R42, F1-D41, F1-I40, F1-Q39, F1-D38, F1-A37, F1-F36, F1-V35, F1-E34, F1-G33, F1-Y32, F1-I31, F1-M30, F1-W29, F1-Y28, F1-P27, F1-M26, F1-Y25, F1-F24, F1-I23, F1-N22, F1-K21, F1-A20, F1-L19, F1-K18, F1-W17, F1-S16, F1-P15, F1-E14, F1-E13, F1-N12, F1-P11, F1-F10, F1-L9, F1-I8, and/or F1-A7 of SEQ ID NO:154. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3c inter TM5-6 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The LTRPC3c polypeptide was determined to comprise several conserved cysteines, at amino acid 94, 210, 265, 419, 517, 582, 599. 821, 941, 1057, 1148, and 1414 of SEQ ID No: 2 (FIGS. 1A-F). Conservation of cysteines at key amino acid residues is indicative of conserved structural features, which may correlate with conservation of protein function and/or activity.
In confirmation of the LTRPC3c representing a member of the transient receptor channel family, the LTRPC3c polypeptide was determined to comprise a predicted TRP domain (EWKFAR) located from about amino acid 985 to about amino acid 990 of SEQ ID NO:6. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of the above referenced polypeptide.
In preferred embodiments, the following TRP domain polypeptide is encompassed by the present invention: VWKFQR (SEQ ID NO:155). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of this LTRPC3c TRP domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
In further confirmation of the LTRPC3c representing a member of the transient receptor channel family, the LTRPC3c polypeptide was determined to comprise a predicted ion transport signature domain located at about amino acid 760 to about amino acid 971 of SEQ ID NO:6. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of the above referenced polypeptide.
In preferred embodiments, the following ion transport signature domain polypeptide is encompassed by the present invention: LGIEKMREILMSEPGKLLQKVKVWLQEYWNVTDLIAILLFSVGMILRLQDQPF RSDGRVIYCVNIIYWYIRLLDIFGVNKYLGPYVMMIGKMMIDMMYFVIIMLV VLMSFGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFADQIDRKQVYDS HTPKSAPCGQNETREDGKIIQLPPCKTGAWIVPAIMACYLLVANILLVNLLIAV F (SEQ ID NO:156). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of this LTRPC3c ion transport signature domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The LTRPC3c polypeptide was determined to comprise a predicted coiled-coil domain located at about amino acid 1075 to about amino acid 1129 of SEQ ID NO:6. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of the above referenced polypeptide.
In preferred embodiments, the following coiled-coil domain polypeptide is encompassed by the present invention: ERIRVTSERVENMSMRLEEVNEREHSMKASLQTVDIRLAQLEDLIGRMATAL ERL (SEQ ID NO:157). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of this LTRPC3c coiled-coil domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The present invention is also directed to polynucleotides comprising at least about 39 nt of the polynucleotide sequence provided as SEQ ID NO:5. Specifically, the present invention is directed to a polynucleotide sequence comprising nucleotides from about nucleotide 1174 to about nucleotide 1212; nucleotides from about nucleotide 1174 to about nucleotide 1215; nucleotides from about nucleotide 1168 to about nucleotide 1209; nucleotides from about nucleotide 1165 to about nucleotide 1209; of SEQ ID NO:5; and/or nucleotides from about nucleotide 1162 to about nucleotide 1209. In this context “about” includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus, or at both termini.
The present invention is also directed to polypeptides comprising at least about 13 amino acids of the polypeptides sequence provided as SEQ ID NO:6. Specifically, the present invention is directed to a polypeptides sequence comprising amino acids from about amino acid 2728 to about amino acid 2766; amino acids from about amino acid 2728 to about amino acid 2769; amino acids from about amino acid 2728 to about amino acid 2772; amino acids from about amino acid 2725 to about amino acid 2766; amino acids from about amino acid 2722 to about amino acid 2766; and/or amino acids from about amino acid 2719 to about amino acid 2766 of SEQ ID NO:6. In this context “about” includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus, or at both termini.
LTRPC3c polypeptides and polynucleotides are useful for diagnosing diseases related to the over and/or under expression of LTRPC3c by identifying mutations in the LTRPC3c gene using LTRPC3c sequences as probes or by determining LTRPC3c protein or mRNA expression levels. LTRPC3c polypeptides will be useful in screens for compounds that affect the activity of the protein. LTRPC3c peptides can also be used for the generation of specific antibodies and as bait in yeast two hybrid screens to find proteins the specifically interact with LTRPC3c.
LTRPC3b polypeptides and polynucleotides are useful for diagnosing diseases related to the over and/or under expression of LTRPC3b by identifying mutations in the LTRPC3b gene using LTRPC3b sequences as probes or by determining LTRPC3b protein or mRNA expression levels. LTRPC3b polypeptides will be useful in screens for compounds that affect the activity of the protein. LTRPC3b peptides can also be used for the generation of specific antibodies and as bait in yeast two hybrid screens to find proteins the specifically interact with LTRPC3b.
Consistent with LTRPC3 representing a transient receptor potential channel, immunohistochemical experiments have shown that LTRPC3 localizes to the cell membrane (see FIG. 17 and Example 6). Specifically, the complete open reading frame of LTRPC3 with a C-terminal HA tag was transiently transfected into HEK 293 cells to assess its cellular localization. The HA-tagged LTRPC3 was detected using a fluorescein-conjugated anti-HA antibody and a laser scanning confocal microscope which produces a green fluorescent signal. The green fluorescent signal was exclusively detected at the subplasma membrane region of the transfected cells (see FIG. 17), which is consistent with LTRPC3 being an integral membrane protein. Under these conditions approximately  ̃70% of cells are expressing LTRPC3. The expression of full-length protein was accessed with immunoblot using an anti-HA antibody and detected as the expected size of  ̃170 kD (data not shown). Cellular localization of the LTRPC3c splice variant is expected to be similarly localized to the subplasma membrane region.
Moreover, physiometric studies have shown that LTRPC3 is a functional Ca2+ permeable channel (see FIGS. 18 and 19, and Example 7). LTRPC3 function was assessed using a Fluorometric Imaging Plate Reader (FLIPRTM) that measures real-time intracellular fluorescence changes. Cells transiently transfected with vector or LTRPC3-HA were loaded with the cytoplasmic Ca2+ indicator Fluoro-4 in a 1 mM Ca2+ solution. Addition of Ca2+ to the media resulted in a concentration-dependent influx of Ca2+ into LTRPC3-expressing cells (FIG. 18; right panels), indicating that LTRPC3 is a functional Ca2+ channel. In contrast, vector-transfected cells showed minimal Ca2+ influx under the same experimental conditions (FIG. 18, left panels). The non-transfected cells were indistinguishable from the vector-transfected cells (data not shown). Therefore, LTRPC3 is a constitutively active channel capable of mediating Ca2+ influx. The Ca2+ influx activity of the LTRPC3c splice variants is expected to be similar to LTRPC3.
To further address the mechanism of LTRPC3-mediated Ca2+ entry, similar Ca2+ addition experiments were performed on transfected cells incubated ( ̃30 min) in a nominally Ca2+-free solution. Previous studies have shown that lowering extracellular Ca2+ concentration below physiological levels can deplete intracellular Ca2+ stores in many cell types including HEK 293 (EMBO J. 17, 4274-4282, (1998)). Incubating vector-transfected HEK 293 cells in a nominally Ca2+-free solution gave rise to Ca2+ entry that was dependent on the concentration of Ca2+ added to the buffers, indicating Ca2+ influx was mediated through endogenous SOCs in HEK293 cells (FIG. 18, left panels). In LTRPC3 cells, the Ca2+ transients triggered by similar Ca2+ treatment were much larger (FIG. 18, right panels). This Ca2+ entry observed in LTRPC3 cells incubated in Ca2+-free media were greater than those observed in 1 mM Ca2+ media, indicating that LTRPC3-mediated Ca2+ entry can be potentiated by the store-depletion. The store-depletion potentiation of LTRPC3-mediated Ca2+ entry is expected to be similar for the LTRPC3c splice variant.
The store-operated mechanism of LTRPC3-mediated Ca2+ influx was tested further by passively depleting Ca2+ stores with thapsigargin (TG), an inhibitor of microsomal Ca2+ ATPases that pumps ions from the cytosol back into the stores. Addition of 2 μM thapsigargin equivalently depleted Ca2+ stores in LTRPC3-HA- and vector-transfected cells (FIG. 19A). Following store depletion with TG, addition of Ca2+ to the buffer induced a much larger Ca2+ entry in LTRPC3 cells compared to the vector control cells. The increased Ca2+ entry of LTRPC3 cells, relative to non-LTRPC3 transfected cells, post store depletion with TG is expected to be similar for the LTRPC3c splice variant.
Receptor-mediated Ca2+ entry was also more pronounced in LTRPC3-HA-transfected cells. Carbachol (CCh) can activate an endogenous muscarinic receptor and trigger IP3 production, leading to store-depletion in HEK 293 cells. The addition of 50 μM of CCh caused a transient and rapid intracellular Ca2+ increase in both LTRPC3- and vector-transfected cells (FIG. 19B). After the store depletion with CCh, adding of Ca2+ to the buffer induced a much larger influx of Ca2+ into LTRPC3 cells, as compared to vector control cells. These results show that after store depletion with TG or CCh LTRPC3-transfected cells exhibit an increased Ca2+ influx when compared to control cells. The increased Ca2+ entry of LTRPC3 cells, relative to non-LTRPC3 transfected cells, post store depletion with TG or CCh is expected to be similar for the LTRPC3c splice variant.
The lanthanides, gadolinium (Gd3+) and lanthanum (La3+), are noneselective Ca2+-permeable channel blockers, often used as part of the characterization of overexpressed TRP channels. Both lanthanides blocked LTRPC3 Ca2+ conductance, although La3+ was more potent (FIG. 19C). In the presence of 1 mM Ca2+ in which endogenous SOCs is minimally activated (FIG. 18A), pre-treatment with 100 μM of La3+ and Gd3+ blocked LTRPC3 Ca2+ currents, stimulated by adding 10 mM Ca2+, by 67 and 39%, respectively. These results indicated that LTRPC3 mediated currents are not non-specific leak currents resulting from protein overexpression.
LTRPC3 is constitutively active but can be potentiated by store-depletion and is partially sensitive to La3+ and Gd3+ blockade. LTRPC3 is believed to represent the first member of the TRPM subfamily that exhibits this store-operated mechanism, although some members of TRPC subfamily have been considered for this role. TRPM1 and TRPM4a are constitutive Ca2+ permeable channels but it is unclear whether they can be stimulated by store-depletion (Proc. Natl. Acad. Sci. U.S.A. 98, 10692-10697, (2001)). Distinct from TRPM4a, TRPM4b is directly activated by changes in intracellular Ca2+ without significant permeation of Ca2+ (Cell 109, 397-401, (2002)). TRPM2 is activated by ADP-ribose, NAD and changes in redox status (Nature 411, 595-599, (2001); Science 293, 1327-1330, (2001); and Mol. Cell 9, 163-173, (2002)). TRPM7 is regulated by Mg2+-ATP and/or PIP2 (Science 291, 1043-1047, (2001); Nature 411, 690-695, (2001); and Nat. Cell Biol. 4, 329-36 (2002)). TRPM8 is activated by cold temperatures and cooling agents (Nature 416, 52-58, (2002); and Cell 108, 705-715, (2002)). Therefore, in conjunction with its fairly restricted tissue expression, which is not observed with any other family members, LTRPC3 may have a unique biological function in human.
Expression profiling designed to measure the steady state mRNA levels encoding the LTRPC3 polypeptide showed predominately high expression levels in kidney. The LTRPC3 polypeptide was also significantly expression in spinal cord, testis, and brain (as shown in FIG. 6).
Moreover, Northern hybridizations of the LTRPC3 mRNA confirmed the predominately high expression levels in kidney, and significant expression levels in testis, and brain (as shown in FIG. 7). The Northern hybridization was not performed on spinal cord tissue.
Expanded analysis of LTRPC3 expression levels by TaqManTM quantitative PCR (see FIG. 12) confirmed that the LTRPC3 polypeptide is expressed in kidney, brain, testis (FIGS. 6 and 7), although higher expression levels were observed in brain than previously appreciated. LTRPC3 mRNA was expressed predominately in the brain, specifically the cerebellum, choroid plexus, the locus coeruleus, the posterior hypothalamus and the substantia nigra. Expression of LTRPC3 was also significantly expressed in the kidney, with higher levels observed in the cortex than in the medulla or pelvis. LTRPC3 was also significantly expressed in the spinal cord, testis, and to a lesser extent in other tissues as shown.
Therefore, LTRPC3c polynucleotides and polypeptides, including modulators or fragments thereof, may be useful in treating, diagnosing, prognosing, and/or preventing a variety of neurological conditions, in particular various choroid plexus neoplasms, choroid plexus papilloma, Alzheimer's disease, prion disorders and multiple sclerosis and movement disorders that involve the cerebellum. Based upon the expression pattern of LTRPC3 in kidney, this novel TRP family member, or a splice variant or polymorphism thereof, may also be the cause solitary metastasis in the choroid plexus, a rare type of carcinoma. For example, it has been shown that out of 15 cases of solitary metastasis of the choroid plexus, five originated from renal cell carcinoma (Neurol. Med. Chir. (Tokyo) 1997 December; 37(12):916-9). Additionally, given the rather selective expression of LTRPC3 in the choroid plexus and renal tissues, it may be possible that altered function of LTRPC3 or a splice variant or polymorphism thereof, may be responsible for solitary metastasis and renal carcinoma. LTRPC3 polynucleotides and polypeptides, including modulators or fragments thereof, may be useful in treating, diagnosing, prognosing, and/or preventing a variety of renal disorders, particularly solitary metastasis in the choroid plexus, and renal carcinoma.
Moreover, an additional analysis of LTRPC3 expression levels by TaqManTM quantitative PCR (see FIG. 13) in disease cells and tissues indicated that the LTRPC3 polypeptide is differentially expressed in renal, testicular, and ovarian tumor tissues. In the renal tumor tissue results, an average of 2 out of 3 matched samples, which represent 3 out of 4 samples total, showed a significant decrease in LTRPC3 steady state RNA levels in tumor compared to control samples. In the testicular tumor tissue results, differential expression of LTRPC3 in testicular cancers was observed with all 5 tumor samples showing a significant reduction in steady-state RNA levels compared to two control samples. In the ovarian tumor tissue results, differential expression of LTRPC3 in ovarian cancers was observed with 3 tumor samples showing a significant reduction in steady-state RNA levels compared to five control samples.
The differential expression of LTRPC3 in tumors relative to normal tissues suggests that loss of LTRPC3 expression during tumor progression might contribute to the metastatic process by altering internal calcium stores in a manner that reflects a loss of cellular control on apoptosis. Restoring LTRPC3c function might provide a novel therapeutic approach to treating certain cancers. Therefore, LTRPC3c polynucleotides and polypeptides, including modulators or fragments thereof, particularly agonists of LTRPC3c activity or expression, may be useful in treating, diagnosing, prognosing, ameloriating, and/or preventing a variety of cancers and proliferative conditions, particularly of the kidney, testis, and ovaries.
Characterization of the LTRPC3 polypeptide of the present invention using antisense oligonucleotides led to the determination that LTRPC3 is involved in the negative modulation of the FEN1 DNA base-excision repair/proliferation modulating protein as described in Example 5 herein.
Specifically, antisense inhibition of LTRPC3 resulted in upregulation of Fen1, a marker of proliferation. As such, LTRPC3 behaves in a manner similar to a tumor suppressor, in that loss results in increases in proliferation. While some increases in the proliferation markers p21 and IkB were also observed, they were less significant, and could be a response to the pro-proliferative effect of LTRPC3 antisense inhibition. Interestingly, expression of LTRPC3 was also observed to be significantly reduced in tumor tissues, relative to normal tissues (see FIG. 13). Specifically, renal, testicular, and ovarian tumor samples showed as much as 12 to 30 fold down regulation of LTRPC3 message levels compared to normal tissues. This reduction of LTRPC3 correlating with the tumor state is consistent with the negative FEN1 modulatory activity and suggests this gene may have antiproliferative activities. Therefore, for modulators of LTRPC3 to be useful for treating cancer, it would be necessary to increase the activity of the gene or gene product in order to have the reverse effect on cancerous cells. Thus, an agonist of LTRPC3 activity or a compound that increased LTRPC3 message levels would be a desired invention for cancer therapy. The same regimen may also be applicable to LTRPC3 splice variants and/or polymorphisms, such as LTRPC3c.
In preferred embodiments, LTRPC3c polynucleotides and polypeptides, including fragments and modulators thereof, are useful for treating, diagnosing, and/or ameliorating DNA-repair deficiencies, particularly base-excision repair deficiencies, Xeroderma pigmentosum, skin cancer, melanoma, UV senstivity, alkylation sensivity, gamma irradiation sensitivity, pyrimidine dimer sensitivity, chemical mutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone, Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, blood vessel dilation aberrations in the skin, blood vessel dilation aberrations in the eye, conditions involving increased levels of apurinic sites, conditions involving increased levels of apyrimidinic sites, conditions involving increased levels of abasic sites, disorders related to aberrant signal transduction, proliferating disorders, and/or cancers.
Moreover, LTRPC3c polynucleotides and polypeptides, including fragments and modulators thereof, are useful for increasing mammalian base excision repair activity, increasing mammalian single-nucleotide base excision repair activity, and/or increasing mammalian long patch base excision repair activity.
In preferred embodiments, antagonists directed against LTRPC3c are useful for treating, diagnosing, and/or ameliorating DNA-repair deficiencies, particularly base-excision repair deficiencies, Xeroderma pigmentosum, skin cancer, melanoma, UV senstivity, alkylation sensivity, gamma irradiation sensitivity, pyrimidine dimer sensitivity, chemical mutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone, Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, blood vessel dilation aberrations in the skin, blood vessel dilation aberrations in the eye, conditions involving increased levels of apurinic sites, conditions involving increased levels of apyrimidinic sites, conditions involving increased levels of abasic sites, disorders related to aberrant signal transduction, proliferating disorders, and/or cancers.
Moreover, antagonists directed against LTRPC3c are useful for increasing mammalian base excision repair activity, increasing mammalian single-nucleotide base excision repair activity, and/or increasing mammalian long patch base excision repair activity.
In preferred embodiments, agonists directed against LTRPC3c are useful for treating, diagnosing, and/or ameliorating, disorders related to aberrant signal transduction, proliferating disorders, and/or cancers, particularly renal cell carcinomas, testicular cancers, and/or ovarian cancers.
The strong homology to human transient receptor potential channels (TRP), combined with the predominate localized expression in kidney tissue of LTRPC3 suggests the LTRPC3c polynucleotides and polypeptides, including modulators or fragments thereof, may be useful in treating, diagnosing, prognosing, and/or preventing renal diseases and/or disorders, which include, but are not limited to: nephritis, renal failure, nephrotic syndrome, urinary tract infection, hematuria, proteinuria, oliguria, polyuria, nocturia, edema, hypertension, electrolyte disorders, sterile pyuria, renal osteodystrophy, large kidneys, renal transport defects, nephrolithiasis, azotemia, anuria, urinary retention, slowing of urinary stream, large prostate, flank tenderness, full bladder sensation after voiding, enuresis, dysuria, bacteriuria, kidney stones, glomerulonephritis, vasculitis, hemolytic uremic syndromes, thrombotic thrombocytopenic purpura, malignant hypertension, casts, tubulointerstitial kidney diseases, renal tubular acidosis, pyelonephritis, hydronephritis, nephrotic syndrome, crush syndrome, and/or renal colic, in addition to Wilm's Tumor Disease, and congenital kidney abnormalities such as horseshoe kidney, polycystic kidney, and Falconi's syndrome.for example.
Several known TRP family members have been identified that are expressed significantly in kidney tissue. These TRP family members include, for example, Trp12 (Wissenbach, U., Bodding, M., Freichel, M., Flockerzi, V, Lett., 485(2-3):127-34, (2000)); OTRPC4 (Strotmann, R., Harteneck, C., Nunnenmacher, K., Schultz, G., Plant, T, D, Nat, Cell, Biol., 2(10):695-702, (2000)); polycystin-L2 (Guo, L., Schreiber, T, H., Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics., 64(3):241-51, (2000)); and EcaC (Hoenderop, J. G., van, der, Kemp, A, W., Hartog, A., van, de, Graaf, S, F., van, Os, C, H., Willems, P, H., Bindels, R, J. J. Biol, Chem., 274(13):8375-8, (1999)).
Thus, the LTRPC3c polynucleotides and polypeptides are expected to share at least some biological activity with TRP family members expressed in kidney cells and tissues, particularly those specifically referenced herein.
The strong homology to human transient receptor potential channels (TRP) proteins, combined with the localized expression in spinal cord and brain of LTRPC3 suggests the LTRPC3c polynucleotides and polypeptides, including modulators or fragments thereof, may be useful in treating, diagnosing, prognosing, and/or preventing neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in the Examples, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
The strong homology to human transient receptor potential channels (TRP), combined with the localized expression in testis tissue of LTRPC3 emphasizes the potential utility for LTRPC3c polynucleotides and polypeptides in treating, diagnosing, prognosing, and/or preventing testicular, in addition to reproductive disorders.
In preferred embodiments, LTRPC3c polynucleotides and polypeptides including agonists and fragments thereof, have uses which include treating, diagnosing, prognosing, and/or preventing the following, non-limiting, diseases or disorders of the testis: spermatogenesis, infertility, Klinefelter's syndrome, XX male, epididymitis, genital warts, germinal cell aplasia, cryptorchidism, varicocele, immotile cilia syndrome, and viral orchitis. The LTRPC3c polynucleotides and polypeptides including agonists and fragments thereof, may also have uses related to modulating testicular development, embryogenesis, reproduction, and in ameliorating, treating, and/or preventing testicular proliferative disorders (e.g., cancers, which include, for example, choriocarcinoma, Nonseminoma, seminona, and testicular germ cell tumors).
Likewise, the localized expression in testis tissue also emphasizes the potential utility for LTRPC3c polynucleotides and polypeptides in treating, diagnosing, prognosing, and/or preventing metabolic diseases and disorders which include the following, not limiting examples: premature puberty, incomplete puberty, Kallman syndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis, congenital adrenal hyperplasia, FSH deficiency, and granulomatous disease, for example.
This gene product may also be useful in assays designed to identify binding agents, as such agents (antagonists) are useful as male contraceptive agents. The testes are also a site of active gene expression of transcripts that is expressed, particularly at low levels, in other tissues of the body. Therefore, this gene product may be expressed in other specific tissues or organs where it may play related functional roles in other processes, such as hematopoiesis, inflammation, bone formation, and kidney function, to name a few possible target indications.
Several known TRP family members have been identified that are expressed significantly in testis tissue. These TRP family members include, for example, polycystin-L2 (Guo, L., Schreiber, T, H., Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics., 64(3):241-51, (2000)); TRP7 (Okada, T., Inoue, R., Yamazaki, K., Maeda, A., Kurosaki, T., Yamakuni, T., Tanaka, I., Shimizu, S., Ikenaka, K., Imoto, K., Mori, Y, J. Biol, Chem., 274(39):27359-70, (1999)); btrp2 (Wissenbach, U., Schroth, G., Philipp, S., Flockerzi, V, Lett., 429(1):61-6, (1998)); Htrp-1 (Zhu, X., Chu, P, B., Peyton, M., Bimbaumer, L, Lett., 373(3):193-8, (1995)); and TRPC1 (Wes, P, D., Chevesich, J., Jeromin, A., Rosenberg, C., Stetten, G., Montell, C, Proc, Natl, Acad, Sci, U,S,A., 92(21):9652-6, (1995)).
Thus, the LTRPC3c polynucleotides and polypeptides are expected to share at least some biological activity with TRP family members expressed in testis cells and tissues, particularly those specifically referenced herein.
As described elsewhere herein, transient receptor potential channel family members have been implicated in modulating cell proliferation, differentiation, migration, activation, exocytosis, muscle contraction, gene expression, apoptosis. signalling, pheromone sensory signaling, smooth muscle tone, pain perception, heat perception, osmosenstivity, and mechanosensitivity. Moreover, transient receptor potential channel family members have been implicated in disorders of the skin, skeletal-muscle, nervous, cardiac, and vascular systems, in addition to the following, non-limiting diseases and disorders, which include, for example, arteriosclerosis, neointimal hypoerplasia, metastatic melanomas, bipolar disorder, nonsyndromic hereditary deafness, Knobloch syndrome, holosencephaly, and various maligancies including prostate cancer.
In preferred embodiments, LTRPC3c polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include, modulating cell proliferation, differentiation, migration, activation, exocytosis, muscle contraction, gene expression, apoptosis. signalling, pheromone sensory signaling, smooth muscle tone, pain perception, heat perception, osmosenstivity, and mechanosensitivity.
In more preferred embodiments, LTRPC3c polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include, treating, ameliorating, preventing, detecting, and/or prognosing various diseases and disorders, particularly the following, non-limiting examples, disorders of the skin, skeletal-muscle, nervous, cardiac, and vascular systems, in addition to the following, non-limiting diseases and disorders, which include, for example, arteriosclerosis, neointimal hypoerplasia, metastatic melanomas, bipolar disorder, nonsyndromic hereditary deafness, Knobloch syndrome, holosencephaly, and various maligancies including prostate cancer.
LTRPC3c polynucleotides and polypeptides of the present invention, including agonists and/or fragments may be involved in intracellular Ca2+ homeostasis which affects various aspects of biological functions including mechano-regulation, pain transduction, vasorelaxation, gene expression, cell cycle and proliferation/apoptosis. Since LTRPC3c is dominantly expressed in kidney, it may particularly play an important role in regulating cytosolic Ca2+ in the renal system, potentially Ca2+ absorption.
The LTRPC3c gene maps to chromosome 9q21.11-21.31 between the two markers D9S1874 and D9S1807. This region is linked to amyotrophic lateral sclerosis with frontotemporal dementia, early-onset pulverulent cataract, infantile nephronophthisis, hypomagnesemia with secondary hypocalcemia (HSH), and familial hemophagocytic lymphohistiocytosis. Therefore, agonists and/or antagonists of the novel LTRPC3c can be used to treat diseases including various forms of neuronal degeneration, neurogenic inflammation, allergy, immunodeficiency/excessive immune activation, visual defects, hearing disorder, pain, cancer, hypertension and other cardiovascular diseases. In addition, the therapeutics may be useful in the treatment of diseases associated with disturbances in Ca2+ homeostasis including osteoporosis, hypercalciuric stone disease, and chronic renal failure.
Moreover, given the selective expression in kidney, LTRPC3c could be considered a candidate gene for HSH, since the phenotype is a renal insufficiency. LTRPC3c chromosomal location is  ̃600 kb downstream of a X;9 translocation breakpoint interval described for one patient with HSH (Hum. Mol. Genet. 6, 1491-1497, (1997)). Recently, two groups reported that a new member of the TRPM subfamily, TRPM6, expressed in both intestinal tissues and kidney, is associated with HSH (Nat. Genet. 31, 166-170, (2002); and Nat. Genet. 31, 171-174 (2002)). Indeed, LTRPC3 is  ̃4 mb 5′ to TRPM6 on 9q21. Although TRPM6 is the first component identified for HSH, this should not preclude involvement of LTRPC3c in the disease. Therefore, it is possible that LTRPC3c may co-localize with TRPM6 and/or forms a heteromultimer with TRPM6 in the kidney.
The kidney plays a major role in Ca2+ homeostasis (reviewed in Physiol. Rev. 75, 429-471, (1995)). LTRPC3c may play a direct role in Ca2+ absorption due to its constitutive Ca2+ permeability. Alternatively, LTRPC3c may be the SOC that regulates Ca2+ absorption. In kidney, Ca2+ absorption is regulated by agonists such as calcitonin, parathyroid hormone and parathyroid hormone-related peptide through their respective G protein-coupled receptors, and downstream SOCs. Additionally, in kidney a SOC has been described (J. Biol. Chem. 276, 25759-25765, (2001)) for the action of angiotensin II, a vasoactive peptide that plays a major role in regulating blood pressure.
In addition, LTRPC3c polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include modulating intracellular Ca++ ion concentrations, Ca++ ion flux, stored intracellular Ca++ ion concentrations, Ca++ ion pump activity, Ca++ ion flow into cell, Ca++ ion flow out of cells, the activation of Ca++ sensitive proteins, the activation of Ca++ sensitive signaling pathways, the activation of kinase-activatible proteins, and the activation of kinase-dependent signaling pathways.
The LTRPC3c polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include modulating proliferation, differentiation, migration, and activation in various cells, tissues, and organisms, and particularly in mammalian kidney, spinal cord, testis, and brain, preferably human. LTRPC3c polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, may be useful in diagnosing, treating, prognosing, and/or preventing renal, neural, and/or proliferative diseases or disorders, particularly of the renal system.
In addition, antagonists of the LTRPC3c polynucleotides and polypeptides, including modulators or fragments thereof, may have uses that include diagnosing, treating, prognosing, and/or preventing diseases or disorders related to transient receptor potential channel activity, which may include renal, neural, reproductive, and/or proliferative diseases or disorders.
Although it is believed the encoded polypeptide may share at least some biological activities with transient receptor potential channel family members, particularly those melastatin1, a number of methods of determining the exact biological function of this clone are either known in the art or are described elsewhere herein. Briefly, the function of this clone may be determined by applying microarray methodology. Nucleic acids corresponding to the LTRPC3c polynucleotides, in addition to, other clones of the present invention, may be arrayed on microchips for expression profiling. Depending on which polynucleotide probe is used to hybridize to the slides, a change in expression of a specific gene may provide additional insight into the function of this gene based upon the conditions being studied. For example, an observed increase or decrease in expression levels when the polynucleotide probe used comes from tissue that has been treated with known transient potential receptor inhibitors, which include, but are not limited to the drugs listed herein or otherwise known in the art, might indicate a function in modulating transient potential receptor function, for example. In the case of LTRPC3c, kidney, spinal cord, testis, and brain, should be used to extract RNA to prepare the probe.
In addition, the function of the protein may be assessed by applying quantitative PCR methodology, for example. Real time quantitative PCR would provide the capability of following the expression of the LTRPC3c gene throughout development, for example. Quantitative PCR methodology requires only a nominal amount of tissue from each developmentally important step is needed to perform such experiments. Therefore, the application of quantitative PCR methodology to refining the biological function of this polypeptide is encompassed by the present invention. Also encompassed by the present invention are quantitative PCR probes corresponding to the polynucleotide sequence provided as SEQ ID NO:5 (FIGS. 3A-F).
The function of the protein may also be assessed through complementation assays in yeast. For example, in the case of the LTRPC3c, transforming yeast deficient in transient receptor potential channel activity with LTRPC3c and assessing their ability to grow would provide convincing evidence the LTRPC3c polypeptide has transient receptor potential channel activity. Additional assay conditions and methods that may be used in assessing the function of the polynucleotides and polypeptides of the present invention are known in the art, some of which are disclosed elsewhere herein.
Alternatively, the biological function of the encoded polypeptide may be determined by disrupting a homologue of this polypeptide in Mice and/or rats and observing the resulting phenotype.
Moreover, the biological function of this polypeptide may be determined by the application of antisense and/or sense methodology and the resulting generation of transgenic mice and/or rats. Expressing a particular gene in either sense or antisense orientation in a transgenic mouse or rat could lead to respectively higher or lower expression levels of that particular gene. Altering the endogenous expression levels of a gene can lead to the observation of a particular phenotype that can then be used to derive indications on the function of the gene. The gene can be either over-expressed or under expressed in every cell of the organism at all times using a strong ubiquitous promoter, or it could be expressed in one or more discrete parts of the organism using a well characterized tissue-specific promoter (e.g., a kidney, spinal cord, testis, and/or brain-specific promoter), or it can be expressed at a specified time of development using an inducible and/or a developmentally regulated promoter.
In the case of LTRPC3c transgenic mice or rats, if no phenotype is apparent in normal growth conditions, observing the organism under diseased conditions (renal, reproductive, neural, or proliferative disorders, etc.) may lead to understanding the function of the gene. Therefore, the application of antisense and/or sense methodology to the creation of transgenic mice or rats to refine the biological function of the polypeptide is encompassed by the present invention.
In preferred embodiments, the following N-terminal LTRPC3c deletion polypeptides are encompassed by the present invention: M1-T1566, Y2-T1566, V3-T1566, R4-T1566, V5-T1566, S6-T1566, F7-T1566, D8-T1566, T9-T1566, K10-T1566, P11-T1566, D12-T1566, L13-T1566, L14-T1566, L15-T1566, H16-T1566, L17-T1566, M18-T1566, T19-T1566, K20-T1566, E21-T1566, W22-T1566, Q23-T1566, L24-T1566, E25-T1566, L26-T1566, P27-T1566, K28-T1566, L29-T1566, L30-T1566, I31-T1566, S32-T1566, V33-T1566, H34-T1566, G35-T1566, G36-T1566, L37-T1566, Q38-T1566, N39-T1566, F40-T1566, E41-T1566, L42-T1566, Q43-T1566, P44-T1566, K45-T1566, L46-T1566, K47-T1566, Q48-T1566, V49-T1566, F50-T1566, G51-T1566, K52-T1566, G53-T1566, L54-T1566, I55-T1566, K56-T1566, A57-T1566, A58-T1566, M59-T1566, T60-T1566, T61-T1566, G62-T1566, A63-T1566, W64-T1566, I65-T1566, F66-T1566, T67-T1566, G68-T1566, G69-T1566, V70-T1566, N71-T1566, T72-T1566, G73-T1566, V74-T1566, I75-T1566, R76-T1566, H77-T1566, V78-T1566, G79-T1566, D80-T1566, A81-T1566, L82-T1566, K83-T1566, D84-T1566, H85-T1566, A86-T1566, S87-T1566, K88-T1566, S89-T1566, R90-T1566, G91-T1566, K92-T1566, I93-T1566, C94-T1566, T95-T1566, I96-T1566, G97-T1566, I98-T1566, A99-T1566, P100-T1566, W101-T1566, G102-T1566, I103-T1566, V104-T1566, E105-T1566, N106-T1566, Q107-T1566, E108-T1566, D109-T1566, L110110-T1566, I11-T1566, G112-T1566, R113-T1566, D114-T1566, V115-T1566, V116-T1566, R117-T1566, P118-T1566, Y119-T1566, Q120-T1566, T121-T1566, M122-T1566, S123-T1566, N124-T1566, P125-T1566, M126-T1566, S127-T1566, K128-T1566, L129-T1566, T130-T1566, V131-T1566, L132-T1566, N133-T1566, S134-T1566, M135-T1566, H136-T1566, S137-T1566, H138-T1566, F139-T1566, I140-T1566, L141-T1566, A142-T1566, D143-T1566, N144-T1566, G145-T1566, T146-T1566, T147-T1566, G148-T1566, K149-T1566, Y150-T1566, G151-T1566, A152-T1566, E153-T1566, V154-T1566, K155-T1566, L156-T1566, R157-T1566, R158-T1566, Q159-T1566, L160-T1566, E161-T1566, K162-T1566, H163-T1566, I164-T1566, S165-T1566, L166-T1566, Q167-T1566, K168-T1566, I169-T1566, N170-T1566, T171-T1566, R172-T1566, I173-T1566, G174-T1566, Q175-T1566, G176-T1566, V177-T1566, P178-T1566, V179-T1566, V180-T1566, A181-T1566, L182-T1566, I183-T1566, V184-T1566, E185-T1566, G186-T1566, G187-T1566, P188-T1566, N189-T1566, V190-T1566, I191-T1566, S192-T1566, I193-T1566, V194-T1566, L195-T1566, E196-T1566, Y197-T1566, L198-T1566, R199-T1566, D200-T1566, T201-T1566, P202-T1566, P203-T1566, V204-T1566, P205-T1566, V206-T1566, V207-T1566, V208-T1566, C209-T1566, D210-T1566, G211-T1566, S212-T1566, G213-T1566, R214-T1566, A215-T1566, S216-T1566, D217-T1566, I218-T1566, L219-T1566, A220-T1566, F221-T1566, G222-T1566, H223-T1566, K224-T1566, Y225-T1566, S226-T1566, E227-T1566, E228-T1566, G229-T1566, G230-T1566, L231-T1566, I232-T1566, N233-T1566, E234-T1566, S235-T1566, L236-T1566, R237-T1566, D238-T1566, Q239-T1566, L240-T1566, L241-T1566, V242-T1566, T243-T1566, I244-T1566, Q245-T1566, K246-T1566, T247-T1566, F248-T1566, T249-T1566, Y250-T1566, T251-T1566, R252-T1566, T253-T1566, Q254-T1566, A255-T1566, Q256-T1566, H257-T1566, L258-T1566, F259-T1566, I260-T1566, I261-T1566, L262-T1566, M263-T1566, E264-T1566, C265-T1566, M266-T1566, K267-T1566, K268-T1566, K269-T1566, E270-T1566, L271-T1566, I272-T1566, T273-T1566, V274-T1566, F275-T1566, R276-T1566, M277-T1566, G278-T1566, S279-T1566, E280-T1566, G281-T1566, H282-T1566, Q283-T1566, D284-T1566, I285-T1566, D286-T1566, L287-T1566, A288-T1566, I289-T1566, L290-T1566, T291-T1566, A292-T1566, L293-T1566, L294-T1566, K295-T1566, G296-T1566, A297-T1566, N298-T1566, A299-T1566, S300-T1566, A301-T1566, P302-T1566, D303-T1566, Q304-T1566, L305-T1566, S306-T1566, L307-T1566, A308-T1566, L309-T1566, A310-T1566, W311-T1566, N312-T1566, R313-T1566, V314-T1566, D315-T1566, I316-T1566, A317-T1566, R318-T1566, S319-T1566, Q320-T1566, I321-T1566, F322-T1566, I323-T1566, Y324-T1566, G325-T1566, Q326-T1566, Q327-T1566, W328-T1566, P329-T1566, V330-T1566, G331-T1566, S332-T1566, L333-T1566, E334-T1566, Q335-T1566, A336-T1566, M337-T1566, L338-T1566, D339-T1566, A340-T1566, L341-T1566, V342-T1566, L343-T1566, D344-T1566, R345-T1566, V346-T1566, D347-T1566, F348-T1566, V349-T1566, K350-T1566, L351-T1566, L352-T1566, I353-T1566, E354-T1566, N355-T1566, G356-T1566, V357-T1566, S358-T1566, M359-T1566, H360-T1566, R361-T1566, F362-T1566, L363-T1566, T364-T1566, I365-T1566, S366-T1566, R367-T1566, L368-T1566, E369-T1566, E370-T1566, L371-T1566, Y372-T1566, N373-T1566, T374-T1566, R375-T1566, H376-T1566, G377-T1566, P378-T1566, S379-T1566, N380-T1566, T381-T1566, L382-T1566, Y383-T1566, H384-T1566, L385-T1566, V386-T1566, R387-T1566, D388-T1566, V389-T1566, K390-T1566, K391-T1566, G392-T1566, N393-T1566, L394-T1566, P395-T1566, P396-T1566, D397-T1566, Y398-T1566, R399-T1566, I400-T1566, S401-T1566, L402-T1566, I403-T1566, D404-T1566, I405-T1566, G406-T1566, L407-T1566, V408-T1566, I409-T1566, E410-T1566, Y411-T1566, L412-T1566, M413-T1566, G414-T1566, G415-T1566, A416-T1566, Y417-T1566, R418-T1566, C419-T1566, N420-T1566, Y421-T1566, T422-T1566, R423-T1566, K424-T1566, R425-T1566, F426-T1566, R427-T1566, T428-T1566, L429-T1566, Y430-T1566, H431-T1566, N432-T1566, L433-T1566, F434-T1566, G435-T1566, P436-T1566, K437-T1566, R438-T1566, P439-T1566, K440-T1566, A441-T1566, L442-T1566, K443-T1566, L444-T1566, L445-T1566, G446-T1566, M447-T1566, E448-T1566, D449-T1566, D450-T1566, I451-T1566, P452-T1566, L453-T1566, R454-T1566, R455-T1566, G456-T1566, R457-T1566, K458-T1566, T459-T1566, T460-T1566, K461-T1566, K462-T1566, R463-T1566, E464-T1566, E465-T1566, E466-T1566, V467-T1566, D468-T1566, I469-T1566, D470-T1566, L471-T1566, D472-T1566, D473-T1566, P474-T1566, E475-T1566, I476-T1566, N477-T1566, H478-T1566, F479-T1566, P480-T1566, F481-T1566, P482-T1566, F483-T1566, H484-T1566, E485-T1566, L486-T1566, M487-T1566, V488-T1566, W489-T1566, A490-T1566, V491-T1566, L492-T1566, M493-T1566, K494-T1566, R495-T1566, Q496-T1566, K497-T1566, M498-T1566, A499-T1566, L500-T1566, F501-T1566, F502-T1566, W503-T1566, Q504-T1566, H505-T1566, G506-T1566, E507-T1566, E508-T1566, A509-T1566, M510-T1566, A511-T1566, K512-T1566, A513-T1566, L514-T1566, V515-T1566, A516-T1566, C517-T1566, K518-T1566, L519-T1566, C520-T1566, K521-T1566, A522-T1566, M523-T1566, A524-T1566, H525-T1566, E526-T1566, A527-T1566, S528-T1566, E529-T1566, N530-T1566, D531-T1566, M532-T1566, V533-T1566, D534-T1566, D535-T1566, I536-T1566, S537-T1566, Q538-T1566, E539-T1566, L540-T1566, N541-T1566, H542-T1566, N543-T1566, S544-T1566, R545-T1566, D546-T1566, F547-T1566, G548-T1566, Q549-T1566, L550-T1566, A551-T1566, V552-T1566, E553-T1566, L554-T1566, L555-T1566, D556-T1566, Q557-T1566, S558-T1566, Y559-T1566, K560-T1566, Q561-T1566, D562-T1566, E563-T1566, Q564-T1566, L565-T1566, A566-T1566, M567-T1566, K568-T1566, L569-T1566, L570-T1566, T571-T1566, Y572-T1566, E573-T1566, L574-T1566, K575-T1566, N576-T1566, W577-T1566, S578-T1566, N579-T1566, A580-T1566, T581-T1566, C582-T1566, L583-T1566, Q584-T1566, L585-T1566, A586-T1566, V587-T1566, A588-T1566, A589-T1566, K590-T1566, H591-T1566, R592-T1566, D593-T1566, F594-T1566, I595-T1566, A596-T1566, H597-T1566, T598-T1566, C599-T1566, S600-T1566, Q601-T1566, M602-T1566, L603-T1566, L604-T1566, T605-T1566, D606-T1566, M607-T1566, W608-T1566, M609-T1566, G610-T1566, R611-T1566, L612-T1566, R613-T1566, M614-T1566, R615-T1566, K616-T1566, N617-T1566, S618-T1566, G619-T1566, L620-T1566, K621-T1566, V622-T1566, I623-T1566, L624-T1566, G625-T1566, I626-T1566, L627-T1566, L628-T1566, P629-T1566, P630-T1566, S631-T1566, I632-T1566, L633-T1566, S634-T1566, L635-T1566, E636-T1566, F637-T1566, K638-T1566, N639-T1566, K640-T1566, D641-T1566, D642-T1566, M643-T1566, P644-T1566, Y645-T1566, M646-T1566, S647-T1566, Q648-T1566, A649-T1566, Q650-T1566, E651-T1566, I652-T1566, H653-T1566, L654-T1566, Q655-T1566, E656-T1566, K657-T1566, E658-T1566, A659-T1566, E660-T1566, E661-T1566, P662-T1566, E663-T1566, K664-T1566, P665-T1566, T666-T1566, K667-T1566, E668-T1566, K669-T1566, E670-T1566, E671-T1566, E672-T1566, D673-T1566, M674-T1566, E675-T1566, L676-T1566, T677-T1566, A678-T1566, M679-T1566, L680-T1566, G681-T1566, R682-T1566, N683-T1566, N684-T1566, G685-T1566, E686-T1566, S687-T1566, S688-T1566, R689-T1566, K690-T1566, K691-T1566, D692-T1566, E693-T1566, E694-T1566, E695-T1566, V696-T1566, Q697-T1566, S698-T1566, K699-T1566, H700-T1566, R701-T1566, L702-T1566, I703-T1566, P704-T1566, L705-T1566, G706-T1566, R707-T1566, K708-T1566, I709-T1566, Y710-T1566, E711-T1566, F712-T1566, Y713-T1566, N714-T1566, A715-T1566, P716-T1566, I717-T1566, V718-T1566, K719-T1566, F720-T1566, W721-T1566, F722-T1566, Y723-T1566, T724-T1566, L725-T1566, A726-T1566, Y727-T1566, I728-T1566, G729-T1566, Y730-T1566, L731-T1566, M732-T1566, L733-T1566, F734-T1566, N735-T1566, Y736-T1566, I737-T1566, V738-T1566, L739-T1566, V740-T1566, K741-T1566, M742-T1566, E743-T1566, R744-T1566, W745-T1566, P746-T1566, S747-T1566, T748-T1566, Q749-T1566, E750-T1566, W751-T1566, I752-T1566, V753-T1566, I754-T1566, S755-T1566, Y756-T1566, I757-T1566, F758-T1566, T759-T1566, L760-T1566, G761-T1566, I762-T1566, E763-T1566, K764-T1566, M765-T1566, R766-T1566, E767-T1566, I768-T1566, L769-T1566, M770-T1566, S771-T1566, E772-T1566, P773-T1566, G774-T1566, K775-T1566, L776-T1566, L777-T1566, Q778-T1566, K779-T1566, V780-T1566, K781-T1566, V782-T1566, W783-T1566, L784-T1566, Q785-T1566, E786-T1566, Y787-T1566, W788-T1566, N789-T1566, V790-T1566, T791-T1566, D792-T1566, L793-T1566, I794-T1566, A795-T1566, I796-T1566, L797-T1566, L798-T1566, F799-T1566, S800-T1566, V801-T1566, G802-T1566, M803-T1566, I804-T1566, L805-T1566, R806-T1566, L807-T1566, Q808-T1566, D809-T1566, Q810-T1566, P811-T1566, F812-T1566, R813-T1566, S814-T1566, D815-T1566, G816-T1566, R817-T1566, V818-T1566, I819-T1566, Y820-T1566, C821-T1566, V822-T1566, N823-T1566, I824-T1566, I825-T1566, Y826-T1566, W827-T1566, Y828-T1566, I829-T1566, R830-T1566, L831-T1566, L832-T1566, D833-T1566, I834-T1566, F835-T1566, G836-T1566, V837-T1566, N838-T1566, K839-T1566, Y840-T1566, L841-T1566, G842-T1566, P843-T1566, Y844-T1566, V845-T1566, M846-T1566, M847-T1566, I848-T1566, G849-T1566, K850-T1566, M851-T1566, M852-T1566, I853-T1566, D854-T1566, M855-T1566, M856-T1566, Y857-T1566, F858-T1566, V859-T1566, I860-T1566, I861-T1566, M862-T1566, L863-T1566, V864-T1566, V865-T1566, L866-T1566, M867-T1566, S868-T1566, F869-T1566, G870-T1566, V871-T1566, A872-T1566, R873-T1566, Q874-T1566, A875-T1566, I876-T1566, L877-T1566, F878-T1566, P879-T1566, N880-T1566, E881-T1566, E882-T1566, P883-T1566, S884-T1566, W885-T1566, K886-T1566, L887-T1566, A888-T1566, K889-T1566, N890-T1566, I891-T1566, F892-T1566, Y893-T1566, M894-T1566, P895-T1566, Y896-T1566, W897-T1566, M898-T1566, I899-T1566, Y900-T1566, G901-T1566, E902-T1566, V903-T1566, F904-T1566, A905-T1566, D906-T1566, Q907-T1566, I908-T1566, D909-T1566, R910-T1566, K911-T1566, Q912-T1566, V913-T1566, Y914-T1566, D915-T1566, S916-T1566, H917-T1566, T918-T1566, P919-T1566, K920-T1566, S921-T1566, and/or A922-T1566 of SEQ ID NO:6. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3c deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3c deletion polypeptides are encompassed by the present invention: M1-T1566, M1-H1565, M1-K1564, M1-S1563, M1-E1562, M1-F1561, M1-S1560, M1-Q1559, M1-F1558, M1-A1557, M1-S1556, M1-T1555, M1-R1554, M1-S1553, M1-L1552, M1-R1551, M1-R1550, M1-M1549, M1-S1548, M1-L1547, M1-S1546, M1-D1545, M1-G1544, M1-R1543, M1-G1542, M1-E1541, M1-P1540, M1-K1539, M1-S1538, M1-S1537, M1-K1536, M1-S1535, M1-R1534, M1-Q1533, M1-F1532, M1-P1531, M1-N1530, M1-Q1529, M1-L1528, M1-S1527, M1-A1526, M1-T1525, M1-N1524, M1-R1523, M1-Q1522, M1-R1521, M1-D1520, M1-L1519, M1-K1518, M1-D1517, M1-S1516, M1-I1515, M1-S1514, M1-F1513, M1-S1512, M1-K1511, M1-R1510, M1-T1509, M1-H1508, M1-A1507, M1-Y1506, M1-P1505, M1-A1504, M1-S1503, M1-P1502, M1-E1501, M1-E1500, M1-A1499, M1-S1498, M1-Y1497, M1-S1496, M1-N1495, M1-A1494, M1-R1493, M1-E1492, M1-I1491, M1-K1490, M1-P1489, M1-V1488, M1-T1487, M1-I1486, M1-N1485, M1-N1484, M1-S1483, M1-L1482, M1-T1481, M1-R1480, M1-E1479, M1-S1478, M1-N1477, M1-D1476, M1-G1475, M1-E1474, M1-Q1473, M1-S1472, M1-S1471, M1-I1470, M1-A1469, M1-I1468, M1-T1467, M1-A1466, M1-R1465, M1-R1464, M1-G1463, M1-K1462, M1-A1461, M1-E1460, M1-N1459, M1-E1458, M1-E1457, M1-S1456, M1-D1455, M1-S1454, M1-S1453, M1-P1452, M1-H1451, M1-S1450, M1-L1449, M1-E1448, M1-A1447, M1-E1446, M1-R1445, M1-E1444, M1-P1443, M1-H1442, M1-C1441, M1-C1440, M1-T1439, M1-L1438, M1-D1437, M1-E1436, M1-V1435, M1-K1434, M1-D1433, M1-G1432, M1-L1431, M1-G1430, M1-G1429, M1-P1428, M1-F1427, M1-A1426, M1-A1425, M1-R1424, M1-D1423, M1-A1422, M1-I1421, M1-A1420, M1-Q1419, M1-P1418, M1-A1417, M1-N1416, M1-V1415, M1-C1414, M1-R1413, M1-T1412, M1-D1411, M1-I1410, M1-C1409, M1-D1408, M1-T1407, M1-I1406, M1-S1405, M1-T1404, M1-Y1403, M1-E1402, M1-A1401, M1-T1400, M1-K1399, M1-V1398, M1-P1397, M1-V1396, M1-G1395, M1-F1394, M1-N1393, M1-A1392, M1-Y1391, M1-Y1390, M1-S1389, M1-R1388, M1-S1387, M1-P1386, M1-S1385, M1-F1384, M1-M1383, M1-F1382, M1-S1381, M1-H1380, M1-S1379, M1-K1378, M1-V1377, M1-I1376, M1-P1375, M1-A1374, M1-E1373, M1-E1372, M1-L1371, M1-L1370, M1-F1369, M1-P1368, M1-T1367, M1-T1366, M1-A1365, M1-L1364, M1-Y1363, M1-R1362, M1-S1361, M1-S1360, M1-K1359, M1-S1358, M1-R1357, M1-E1356, M1-I1355, M1-T1354, M1-H1353, M1-Y1352, M1-M1351, M1-P1350, M1-P1349, M1-E1348, M1-S1347, M1-D1346, M1-W1345, M1-P1344, M1-N1343, M1-Q1342, M1-C1341, M1-E1340, M1-P1339, M1-L1338, M1-H1337, M1-T1336, M1-Y1335, M1-D1334, M1-S1333, M1-S1332, M1-F1331, M1-S1330, M1-R1329, M1-T1328, M1-D1327, M1-M1326, M1-S1325, M1-T1324, M1-I1323, M1-D1322, M1-E1321, M1-F1320, M1-D1319, M1-I1318, M1-S1317, M1-R1316, M1-S1315, M1-P1314, M1-P1313, M1-R1312, M1-D1311, M1-T1310, M1-P1309, M1-A1308, M1-L1307, M1-T1306, M1-A1305, M1-Y1304, M1-A1303, M1-S1302, M1-S1301, M1-S1300, M1-P1299, M1-A1298, M1-T1297, M1-S1296, M1-P1295, M1-V1294, M1-P1293, M1-T1292, M1-S1291, M1-F1290, M1-S1289, M1-P1288, M1-E1287, M1-G1286, M1-L1285, M1-G1284, M1-L1283, M1-I1282, M1-N1281, M1-V1280, M1-S1279, M1-N1278, M1-D1277, M1-L1276, M1-P1275, M1-D1274, M1-I1273, M1-D1272, M1-C1271, M1-H1270, M1-L1269, M1-E1268, M1-D1267, M1-M1266, M1-A1265, M1-S1264, M1-V1263, M1-Y1262, M1-I1261, M1-D1260, M1-I1259, M1-C1258, M1-S1257, M1-S1256, M1-P1255, M1-R1254, M1-R1253, M1-S1252, M1-D1251, M1-P1250, M1-V1249, M1-I1248, M1-A1247, M1-L1246, M1-T1245, M1-N1244, M1-A1243, M1-P1242, M1-A1241, M1-A1240, M1-P1239, M1-A1238, M1-K1237, M1-P1236, M1-E1235, M1-K1234, M1-A1233, M1-V1232, M1-S1231, M1-H1230, M1-S1229, M1-S1228, M1-T1227, M1-A1226, M1-R1225, M1-H1224, M1-L1223, M1-S1222, M1-L1221, M1-S1220, M1-R1219, M1-E1218, M1-K1217, M1-F1216, M1-I1215, M1-S1214, M1-E1213, M1-L1212, M1-K1211, M1-E1210, M1-I1209, M1-G1208, M1-G1207, M1-K1206, M1-D1205, M1-K1204, M1-M1203, M1-N1202, M1-V1201, M1-S1200, M1-Y1199, M1-F1198, M1-S1197, M1-H1196, M1-S1195, M1-R1194, M1-M1193, M1-R1192, M1-P1191, M1-M1190, M1-L1189, M1-T1188, M1-P1187, M1-S1186, M1-T1185, M1-P1184, M1-S1183, M1-M1182, M1-T1181, M1-E1180, M1-E1179, M1-G1178, M1-A1177, M1-P1176, M1-D1175, M1-I1174, M1-S1173, M1-E1172, M1-Q1171, M1-L1170, M1-K1169, M1-F1168, M1-T1167, M1-N1166, M1-G1165, M1-E1164, M1-Q1163, M1-S1162, M1-N1161, M1-F1160, M1-S1159, M1-S1158, M1-Q1157, M1-R1156, M1-V1155, M1-I1154, M1-Y1153, M1-A1152, M1-A1151, M1-D1150, M1-T1149, M1-C1148, M1-D1147, M1-S1146, M1-S1145, M1-T1144, M1-R1143, M1-S1142, M1-R1141, M1-I1140, M1-K1139, M1-N1138, M1-S1137, M1-E1136, M1-A1135, M1-R1134, M1-E1133, M1-L1132, M1-G1131, M1-T1130, M1-L1129, M1-R1128, M1-E1127, M1-L1126, M1-A1125, M1-T1124, M1-A1123, M1-M1122, M1-R1121, M1-G1120, M1-I1119, M1-L1118, M1-D1117, M1-E1116, M1-L1115, M1-Q1114, M1-A1113, M1-L1112, M1-R1111, M1-I1110, M1-D1109, M1-V1108, M1-T1107, M1-Q1106, M1-L1105, M1-S1104, M1-A1103, M1-K1102, M1-M111, M1-S1100, M1-H1099, M1-E1098, M1-R1097, M1-E1096, M1-N1095, M1-V1094, M1-E1093, M1-E1092, M1-L1091, M1-R1090, M1-M1089, M1-S1088, M1-M1087, M1-N1086, M1-E1085, M1-V1084, M1-R1083, M1-E1082, M1-S1081, M1-T1080, M1-V1079, M1-R1078, M1-I1077, M1-R1076, M1-E1075, M1-D1074, M1-N1073, M1-S1072, M1-S1071, M1-N1070, M1-F1069, M1-R1068, M1-D1067, M1-D1066, M1-K1065, M1-E1064, M1-R1063, M1-F1062, M1-Y1061, M1-E1060, M1-E1059, M1-I1058, M1-C1057, M1-Q1056, M1-E1055, M1-E1054, M1-F1053, M1-D1052, M1-H1051, M1-V1050, M1-K1049, M1-K1048, M1-L1047, M1-E1046, M1-D1045, M1-D1044, M1-T1043, M1-I1042, M1-F1041, M1-L1040, M1-K1039, M1-L1038, M1-G1037, M1-Y1036, M1-D1035, M1-R1034, M1-E1033, M1-D1032, M1-P1031, M1-D1030, M1-S1029, M1-E1028, M1-H1027, M1-K1026, M1-R1025, M1-W1024, M1-R1023, M1-C1022, M1-C1021, M1-L1020, M1-H1019, M1-Q1018, M1-F1017, M1-I1016, M1-M1015, M1-T1014, M1-M1013, M1-H1012, M1-S1011, M1-F1010, M1-I1009, M1-I1008, M1-L1007, M1-P1006, M1-P1005, M1-P1004, M1-L1003, M1-V1002, M1-P1001, M1-R1000, M1-E999, M1-H998, M1-F997, M1-T996, M1-M995, M1-I994, M1-L993, M1-Q992, M1-Y991, M1-R990, M1-Q989, M1-F988, M1-K987, M1-W986, M1-V985, M1-Q984, M1-N983, M1-S982, M1-I981, M1-S980, M1-K979, M1-V978, M1-E977, M1-F976, M1-F975, M1-T974, M1-N973, M1-N972, M1-F971, M1-V970, M1-A969, M1-I968, M1-L967, M1-L966, M1-N965, M1-V964, M1-L963, M1-L962, M1-I961, M1-N960, M1-A959, M1-V958, M1-L957, M1-L956, M1-Y955, M1-C954, M1-A953, M1-M952, M1-I951, M1-A950, M1-P949, M1-V948, M1-I947, M1-W946, M1-A945, M1-G944, M1-T943, M1-K942, M1-C941, M1-P940, M1-P939, M1-L938, M1-Q937, M1-I936, M1-I935, M1-K934, M1-G933, M1-D932, M1-E931, M1-R930, M1-T929, M1-E928, M1-N927, M1-Q926, M1-G925, M1-C924, M1-P923, M1-A922, M1-S921, M1-K920, M1-P919, M1-T918, M1-H917, M1-S916, M1-D915, M1-Y914, M1-V913, M1-Q912, M1-K911, and/or M1-R910 of SEQ ID NO:6. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3c deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
Alternatively, preferred polypeptides of the present invention may comprise polypeptide sequences corresponding to, for example, internal regions of the LTRPC3c polypeptide (e.g., any combination of both N- and C-terminal LTRPC3c polypeptide deletions) of SEQ ID NO:6. For example, internal regions could be defined by the equation: amino acid NX to amino acid CX, wherein NX refers to any N-terminal deletion polypeptide amino acid of LTRPC3c (SEQ ID NO:6), and where CX refers to any C-terminal deletion polypeptide amino acid of LTRPC3c (SEQ ID NO:6). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.
The LTRPC3c polypeptides of the present invention were determined to comprise several phosphorylation sites based upon the Motif algorithm (Genetics Computer Group, Inc.). The phosphorylation of such sites may regulate some biological activity of the LTRPC3c polypeptide. For example, phosphorylation at specific sites may be involved in regulating the proteins ability to associate or bind to other molecules (e.g., proteins, ligands, substrates, DNA, etc.). In the present case, phosphorylation may modulate the ability of the LTRPC3c polypeptide to associate with other potassium channel alpha subunits, beta subunits, or its ability to modulate potassium channel function.
Specifically, the LTRPC3c polypeptide was predicted to comprise two tyrosine phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). Such sites are phosphorylated at the tyrosine amino acid residue. The consensus pattern for tyrosine phosphorylation sites are as follows: [RK]-x(2)-[DE]-x(3)-Y, or [RK]-x(3)-[DE]-x(2)-Y, where Y represents the phosphorylation site and ‘x’ represents an intervening amino acid residue. Additional information specific to tyrosine phosphorylation sites can be found in Patschinsky T., Hunter T., Esch F. S., Cooper J. A., Sefton B. M., Proc. Natl. Acad. Sci. U.S.A. 79:973-977 (1982); Hunter T., J. Biol. Chem. 257:4843-4848 (1982), and Cooper J. A., Esch F. S., Taylor S. S., Hunter T., J. Biol. Chem. 259:7835-7841(1984), which are hereby incorporated herein by reference.
In preferred embodiments, the following tyrosine phosphorylation site polypeptides are encompassed by the present invention: LSLEFKNKDDMPYMSQAQ (SEQ ID NO:158), and/or VMMIGKMMIDMMYFVIIM (SEQ ID NO:159). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these LTRPC3c tyrosine phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The LTRPC3c polypeptide was predicted to comprise twenty four PKC phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). In vivo, protein kinase C exhibits a preference for the phosphorylation of serine or threonine residues. The PKC phosphorylation sites have the following consensus pattern: [ST]-x-[RK], where S or T represents the site of phosphorylation and ‘x’ an intervening amino acid residue. Additional information regarding PKC phosphorylation sites can be found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem. 161:177-184 (1986), and Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem. 260:12492-12499 (1985); which are hereby incorporated by reference herein.
In preferred embodiments, the following PKC phosphorylation site polypeptides are encompassed by the present invention: ADNGTTGKYGAEV (SEQ ID NO:160), VVCDGSGRASDIL (SEQ ID NO:161), GLINESLRDQLLV (SEQ ID NO:162), YRCNYTRKRFRTL (SEQ ID NO:163), RRGRKTTKKREEE (SEQ ID NO:164), RGRKTTKKREEEV (SEQ ID NO:165), ELLDQSYKQDEQL (SEQ ID NO:166), RNNGESSRKKDEE (SEQ ID NO:167), NNGESSRKKDEEE (SEQ ID NO:168), PNEEPSWKLAKNI (SEQ ID NO:169), VYDSHTPKSAPCG (SEQ ID NO:170), RIRVTSERVENMS (SEQ ID NO:171), RVENMSMRLEEVN (SEQ ID NO:172), NEREHSMKASLQT (SEQ ID NO:173), LERAESNKIRSRT (SEQ ID NO:174), SQEGNTFKLQESI (SEQ ID NO:175), AIVPDSRRPSSCI (SEQ ID NO:176), ATLAPTDRPPSRS (SEQ ID NO:177), IERSKSSRYLATT (SEQ ID NO:178), QEGDNSERTLSNN (SEQ ID NO:179), APYAHTRKSFSIS (SEQ ID NO:180), KSFSISDKLDRQR (SEQ ID NO:181), FQRSKSSKPEGRG (SEQ ID NO:182), and/or RGDSLSMRRLSRT (SEQ ID NO:183.). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these LTRPC3c PKC phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The present invention also encompasses immunogenic and/or antigenic epitopes of the LTRPC3c polypeptide.
The LTRPC3c polypeptide has been shown to comprise twelve glycosylation sites according to the Motif algorithm (Genetics Computer Group, Inc.). As discussed more specifically herein, protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.
Asparagine phosphorylation sites have the following consensus pattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation site. However, it is well known that that potential N-glycosylation sites are specific to the consensus sequence Asn-Xaa-Ser/Thr. However, the presence of the consensus tripeptide is not sufficient to conclude that an asparagine residue is glycosylated, due to the fact that the folding of the protein plays an important role in the regulation of N-glycosylation. It has been shown that the presence of proline between Asn and Ser/Thr will inhibit N-glycosylation; this has been confirmed by a recent statistical analysis of glycosylation sites, which also shows that about 50% of the sites that have a proline C-terminal to Ser/Thr are not glycosylated. Additional information relating to asparagine glycosylation may be found in reference to the following publications, which are hereby incorporated by reference herein: Marshall R. D., Annu. Rev. Biochem. 41:673-702 (1972); Pless D. D., Lennarz W. J., Proc. Natl. Acad. Sci. U.S.A. 74:134-138 (1977); Bause E., Biochem. J. 209:331-336 (1983); Gavel Y., von Heijne G., Protein Eng. 3:433-442 (1990); and Miletich J. P., Broze G. J. Jr., J. Biol. Chem. 265:11397-11404 (1990).
In preferred embodiments, the following asparagine glycosylation site polypeptides are encompassed by the present invention: FILADNGTTGKYGA (SEQ ID NO:184), EGGLINESLRDQLL (SEQ ID NO:185), LLKGANASAPDQLS (SEQ ID NO:186), GAYRCNYTRKRFRT (SEQ ID NO:187), TYELKNWSNATCLQ (SEQ ID NO:188), LKNWSNATCLQLAV (SEQ ID NO:189), LQEYWNVTDLIAIL (SEQ ID NO:190), APCGQNETREDGKI (SEQ ID NO:191), LIAVFNNTFFEVKS (SEQ ID NO:192), KDDRFNSSNDERIR (SEQ ID NO:193), SERVENMSMRLEEV (SEQ ID NO:194), and/or RTLSNNITVPKIER (SEQ ID NO:195). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these LTRPC3c asparagine glycosylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The LTRPC3c polypeptide has been shown to comprise one RGD cell attachment site domain according to the Motif algorithm (Genetics Computer Group, Inc.). The sequence Arg-Gly-Asp, found in fibronectin, is crucial for its interaction with its cell surface receptor, an integrin. What has been called the ‘RGD’ tripeptide is also found in the sequences of a number of other proteins, where it has been shown to play a role in cell adhesion. Non-limiting examples of these proteins are the following: some forms of collagens, fibrinogen, vitronectin, von Willebrand factor (VWF), snake disintegrins, and slime mold discoidins. The ‘RGD’ tripeptide is also found in other proteins where it may serve the same purpose. A consensus pattern for RGD cell attachment sites is the following: R-G-D. Additional information relating to RGD cell attachment site domains may be found in reference to the following publications, which are hereby incorporated by reference herein: Ruoslahti E., Pierschbacher M. D., Cell 44:517-518 (1986); and d'Souza S. E., Ginsberg M. H., Plow E. F., Trends Biochem. Sci. 16:246-250 (1991).
In preferred embodiments, the following RGD cell attachment site domain polypeptide is encompassed by the present invention: SKPEGRGDSLSMR (SEQ ID NO:196). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this RGD cell attachment site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The LTRPC3c polypeptide has been shown to comprise one aminoacyl-transfer RNA synthetases class-II domain according to the Motif algorithm (Genetics Computer Group, Inc.). Aminoacyl-tRNA synthetases (EC 6.1.1.-) are a group of enzymes which activate amino acids and transfer them to specific tRNA molecules as the first step in protein biosynthesis. In prokaryotic organisms there are at least twenty different types of aminoacyl-tRNA synthetases, one for each different amino acid. In eukaryotes there are generally two aminoacyl-tRNA synthetases for each different amino acid: one cytosolic form and a mitochondrial form. While all these enzymes have a common function, they are widely diverse in terms of subunit size and of quaternary structure.
The synthetases specific for alanine, asparagine, aspartic acid, glycine, histidine, lysine, phenylalanine, proline, serine, and threonine are referred to as class-II synthetases and probably have a common folding pattern in their catalytic domain for the binding of ATP and amino acid which is different to the Rossmann fold observed for the class I synthetases.
Class-II tRNA synthetases do not share a high degree of similarity, however at least three conserved regions are present.
The consensus pattern for aminoacyl-transfer RNA synthetases class-II domains are as follows: [FYH]-R-x-[DE]-x(4,12)-[RH]-x(3)-F-x(3)-[DE]; and [GSTALVF]-{DENQHRKP}-[GSTA]-[LIVMF]-[DE]-R-[LIVMF]-x-[LIVMSTAG]-[LIVMFY], where ‘x’ represents an intervening amino acid residue.
Additional information specific to aminoacyl-transfer RNA synthetases class-II domains may be found in reference to the following publications, Schimmel P., Annu. Rev. Biochem. 56:125-158 (1987); Delarue M., Moras D., BioEssays 15:675-687 (1993); Schimmel P., Trends Biochem. Sci. 16:1-3 (1991); Nagel G. M., Doolittle R. F., Proc. Natl. Acad. Sci. U.S.A. 88:8121-8125 (1991); Cusack S., Haertlein M., Leberman R., Nucleic Acids Res. 19:3489-3498 (1991); Cusack S., Biochimie 75:1077-1081 (1993); Cusack S., Berthet-Colominas C., Haertlein M., Nassar N., Leberman R., Nature 347:249-255 (1990); and Leveque F., Plateau P., Dessen P., Blanquet S., Nucleic Acids Res. 18:305-312 (1990); which are hereby incorporated herein by reference in their entirety.
In preferred embodiments, the following aminoacyl-transfer RNA synthetases class-II domain polypeptide is encompassed by the present invention: LIGRMATALERLTGLERAES (SEQ ID NO:197). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this RGD cell attachment site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The LTRPC3c polypeptide has been shown to comprise three amidation sites according to the Motif algorithm (Genetics Computer Group, Inc.). The precursor of hormones and other active peptides which are C-terminally amidated is always directly followed by a glycine residue which provides the amide group, and most often by at least two consecutive basic residues (Arg or Lys) which generally function as an active peptide precursor cleavage site. Although all amino acids can be amidated, neutral hydrophobic residues such as Val or Phe are good substrates, while charged residues such as Asp or Arg are much less reactive. A consensus pattern for amidation sites is the following: x-G-[RK]-[RK], wherein “X” represents the amidation site. Additional information relating to asparagine glycosylation may be found in reference to the following publications, which are hereby incorporated by reference herein: Kreil G., Meth. Enzymol. 106:218-223(1984); and Bradbury A. F., Smyth D. G., Biosci. Rep. 7:907-916 (1987).
In preferred embodiments, the following amidation site polypeptides are encompassed by the present invention: DIPLRRGRKTTKKR (SEQ ID NO:198), HRLIPLGRKIYEFY (SEQ ID NO:199), and/or EENEAKGRRATIAI (SEQ ID NO:200). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these LTRPC3c amidation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:5 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 4687 of SEQ ID NO:5, b is an integer between 15 to 4701, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:5, and where b is greater than or equal to a+14.
Features of the Polypeptide Encoded by Gene No:4
The polypeptide of this gene provided as SEQ ID NO:10 (FIGS. 4A-F), encoded by the polynucleotide sequence according to SEQ ID NO:8 (FIGS. 4A-F), and/or encoded by the polynucleotide contained within the deposited clone, LTRPC3e, has significant homology at the nucleotide and amino acid level to the human melastatin 1 protein (Melastatin1; Genbank Accession No. gi|3243075; SEQ ID NO:11). An alignment of the LTRPC3e polypeptide with this protein is provided in FIGS. 5A-D.
The LTRPC3e polypeptide was determined to share 65.5% identity and 73.3% similarity with the human melastatin 1 protein (Melastatin1; Genbank Accession No. gi|3243075; SEQ ID NO:11) as shown in FIG. 8.
The LTRPC3e protein is believed to represent a member of a new class of protein kinases referred to as alpha kinases (Curr. Biol. 9 (2), R43-R45 (1999)). These kinases represent a novel type of signaling molecule comprising both a catalytic protein kinase domain, in addition to, an ion channel domain. This family is commonly referred to as the transient receptor potential channel (TRP) family. to Melastatin1 defines a separate subfamily of TRP channels referred to as TRPM (melastatin1). TRPM family members are characteristic of their unusually long cytoplasmic tails at both ends of the channel domain and some of the family members contain an enzyme domain at the C-terminal region. Thus, LTRPC3 represents a novel member of the TRPM subfamily.
The melastatin1 protein is believed to be negatively associated with the incidence of melanoma based upon its inverse correlative expression in highly aggressive melanomas (Genomics 54 (1), 116-123 (1998)). Thus, overexpression of melastatin1 could represent a novel therapeutic in the treatment of melanoma and potentially other cancers.
Based upon the observed homology, the polypeptide of the present invention is expected to share at least some biological activity with other transient receptor potential channel family members, more specifically with the melastatin1 protein, in addition to, other transient receptor potential channel family members referenced elsewhere herein or otherwise known in the art.
Most of the known transient receptor potential channel family members, possess one or more transmembrane domains. Likewise, the LTRPC3e polypeptide has been determined to comprise six transmembrane domains (TM1-TM6) as shown in FIGS. 4A-F. The transmembrane domains are located from about amino acid 622 to about amino acid 639 (TM1), from about amino acid 719 to about amino acid 736 (TM2), from about amino acid 792 to about amino acid 805 (TM3), from about amino acid 819 to about amino acid 836 (TM4), from about amino acid 853 to about amino acid 870 (TM5), and/or from about amino acid 940 to about amino acid 960 (TM6) of SEQ ID NO:9. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of the above referenced polypeptide.
In preferred embodiments, the following transmembrane domain polypeptides are encompassed by the present invention: LKVILGILLPPSILSLEF (SEQ ID NO:44), IVKFWFYTLAYIGYLMLF (SEQ ID NO:45), VTDLIAILLFSVGM (SEQ ID NO:46), RVIYCVNIIYWYIRLLDI (SEQ ID NO:47), MMIDMMYFVIIMLVVLMS (SEQ ID NO:48), and/or AIMACYLLVANILLVNLLIAV (SEQ ID NO:49). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the LTRPC3e transmembrane polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The present invention also encompasses polypeptides corresponding to the regions between each transmembrane domain of LTRPC3e, referred to herein as inter-transmembrane domain polypeptides (inter TM1-2, inter TM2-3, inter TM3-4, inter TM4-5, and inter TM5-6). Such regions are typically solvent accessible (either extracellularly or intracellularly). Therefore, these regions are amenable to the development of agonists and/or antagonists to the LTRPC3e polypeptide, particularly for the development of antibodies specific to these regions.
In preferred embodiments, the following inter-transmembrane domain polypeptides are encompassed by the present invention: KNKDDMPYMSQAQEIHLQEKEAEEPEKPTKEKEEEDMELTAMLGRNNGESS RKKDEEEVQSKHRLIPLGRKIYEFYNAP (SEQ ID NO:201), NYIVLVKMERWPSTQEWIVISYIFTLGIEKMREILMSEPGKLLQKVKVWLQEY WN (SEQ ID NO:202), ILRLQDQPFRSDG (SEQ ID NO:203), FGVNKYLGPYVMMIGK (SEQ ID NO:204), and/or FGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFADQIDPPCGQNETRED GKIIQLPPCKTGAWIVP (SEQ ID NO:205). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of the LTRPC3e inter-transmembrane polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3e inter TM1-2 domain deletion polypeptides are encompassed by the present invention: K1-P79, N2-P79, K3-P79, D4-P79, D5-P79, M6-P79, P7-P79, Y8-P79, M9-P79, S10-P79, Q11-P79, A12-P79, Q13-P79, E14-P79, I15-P79, H16-P79, L17-P79, Q18-P79, E19-P79, K20-P79, E21-P79, A22-P79, E23-P79, E24-P79, P25-P79, E26-P79, K27-P79, P28-P79, T29-P79, K30-P79, E31-P79, K32-P79, E33-P79, E34-P79, E35-P79, D36-P79, M37-P79, E38-P79, L39-P79, T40-P79, A41-P79, M42-P79, L43-P79, G44-P79, R45-P79, N46-P79, N47-P79, G48-P79, E49-P79, S50-P79, S51-P79, R52-P79, K53-P79, K54-P79, D55-P79, E56-P79, E57-P79, E58-P79, V59-P79, Q60-P79, S61-P79, K62-P79, H63-P79, R64-P79, L65-P79, I66-P79, P67-P79, L68-P79, G69-P79, R70-P79, K71-P79, I72-P79, and/or Y73-P79 of SEQ ID NO:201. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3e inter TM1-2 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3e inter TM1-2 domain deletion polypeptides are encompassed by the present invention: K1-P79, K1-A78, K1-N77, K1-Y76, K1-F75, K1-E74, K1-Y73, K1-I72, K1-K71, K1-R70, K1-G69, K1-L68, K1-P67, K1-I66, K1-L65, K1-R64, K1-H63, K1-K62, K1-S61, K1-Q60, K1-V59, K1-E58, K1-E57, K1-E56, K1-D55, K1-K54, K1-K53, K1-R52, K1-S51, K1-S50, K1-E49, K1-G48, K1-N47, K1-N46, K1-R45, K1-G44, K1-L43, K1-M42, K1-A41, K1-T40, K1-L39, K1-E38, K1-M37, K1-D36, K1-E35, K1-E34, K1-E33, K1-K32, K1-E31, K1-K30, K1-T29, K1-P28, K1-K27, K1-E26, K1-P25, K1-E24, K1-E23, K1-A22, K1-E21, K1-K20, K1-E19, K1-Q18, K1-L17, K1-H16, K1-I15, K1-E14, K1-Q13, K1-A12, K1-Q11, K1-S10, K1-M9, K1-Y8, and/or K1-P7 of SEQ ID NO:201. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3e inter TM1-2 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3e inter TM2-3 domain deletion polypeptides are encompassed by the present invention: F1-N56, N2-N56, Y3-N56, I4-N56, V5-N56, L6-N56, V7-N56, K8-N56, M9-N56, E10-N56, R11-N56, W12-N56, P13-N56, S14-N56, T15-N56, Q16-N56, E17-N56, W18-N56, I19-N56, V20-N56, I21-N56, S22-N56, Y23-N56, I24-N56, F25-N56, T26-N56, L27-N56, G28-N56, I29-N56, E30-N56, K31-N56, M32-N56, R33-N56, E34-N56, I35-N56, L36-N56, M37-N56, S38-N56, E39-N56, P40-N56, G41-N56, K42-N56, L43-N56, L44-N56, Q45-N56, K46-N56, V47-N56, K48-N56, V49-N56, and/or W50-N56 of SEQ ID NO:202. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3e inter TM2-3 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3e inter TM2-3 domain deletion polypeptides are encompassed by the present invention: F1-N56, F1-W55, F1-Y54, F1-E53, F1-Q52, F1-L51, F1-W50, F1-V49, F1-K48, F1-V47, F1-K46, F1-Q45, F1-L44, F1-L43, F1-K42, F1-G41, F1-P40, F1-E39, F1-S38, F1-M37, F1-L36, F1-I35, F1-E34, F1-R33, F1-M32, F1-K31, F1-E30, F1-I29, F1-G28, F1-L27, F1-T26, F1-F25, F1-I24, F1-Y23, F1-S22, F1-I21, F1-V20, F1-I19, F1-W18, F1-E17, F1-Q16, F1-T15, F1-S14, F1-P13, F1-W12, F1-R11, F1-E10, F1-M9, F1-K8, and/or F1-V7 of SEQ ID NO:202. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3e inter TM2-3 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3e inter TM3-4 domain deletion polypeptides are encompassed by the present invention: I1-G13, L2-G13, R3-G13, L4-G13, Q5-G13, D6-G13, and/or Q7-G13 of SEQ ID NO:203. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3e inter TM3-4 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3e inter TM3-4 domain deletion polypeptides are encompassed by the present invention: I1-G13, I1-D12, I1-S11, I1-R10, I1-F9, I1-P8, and/or I1-Q7 of SEQ ID NO:203. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3e inter TM3-4 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3e inter TM4-5 domain deletion polypeptides are encompassed by the present invention: F1-K16, G2-K16, V3-K16, N4-K16, K5-K16, Y6-K16, L7-K16, G8-K16, P9-K16, and/or Y10-K16 of SEQ ID NO:204. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3e inter TM4-5 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3e inter TM4-5 domain deletion polypeptides are encompassed by the present invention: F1-K16, F1-G15, F1-I14, F1-M13, F1-M12, F1-V11, F1-Y10, F1-P9, F1-G8, and/or F1-L7 of SEQ ID NO:204. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3e inter TM4-5 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following N-terminal LTRPC3e inter TM5-6 domain deletion polypeptides are encompassed by the present invention: F1-P81, G2-P81, V3-P81, A4-P81, R5-P81, Q6-P81, A7-P81, I8-P81, L9-P81, F10-P81, P11-P81, N12-P81, E13-P81, E14-P81, P15-P81, S16-P81, W17-P81, K18-P81, L19-P81, A20-P81, K21-P81, N22-P81, I23-P81, F24-P81, Y25-P81, M26-P81, P27-P81, Y28-P81, W29-P81, M30-P81, I31-P81, Y32-P81, G33-P81, E34-P81, V35-P81, F36-P81, A37-P81, D38-P81, Q39-P81, I40-P81, D41-P81, R42-P81, K43-P81, Q44-P81, V45-P81, Y46-P81, D47-P81, S48-P81, H49-P81, T50-P81, P51-P81, K52-P81, S53-P81, A54-P81, P55-P81, C56-P81, G57-P81, Q58-P81, N59-P81, E60-P81, T61-P81, R62-P81, E63-P81, D64-P81, G65-P81, K66-P81, I67-P81, I68-P81, Q69-P81, L70-P81, P71-P81, P72-P81, C73-P81, K74-P81, and/or T75-P81 of SEQ ID NO:205. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3e inter TM5-6 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3e inter TM5-6 domain deletion polypeptides are encompassed by the present invention: F1-P81, F1-V80, F1-I79, F1-W78, F1-A77, F1-G76, F1-T75, F1-K74, F1-C73, F1-P72, F1-P71, F1-L70, F1-Q69, F1-I68, F1-I67, F1-K66, F1-G65, F1-D64, F1-E63, F1-R62, F1-T61, F1-E60, F1-N59, F1-Q58, F1-G57, F1-C56, F1-P55, F1-A54, F1-S53, F1-K52, F1-P51, F1-T50, F1-H49, F1-S48, F1-D47, F1-Y46, F1-V45, F1-Q44, F1-K43, F1-R42, F1-D41, F1-I40, F1-Q39, F1-D38, F1-A37, F1-F36, F1-V35, F1-E34, F1-G33, F1-Y32, F1-I31, F1-M30, F1-W29, F1-Y28, F1-P27, F1-M26, F1-Y25, F1-F24, F1-I23, F1-N22, F1-K21, F1-A20, F1-L19, F1-K18, F1-W17, F1-S16, F1-P15, F1-E14, F1-E13, F1-N12, F1-P11, F1-F10, F1-L9, F1-I8, and/or F1-A7 of SEQ ID NO:205. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3e inter TM5-6 domain deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The LTRPC3e polypeptide was determined to comprise several conserved cysteines, at amino acid 94, 209, 265, 431, 519, 584, 601, 823, 914, 931, 944, 1047, 1138, and 1404 of SEQ ID NO:9 (FIGS. 4A-F). Conservation of cysteines at key amino acid residues is indicative of conserved structural features, which may correlate with conservation of protein function and/or activity.
In confirmation of the LTRPC3e representing a member of the transient receptor channel family, the LTRPC3e polypeptide was determined to comprise a predicted TRP domain (EWKFAR) located from about amino acid 975 to about amino acid 980 of SEQ ID NO:9. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of the above referenced polypeptide.
In preferred embodiments, the following TRP domain polypeptide is encompassed by the present invention: VWKFQR (SEQ ID NO:206). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of this LTRPC3e TRP domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
In further confirmation of the LTRPC3e representing a member of the transient receptor channel family, the LTRPC3e polypeptide was determined to comprise a predicted ion transport signature domain located at about amino acid 750 to about amino acid 961 of SEQ ID NO:9. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of the above referenced polypeptide.
In preferred embodiments, the following ion transport signature domain polypeptide is encompassed by the present invention: TQEWIVISYIFTLGIEKMREILMSEPGKLLQKVKVWLQEYWNVTDLIAILLFSV GMILRLQDQPFRSDGRVIYCVNIIYWYIRLLDIFGVNKYLGPYVMMIGKMMID MMYFVIIMLVVLMSFGVARQAILFPNEEPSWKLAKNIFYMPYWMIYGEVFAD QIDPPCGQNETREDGKIIQLPPCKTGAWIVPAIMACYLLVANILLVNLLIAVF (SEQ ID NO:207). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of this LTRPC3e ion transport signature domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The LTRPC3e polypeptide was determined to comprise a predicted coiled-coil domain located at about amino acid 1065 to about amino acid 1119 of SEQ ID NO:9. In this context, the term “about” may be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids beyond the N-Terminus and/or C-terminus of the above referenced polypeptide.
In preferred embodiments, the following coiled-coil domain polypeptide is encompassed by the present invention: ERIRVTSERVENMSMRLEEVNEREHSMKASLQTVDIRLAQLEDLIGRMATAL ERL (SEQ ID NO:208). Polynucleotides encoding this polypeptide are also provided. The present invention also encompasses the use of this LTRPC3e coiled-coil domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The present invention is also directed to polynucleotides comprising at least about 45 nt of the polynucleotide sequence provided as SEQ ID NO:8. Specifically, the present invention is directed to a polynucleotide sequence comprising nucleotides from about nucleotide 1174 to about nucleotide 1212; nucleotides from about nucleotide 1174 to about nucleotide 1215; nucleotides from about nucleotide 1168 to about nucleotide 1209; nucleotides from about nucleotide 1165 to about nucleotide 1209; of SEQ ID NO:8; and/or nucleotides from about nucleotide 1162 to about nucleotide 1209. In this context “about” includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus, or at both termini.
The present invention is also directed to polypeptides comprising at least about 15 amino acids of the polypeptides sequence provided as SEQ ID NO:9. Specifically, the present invention is directed to a polypeptides sequence comprising amino acids from about amino acid 392 to about amino acid 404; amino acids from about amino acid 392 to about amino acid 405; amino acids from about amino acid 392 to about amino acid 406; amino acids from about amino acid 390 to about amino acid 403; amino acids from about amino acid 389 to about amino acid 403; and/or amino acids from about amino acid 388 to about amino acid 403 of SEQ ID NO:9. In this context “about” includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus, or at both termini.
LTRPC3e polypeptides and polynucleotides are useful for diagnosing diseases related to the over and/or under expression of LTRPC3e by identifying mutations in the LTRPC3e gene using LTRPC3e sequences as probes or by determining LTRPC3e protein or mRNA expression levels. LTRPC3e polypeptides will be useful in screens for compounds that affect the activity of the protein. LTRPC3e peptides can also be used for the generation of specific antibodies and as bait in yeast two hybrid screens to find proteins the specifically interact with LTRPC3e.
Consistent with LTRPC3 representing a transient receptor potential channel, immunohistochemical experiments have shown that LTRPC3 localizes to the cell membrane (see FIG. 17 and Example 6). Specifically, the complete open reading frame of LTRPC3 with a C-terminal HA tag was transiently transfected into HEK 293 cells to assess its cellular localization. The HA-tagged LTRPC3 was detected using a fluorescein-conjugated anti-HA antibody and a laser scanning confocal microscope which produces a green fluorescent signal. The green fluorescent signal was exclusively detected at the subplasma membrane region of the transfected cells (see FIG. 17), which is consistent with LTRPC3 being an integral membrane protein. Under these conditions approximately  ̃70% of cells are expressing LTRPC3. The expression of full-length protein was accessed with immunoblot using an anti-HA antibody and detected as the expected size of  ̃170 kD (data not shown). Cellular localization of the LTRPC3d splice variant is expected to be similarly localized to the subplasma membrane region.
Moreover, physiometric studies have shown that LTRPC3 is a functional Ca2+ permeable channel (see FIGS. 18 and 19, and Example 7). LTRPC3 function was assessed using a Fluorometric Imaging Plate Reader (FLIPRTM) that measures real-time intracellular fluorescence changes. Cells transiently transfected with vector or LTRPC3-HA were loaded with the cytoplasmic Ca2+ indicator Fluoro-4 in a 1 mM Ca2+ solution. Addition of Ca2+ to the media resulted in a concentration-dependent influx of Ca2+ into LTRPC3-expressing cells (FIG. 18; right panels), indicating that LTRPC3 is a functional Ca2+ channel. In contrast, vector-transfected cells showed minimal Ca2+ influx under the same experimental conditions (FIG. 18, left panels). The non-transfected cells were indistinguishable from the vector-transfected cells (data not shown). Therefore, LTRPC3 is a constitutively active channel capable of mediating Ca2+ influx. The Ca2+ influx activity of the LTRPC3d splice variants is expected to be similar to LTRPC3.
To further address the mechanism of LTRPC3-mediated Ca2+ entry, similar Ca2+ addition experiments were performed on transfected cells incubated ( ̃30 min) in a nominally Ca2+-free solution. Previous studies have shown that lowering extracellular Ca2+ concentration below physiological levels can deplete intracellular Ca2+ stores in many cell types including HEK 293 (EMBO J. 17, 4274-4282, (1998)). Incubating vector-transfected HEK 293 cells in a nominally Ca2+-free solution gave rise to Ca2+ entry that was dependent on the concentration of Ca2+ added to the buffers, indicating Ca2+ influx was mediated through endogenous SOCs in HEK293 cells (FIG. 18, left panels). In LTRPC3 cells, the Ca2+ transients triggered by similar Ca2+ treatment were much larger (FIG. 18, right panels). This Ca2+ entry observed in LTRPC3 cells incubated in Ca2+-free media were greater than those observed in 1 mM Ca2+ media, indicating that LTRPC3-mediated Ca2+ entry can be potentiated by the store-depletion. The store-depletion potentiation of LTRPC3-mediated Ca2+ entry is expected to be similar for the LTRPC3d splice variant.
The store-operated mechanism of LTRPC3-mediated Ca2+ influx was tested further by passively depleting Ca2+ stores with thapsigargin (TG), an inhibitor of microsomal Ca2+ ATPases that pumps ions from the cytosol back into the stores. Addition of 2 μM thapsigargin equivalently depleted Ca2+ stores in LTRPC3-HA- and vector-transfected cells (FIG. 19A). Following store depletion with TG, addition of Ca2+ to the buffer induced a much larger Ca2+ entry in LTRPC3 cells compared to the vector control cells. The increased Ca2+ entry of LTRPC3 cells, relative to non-LTRPC3 transfected cells, post store depletion with TG is expected to be similar for the LTRPC3d splice variant.
Receptor-mediated Ca2+ entry was also more pronounced in LTRPC3-HA-transfected cells. Carbachol (CCh) can activate an endogenous muscarinic receptor and trigger IP3 production, leading to store-depletion in HEK 293 cells. The addition of 50 μM of CCh caused a transient and rapid intracellular Ca2+ increase in both LTRPC3- and vector-transfected cells (FIG. 19B). After the store depletion with CCh, adding of Ca2+ to the buffer induced a much larger influx of Ca2+ into LTRPC3 cells, as compared to vector control cells. These results show that after store depletion with TG or CCh LTRPC3-transfected cells exhibit an increased Ca2+ influx when compared to control cells. The increased Ca2+ entry of LTRPC3 cells, relative to non-LTRPC3 transfected cells, post store depletion with TG or CCh is expected to be similar for the LTRPC3d splice variant.
The lanthanides, gadolinium (Gd3+) and lanthanum (La3+), are noneselective Ca2+-permeable channel blockers, often used as part of the characterization of overexpressed TRP channels. Both lanthanides blocked LTRPC3 Ca2+ conductance, although La3+ was more potent (FIG. 19C). In the presence of 1 mM Ca2+ in which endogenous SOCs is minimally activated (FIG. 18A), pre-treatment with 100 μM of La3+ and Gd3+ blocked LTRPC3 Ca2+ currents, stimulated by adding 10 mM Ca2+, by 67 and 39%, respectively. These results indicated that LTRPC3 mediated currents are not non-specific leak currents resulting from protein overexpression.
LTRPC3 is constitutively active but can be potentiated by store-depletion and is partially sensitive to La3+ and Gd3+ blockade. LTRPC3 is believed to represent the first member of the TRPM subfamily that exhibits this store-operated mechanism, although some members of TRPC subfamily have been considered for this role. TRPM1 and TRPM4a are constitutive Ca2+ permeable channels but it is unclear whether they can be stimulated by store-depletion (Proc. Natl. Acad. Sci. U.S.A. 98, 10692-10697, (2001)). Distinct from TRPM4a, TRPM4b is directly activated by changes in intracellular Ca2+ without significant permeation of Ca2+ (Cell 109, 397-401, (2002)). TRPM2 is activated by ADP-ribose, NAD and changes in redox status (Nature 411, 595-599, (2001); Science 293, 1327-1330, (2001); and Mol. Cell 9, 163-173, (2002)). TRPM7 is regulated by Mg2+-ATP and/or PIP2 (Science 291, 1043-1047, (2001); Nature 411, 690-695, (2001); and Nat. Cell Biol. 4, 329-36 (2002)). TRPM8 is activated by cold temperatures and cooling agents (Nature 416, 52-58, (2002); and Cell 108, 705-715, (2002)). Therefore, in conjunction with its fairly restricted tissue expression, which is not observed with any other family members, LTRPC3 may have a unique biological function in human.
Expression profiling designed to measure the steady state mRNA levels encoding the LTRPC3 polypeptide showed predominately high expression levels in kidney. The LTRPC3 polypeptide was also significantly expression in spinal cord, testis, and brain (as shown in FIG. 6).
Moreover, Northern hybridizations of the LTRPC3 mRNA confirmed the predominately high expression levels in kidney, and significant expression levels in testis, and brain (as shown in FIG. 7). The Northern hybridization was not performed on spinal cord tissue.
Expanded analysis of LTRPC3 expression levels by TaqManTM quantitative PCR (see FIG. 12) confirmed that the LTRPC3 polypeptide is expressed in kidney, brain, testis (FIGS. 6 and 7), although higher expression levels were observed in brain than previously appreciated. LTRPC3 mRNA was expressed predominately in the brain, specifically the cerebellum, choroid plexus, the locus coeruleus, the posterior hypothalamus and the substantia nigra. Expression of LTRPC3 was also significantly expressed in the kidney, with higher levels observed in the cortex than in the medulla or pelvis. LTRPC3 was also significantly expressed in the spinal cord, testis, and to a lesser extent in other tissues as shown.
Therefore, LTRPC3e polynucleotides and polypeptides, including modulators or fragments thereof, may be useful in treating, diagnosing, prognosing, and/or preventing a variety of neurological conditions, in particular various choroid plexus neoplasms, choroid plexus papilloma, Alzheimer's disease, prion disorders and multiple sclerosis and movement disorders that involve the cerebellum. Based upon the expression pattern of LTRPC3 in kidney, this novel TRP family member, or a splice variant or polymorphism thereof, may also be the cause solitary metastasis in the choroid plexus, a rare type of carcinoma. For example, it has been shown that out of 15 cases of solitary metastasis of the choroid plexus, five originated from renal cell carcinoma (Neurol. Med. Chir. (Tokyo) 1997 December; 37(12):916-9). Additionally, given the rather selective expression of LTRPC3 in the choroid plexus and renal tissues, it may be possible that altered function of LTRPC3 or a splice variant or polymorphism thereof, may be responsible for solitary metastasis and renal carcinoma. LTRPC3 polynucleotides and polypeptides, including modulators or fragments thereof, may be useful in treating, diagnosing, prognosing, and/or preventing a variety of renal disorders, particularly solitary metastasis in the choroid plexus, and renal carcinoma.
Moreover, an additional analysis of LTRPC3 expression levels by TaqManTM quantitative PCR (see FIG. 13) in disease cells and tissues indicated that the LTRPC3 polypeptide is differentially expressed in renal, testicular, and ovarian tumor tissues. In the renal tumor tissue results, an average of 2 out of 3 matched samples, which represent 3 out of 4 samples total, showed a significant decrease in LTRPC3 steady state RNA levels in tumor compared to control samples. In the testicular tumor tissue results, differential expression of LTRPC3 in testicular cancers was observed with all 5 tumor samples showing a significant reduction in steady-state RNA levels compared to two control samples. In the ovarian tumor tissue results, differential expression of LTRPC3 in ovarian cancers was observed with 3 tumor samples showing a significant reduction in steady-state RNA levels compared to five control samples.
The differential expression of LTRPC3 in tumors relative to normal tissues suggests that loss of LTRPC3 expression during tumor progression might contribute to the metastatic process by altering internal calcium stores in a manner that reflects a loss of cellular control on apoptosis. Restoring LTRPC3e function might provide a novel therapeutic approach to treating certain cancers. Therefore, LTRPC3e polynucleotides and polypeptides, including modulators or fragments thereof, particularly agonists of LTRPC3e activity or expression, may be useful in treating, diagnosing, prognosing, ameloriating, and/or preventing a variety of cancers and proliferative conditions, particularly of the kidney, testis, and ovaries.
Characterization of the LTRPC3 polypeptide of the present invention using antisense oligonucleotides led to the determination that LTRPC3 is involved in the negative modulation of the FEN1 DNA base-excision repair/proliferation modulating protein as described in Example 5 herein.
Specifically, antisense inhibition of LTRPC3 resulted in upregulation of Fen1, a marker of proliferation. As such, LTRPC3 behaves in a manner similar to a tumor suppressor, in that loss results in increases in proliferation. While some increases in the proliferation markers p21 and IkB were also observed, they were less significant, and could be a response to the pro-proliferative effect of LTRPC3 antisense inhibition. Interestingly, expression of LTRPC3 was also observed to be significantly reduced in tumor tissues, relative to normal tissues (see FIG. 13). Specifically, renal, testicular, and ovarian tumor samples showed as much as 12 to 30 fold down regulation of LTRPC3 message levels compared to normal tissues. This reduction of LTRPC3 correlating with the tumor state is consistent with the negative FEN1 modulatory activity and suggests this gene may have antiproliferative activities. Therefore, for modulators of LTRPC3 to be useful for treating cancer, it would be necessary to increase the activity of the gene or gene product in order to have the reverse effect on cancerous cells. Thus, an agonist of LTRPC3 activity or a compound that increased LTRPC3 message levels would be a desired invention for cancer therapy. The same regimen may also be applicable to LTRPC3 splice variants and/or polymorphisms, such as LTRPC3e.
In preferred embodiments, LTRPC3e polynucleotides and polypeptides, including fragments and modulators thereof, are useful for treating, diagnosing, and/or ameliorating DNA-repair deficiencies, particularly base-excision repair deficiencies, Xeroderma pigmentosum, skin cancer, melanoma, UV senstivity, alkylation sensivity, gamma irradiation sensitivity, pyrimidine dimer sensitivity, chemical mutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone, Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, blood vessel dilation aberrations in the skin, blood vessel dilation aberrations in the eye, conditions involving increased levels of apurinic sites, conditions involving increased levels of apyrimidinic sites, conditions involving increased levels of abasic sites, disorders related to aberrant signal transduction, proliferating disorders, and/or cancers.
Moreover, LTRPC3e polynucleotides and polypeptides, including fragments and modulators thereof, are useful for increasing mammalian base excision repair activity, increasing mammalian single-nucleotide base excision repair activity, and/or increasing mammalian long patch base excision repair activity.
In preferred embodiments, antagonists directed against LTRPC3e are useful for treating, diagnosing, and/or ameliorating DNA-repair deficiencies, particularly base-excision repair deficiencies, Xeroderma pigmentosum, skin cancer, melanoma, UV senstivity, alkylation sensivity, gamma irradiation sensitivity, pyrimidine dimer sensitivity, chemical mutagenes, lymphomas, leukemias, photosensitivity, Bloom's syndrone, Fanconi's anemia, ataxia telangiectasia, chromosomal aberrations, blood vessel dilation aberrations in the skin, blood vessel dilation aberrations in the eye, conditions involving increased levels of apurinic sites, conditions involving increased levels of apyrimidinic sites, conditions involving increased levels of abasic sites, disorders related to aberrant signal transduction, proliferating disorders, and/or cancers.
Moreover, antagonists directed against LTRPC3e are useful for increasing mammalian base excision repair activity, increasing mammalian single-nucleotide base excision repair activity, and/or increasing mammalian long patch base excision repair activity.
In preferred embodiments, agonists directed against LTRPC3e are useful for treating, diagnosing, and/or ameliorating, disorders related to aberrant signal transduction, proliferating disorders, and/or cancers, particularly renal cell carcinomas, testicular cancers, and/or ovarian cancers.
The strong homology to human transient receptor potential channels (TRP), combined with the predominate localized expression in kidney tissue of LTRPC3 suggests the LTRPC3e polynucleotides and polypeptides, including modulators or fragments thereof, may be useful in treating, diagnosing, prognosing, and/or preventing renal diseases and/or disorders, which include, but are not limited to: nephritis, renal failure, nephrotic syndrome, urinary tract infection, hematuria, proteinuria, oliguria, polyuria, nocturia, edema, hypertension, electrolyte disorders, sterile pyuria, renal osteodystrophy, large kidneys, renal transport defects, nephrolithiasis, azotemia, anuria, urinary retention, slowing of urinary stream, large prostate, flank tenderness, full bladder sensation after voiding, enuresis, dysuria, bacteriuria, kidney stones, glomerulonephritis, vasculitis, hemolytic uremic syndromes, thrombotic thrombocytopenic purpura, malignant hypertension, casts, tubulointerstitial kidney diseases, renal tubular acidosis, pyelonephritis, hydronephritis, nephrotic syndrome, crush syndrome, and/or renal colic, in addition to Wilm's Tumor Disease, and congenital kidney abnormalities such as horseshoe kidney, polycystic kidney, and Falconi's syndrome.for example.
Several known TRP family members have been identified that are expressed significantly in kidney tissue. These TRP family members include, for example, Trp12 (Wissenbach, U., Bodding, M., Freichel, M., Flockerzi, V, Lett., 485(2-3):127-34, (2000)); OTRPC4 (Strotmann, R., Harteneck, C., Nunnenmacher, K., Schultz, G., Plant, T, D, Nat, Cell, Biol., 2(10):695-702, (2000)); polycystin-L2 (Guo, L., Schreiber, T, H., Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics., 64(3):241-51, (2000)); and EcaC (Hoenderop, J. G., van, der, Kemp, A, W., Hartog, A., van, de, Graaf, S, F., van, Os, C, H., Willems, P, H., Bindels, R, J. J. Biol, Chem., 274(13):8375-8, (1999)).
Thus, the LTRPC3e polynucleotides and polypeptides are expected to share at least some biological activity with TRP family members expressed in kidney cells and tissues, particularly those specifically referenced herein.
The strong homology to human transient receptor potential channels (TRP) proteins, combined with the localized expression in spinal cord and brain of LTRPC3 suggests the LTRPC3e polynucleotides and polypeptides, including modulators or fragments thereof, may be useful in treating, diagnosing, prognosing, and/or preventing neurodegenerative disease states, behavioral disorders, or inflammatory conditions. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in the Examples, and elsewhere herein. Briefly, the uses include, but are not limited to the detection, treatment, and/or prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, depression, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, elevated expression of this gene product in regions of the brain indicates it plays a role in normal neural function. Potentially, this gene product is involved in synapse formation, neurotransmission, learning, cognition, homeostasis, or neuronal differentiation or survival. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.
The strong homology to human transient receptor potential channels (TRP), combined with the localized expression in testis tissue of LTRPC3 emphasizes the potential utility for LTRPC3e polynucleotides and polypeptides in treating, diagnosing, prognosing, and/or preventing testicular, in addition to reproductive disorders.
In preferred embodiments, LTRPC3e polynucleotides and polypeptides including agonists and fragments thereof, have uses which include treating, diagnosing, prognosing, and/or preventing the following, non-limiting, diseases or disorders of the testis: spermatogenesis, infertility, Klinefelter's syndrome, XX male, epididymitis, genital warts, germinal cell aplasia, cryptorchidism, varicocele, immotile cilia syndrome, and viral orchitis. The LTRPC3e polynucleotides and polypeptides including agonists and fragments thereof, may also have uses related to modulating testicular development, embryogenesis, reproduction, and in ameliorating, treating, and/or preventing testicular proliferative disorders (e.g., cancers, which include, for example, choriocarcinoma, Nonseminoma, seminona, and testicular germ cell tumors).
Likewise, the localized expression in testis tissue also emphasizes the potential utility for LTRPC3e polynucleotides and polypeptides in treating, diagnosing, prognosing, and/or preventing metabolic diseases and disorders which include the following, not limiting examples: premature puberty, incomplete puberty, Kallman syndrome, Cushing's syndrome, hyperprolactinemia, hemochromatosis, congenital adrenal hyperplasia, FSH deficiency, and granulomatous disease, for example.
This gene product may also be useful in assays designed to identify binding agents, as such agents (antagonists) are useful as male contraceptive agents. The testes are also a site of active gene expression of transcripts that is expressed, particularly at low levels, in other tissues of the body. Therefore, this gene product may be expressed in other specific tissues or organs where it may play related functional roles in other processes, such as hematopoiesis, inflammation, bone formation, and kidney function, to name a few possible target indications.
Several known TRP family members have been identified that are expressed significantly in testis tissue. These TRP family members include, for example, polycystin-L2 (Guo, L., Schreiber, T, H., Weremowicz, S., Morton, C, C., Lee, C., Zhou, J. Genomics., 64(3):241-51, (2000)); TRP7 (Okada, T., Inoue, R., Yamazaki, K., Maeda, A., Kurosaki, T., Yamakuni, T., Tanaka, I., Shimizu, S., Ikenaka, K., Imoto, K., Mori, Y, J. Biol, Chem., 274(39):27359-70, (1999)); btrp2 (Wissenbach, U., Schroth, G., Philipp, S., Flockerzi, V, Lett., 429(1):61-6, (1998)); Htrp-1 (Zhu, X., Chu, P, B., Peyton, M., Bimbaumer, L, Lett., 373(3):193-8, (1995)); and TRPC1 (Wes, P, D., Chevesich, J., Jeromin, A., Rosenberg, C., Stetten, G., Montell, C, Proc, Natl, Acad, Sci, U,S,A., 92(21):9652-6, (1995)).
Thus, the LTRPC3e polynucleotides and polypeptides are expected to share at least some biological activity with TRP family members expressed in testis cells and tissues, particularly those specifically referenced herein.
As described elsewhere herein, transient receptor potential channel family members have been implicated in modulating cell proliferation, differentiation, migration, activation, exocytosis, muscle contraction, gene expression, apoptosis. signalling, pheromone sensory signaling, smooth muscle tone, pain perception, heat perception, osmosenstivity, and mechanosensitivity. Moreover, transient receptor potential channel family members have been implicated in disorders of the skin, skeletal-muscle, nervous, cardiac, and vascular systems, in addition to the following, non-limiting diseases and disorders, which include, for example, arteriosclerosis, neointimal hypoerplasia, metastatic melanomas, bipolar disorder, nonsyndromic hereditary deafness, Knobloch syndrome, holosencephaly, and various maligancies including prostate cancer.
In preferred embodiments, LTRPC3e polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include, modulating cell proliferation, differentiation, migration, activation, exocytosis, muscle contraction, gene expression, apoptosis. signalling, pheromone sensory signaling, smooth muscle tone, pain perception, heat perception, osmosenstivity, and mechanosensitivity.
In more preferred embodiments, LTRPC3e polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include, treating, ameliorating, preventing, detecting, and/or prognosing various diseases and disorders, particularly the following, non-limiting examples, disorders of the skin, skeletal-muscle, nervous, cardiac, and vascular systems, in addition to the following, non-limiting diseases and disorders, which include, for example, arteriosclerosis, neointimal hypoerplasia, metastatic melanomas, bipolar disorder, nonsyndromic hereditary deafness, Knobloch syndrome, holosencephaly, and various maligancies including prostate cancer.
LTRPC3e polynucleotides and polypeptides of the present invention, including agonists and/or fragments may be involved in intracellular Ca2+ homeostasis which affects various aspects of biological functions including mechano-regulation, pain transduction, vasorelaxation, gene expression, cell cycle and proliferation/apoptosis. Since LTRPC3e is dominantly expressed in kidney, it may particularly play an important role in regulating cytosolic Ca2+ in the renal system, potentially Ca2+ absorption.
The LTRPC3e gene maps to chromosome 9q21.11-21.31 between the two markers D9S1874 and D9S1807. This region is linked to amyotrophic lateral sclerosis with frontotemporal dementia, early-onset pulverulent cataract, infantile nephronophthisis, hypomagnesemia with secondary hypocalcemia (HSH), and familial hemophagocytic lymphohistiocytosis. Therefore, agonists and/or antagonists of the novel LTRPC3e can be used to treat diseases including various forms of neuronal degeneration, neurogenic inflammation, allergy, immunodeficiency/excessive immune activation, visual defects, hearing disorder, pain, cancer, hypertension and other cardiovascular diseases. In addition, the therapeutics may be useful in the treatment of diseases associated with disturbances in Ca2+ homeostasis including osteoporosis, hypercalciuric stone disease, and chronic renal failure.
Moreover, given the selective expression in kidney, LTRPC3e could be considered a candidate gene for HSH, since the phenotype is a renal insufficiency. LTRPC3e chromosomal location is  ̃600 kb downstream of a X;9 translocation breakpoint interval described for one patient with HSH (Hum. Mol. Genet. 6, 1491-1497, (1997)). Recently, two groups reported that a new member of the TRPM subfamily, TRPM6, expressed in both intestinal tissues and kidney, is associated with HSH (Nat. Genet. 31, 166-170, (2002); and Nat. Genet. 31, 171-174 (2002)). Indeed, LTRPC3 is  ̃4 mb 5′ to TRPM6 on 9q21. Although TRPM6 is the first component identified for HSH, this should not preclude involvement of LTRPC3e in the disease. Therefore, it is possible that LTRPC3e may co-localize with TRPM6 and/or forms a heteromultimer with TRPM6 in the kidney.
The kidney plays a major role in Ca2+ homeostasis (reviewed in Physiol. Rev. 75, 429-471, (1995)). LTRPC3e may play a direct role in Ca2+ absorption due to its constitutive Ca2+ permeability. Alternatively, LTRPC3e may be the SOC that regulates Ca2+ absorption. In kidney, Ca2+ absorption is regulated by agonists such as calcitonin, parathyroid hormone and parathyroid hormone-related peptide through their respective G protein-coupled receptors, and downstream SOCs. Additionally, in kidney a SOC has been described (J. Biol. Chem. 276, 25759-25765, (2001)) for the action of angiotensin II, a vasoactive peptide that plays a major role in regulating blood pressure.
In addition, LTRPC3e polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include modulating intracellular Ca++ ion concentrations, Ca++ ion flux, stored intracellular Ca++ ion concentrations, Ca++ ion pump activity, Ca++ ion flow into cell, Ca++ ion flow out of cells, the activation of Ca++ sensitive proteins, the activation of Ca++ sensitive signaling pathways, the activation of kinase-activatible proteins, and the activation of kinase-dependent signaling pathways.
The LTRPC3e polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, have uses that include modulating proliferation, differentiation, migration, and activation in various cells, tissues, and organisms, and particularly in mammalian kidney, spinal cord, testis, and brain, preferably human. LTRPC3e polynucleotides and polypeptides of the present invention, including agonists and/or fragments thereof, may be useful in diagnosing, treating, prognosing, and/or preventing renal, neural, and/or proliferative diseases or disorders, particularly of the renal system.
In addition, antagonists of the LTRPC3e polynucleotides and polypeptides, including modulators or fragments thereof, may have uses that include diagnosing, treating, prognosing, and/or preventing diseases or disorders related to transient receptor potential channel activity, which may include renal, neural, reproductive, and/or proliferative diseases or disorders.
Although it is believed the encoded polypeptide may share at least some biological activities with transient receptor potential channel family members, particularly those melastatin1, a number of methods of determining the exact biological function of this clone are either known in the art or are described elsewhere herein. Briefly, the function of this clone may be determined by applying microarray methodology. Nucleic acids corresponding to the LTRPC3e polynucleotides, in addition to, other clones of the present invention, may be arrayed on microchips for expression profiling. Depending on which polynucleotide probe is used to hybridize to the slides, a change in expression of a specific gene may provide additional insight into the function of this gene based upon the conditions being studied. For example, an observed increase or decrease in expression levels when the polynucleotide probe used comes from tissue that has been treated with known transient potential receptor inhibitors, which include, but are not limited to the drugs listed herein or otherwise known in the art, might indicate a function in modulating transient potential receptor function, for example. In the case of LTRPC3e, kidney, spinal cord, testis, and brain, should be used to extract RNA to prepare the probe.
In addition, the function of the protein may be assessed by applying quantitative PCR methodology, for example. Real time quantitative PCR would provide the capability of following the expression of the LTRPC3e gene throughout development, for example. Quantitative PCR methodology requires only a nominal amount of tissue from each developmentally important step is needed to perform such experiments. Therefore, the application of quantitative PCR methodology to refining the biological function of this polypeptide is encompassed by the present invention. Also encompassed by the present invention are quantitative PCR probes corresponding to the polynucleotide sequence provided as SEQ ID NO:8 (FIGS. 4A-F).
The function of the protein may also be assessed through complementation assays in yeast. For example, in the case of the LTRPC3e, transforming yeast deficient in transient receptor potential channel activity with LTRPC3e and assessing their ability to grow would provide convincing evidence the LTRPC3e polypeptide has transient receptor potential channel activity. Additional assay conditions and methods that may be used in assessing the function of the polynucleotides and polypeptides of the present invention are known in the art, some of which are disclosed elsewhere herein.
Alternatively, the biological function of the encoded polypeptide may be determined by disrupting a homologue of this polypeptide in Mice and/or rats and observing the resulting phenotype.
Moreover, the biological function of this polypeptide may be determined by the application of antisense and/or sense methodology and the resulting generation of transgenic mice and/or rats. Expressing a particular gene in either sense or antisense orientation in a transgenic mouse or rat could lead to respectively higher or lower expression levels of that particular gene. Altering the endogenous expression levels of a gene can lead to the observation of a particular phenotype that can then be used to derive indications on the function of the gene. The gene can be either over-expressed or under expressed in every cell of the organism at all times using a strong ubiquitous promoter, or it could be expressed in one or more discrete parts of the organism using a well characterized tissue-specific promoter (e.g., a kidney, spinal cord, testis, and/or brain-specific promoter), or it can be expressed at a specified time of development using an inducible and/or a developmentally regulated promoter.
In the case of LTRPC3e transgenic mice or rats, if no phenotype is apparent in normal growth conditions, observing the organism under diseased conditions (renal, reproductive, neural, or proliferative disorders, etc.) may lead to understanding the function of the gene. Therefore, the application of antisense and/or sense methodology to the creation of transgenic mice or rats to refine the biological function of the polypeptide is encompassed by the present invention.
In preferred embodiments, the following N-terminal LTRPC3e deletion polypeptides are encompassed by the present invention: M1-T1556, Y2-T1556, V3-T1556, R4-T1556, V5-T1556, S6-T1556, F7-T1556, D8-T1556, T9-T1556, K10-T1556, P11-T1556, D12-T1556, L13-T1556, L14-T1556, L15-T1556, H16-T1556, L17-T1556, M18-T1556, T19-T1556, K20-T1556, E21-T1556, W22-T1556, Q23-T1556, L24-T1556, E25-T1556, L26-T1556, P27-T1556, K28-T1556, L29-T1556, L30-T1556, I31-T1556, S32-T1556, V33-T1556, H34-T1556, G35-T1556, G36-T1556, L37-T1556, Q38-T1556, N39-T1556, F40-T1556, E41-T1556, L42-T1556, Q43-T1556, P44-T1556, K45-T1556, L46-T1556, K47-T1556, Q48-T1556, V49-T1556, F50-T1556, G51-T1556, K52-T1556, G53-T1556, L54-T1556, I55-T1556, K56-T1556, A57-T1556, A58-T1556, M59-T1556, T60-T1556, T61-T1556, G62-T1556, A63-T1556, W64-T1556, I65-T1556, F66-T1556, T67-T1556, G68-T1556, G69-T1556, V70-T1556, N71-T1556, T72-T1556, G73-T1556, V74-T1556, I75-T1556, R76-T1556, H77-T1556, V78-T1556, G79-T1556, D80-T1556, A81-T1556, L82-T1556, K83-T1556, D84-T1556, H85-T1556, A86-T1556, S87-T1556, K88-T1556, S89-T1556, R90-T1556, G91-T1556, K92-T1556, I93-T1556, C94-T1556, T95-T1556, I96-T1556, G97-T1556, I98-T1556, A99-T1556, P100-T1556, W101-T1556, G102-T1556, I103-T1556, V104-T1556, E105-T1556, N106-T1556, Q107-T1556, E108-T1556, D109-T1556, L110-T1556, I111-T1556, G112-T1556, R113-T1556, D114-T1556, V115-T1556, V116-T1556, R117-T1556, P118-T1556, Y119-T1556, Q120-T1556, T121-T1556, M122-T1556, S123-T1556, N124-T1556, P125-T1556, M126-T1556, S127-T1556, K128-T1556, L129-T1556, T130-T1556, V131-T1556, L132-T1556, N133-T1556, S134-T1556, M135-T1556, H136-T1556, S137-T1556, H138-T1556, F139-T1556, I140-T1556, L141-T1556, A142-T1556, D143-T1556, N144-T1556, G145-T1556, T146-T1556, T147-T1556, G148-T1556, K149-T1556, Y150-T1556, G151-T1556, A152-T1556, E153-T1556, V154-T1556, K155-T1556, L156-T1556, R157-T1556, R158-T1556, Q159-T1556, L160-T1556, E161-T1556, K162-T1556, H163-T1556, I164-T1556, S165-T1556, L166-T1556, Q167-T1556, K168-T1556, I169-T1556, N170-T1556, T171-T1556, R172-T1556, I173-T1556, G174-T1556, Q175-T1556, G176-T1556, V177-T1556, P178-T1556, V179-T1556, V180-T1556, A181-T1556, L182-T1556, I183-T1556, V184-T1556, E185-T1556, G186-T1556, G187-T1556, P188-T1556, N189-T1556, V190-T1556, I191-T1556, S192-T1556, I193-T1556, V194-T1556, L195-T1556, E196-T1556, Y197-T1556, L198-T1556, R199-T1556, D200-T1556, T201-T1556, P202-T1556, P203-T1556, V204-T1556, P205-T1556, V206-T1556, V207-T1556, V208-T1556, C209-T1556, D210-T1556, G211-T1556, S212-T1556, G213-T1556, R214-T1556, A215-T1556, S216-T1556, D217-T1556, I218-T1556, L219-T1556, A220-T1556, F221-T1556, G222-T1556, H223-T1556, K224-T1556, Y225-T1556, S226-T1556, E227-T1556, E228-T1556, G229-T1556, G230-T1556, L231-T1556, I232-T1556, N233-T1556, E234-T1556, S235-T1556, L236-T1556, R237-T1556, D238-T1556, Q239-T1556, L240-T1556, L241-T1556, V242-T1556, T243-T1556, I244-T1556, Q245-T1556, K246-T1556, T247-T1556, F248-T1556, T249-T1556, Y250-T1556, T251-T1556, R252-T1556, T253-T1556, Q254-T1556, A255-T1556, Q256-T1556, H257-T1556, L258-T1556, F259-T1556, I260-T1556, I261-T1556, L262-T1556, M263-T1556, E264-T1556, C265-T1556, M266-T1556, K267-T1556, K268-T1556, K269-T1556, E270-T1556, L271-T1556, I272-T1556, T273-T1556, V274-T1556, F275-T1556, R276-T1556, M277-T1556, G278-T1556, S279-T1556, E280-T1556, G281-T1556, H282-T1556, Q283-T1556, D284-T1556, I285-T1556, D286-T1556, L287-T1556, A288-T1556, I289-T1556, L290-T1556, T291-T1556, A292-T1556, L293-T1556, L294-T1556, K295-T1556, G296-T1556, A297-T1556, N298-T1556, A299-T1556, S300-T1556, A301-T1556, P302-T1556, D303-T1556, Q304-T1556, L305-T1556, S306-T1556, L307-T1556, A308-T1556, L309-T1556, A310-T1556, W311-T1556, N312-T1556, R313-T1556, V314-T1556, D315-T1556, I316-T1556, A317-T1556, R318-T1556, S319-T1556, Q320-T1556, I321-T1556, F322-T1556, I323-T1556, Y324-T1556, G325-T1556, Q326-T1556, Q327-T1556, W328-T1556, P329-T1556, V330-T1556, G331-T1556, S332-T1556, L333-T1556, E334-T1556, Q335-T1556, A336-T1556, M337-T1556, L338-T1556, D339-T1556, A340-T1556, L341-T1556, V342-T1556, L343-T1556, D344-T1556, R345-T1556, V346-T1556, D347-T1556, F348-T1556, V349-T1556, K350-T1556, L351-T1556, L352-T1556, I353-T1556, E354-T1556, N355-T1556, G356-T1556, V357-T1556, S358-T1556, M359-T1556, H360-T1556, R361-T1556, F362-T1556, L363-T1556, T364-T1556, I365-T1556, S366-T1556, R367-T1556, L368-T1556, E369-T1556, E370-T1556, L371-T1556, Y372-T1556, N373-T1556, T374-T1556, R375-T1556, H376-T1556, G377-T1556, P378-T1556, S379-T1556, N380-T1556, T381-T1556, L382-T1556, Y383-T1556, H384-T1556, L385-T1556, V386-T1556, R387-T1556, D388-T1556, V389-T1556, K390-T1556, K391-T1556, R392-T1556, E393-T1556, Y394-T1556, P395-T1556, G396-T1556, F397-T1556, G398-T1556, W399-T1556, I400-T1556, Y401-T1556, F402-T1556, K403-T1556, G404-T1556, N405-T1556, L406-T1556, P407-T1556, P408-T1556, D409-T1556, Y410-T1556, R411-T1556, I412-T1556, S413-T1556, L414-T1556, I415-T1556, D416-T1556, I417-T1556, G418-T1556, L419-T1556, V420-T1556, I421-T1556, E422-T1556, Y423-T1556, L424-T1556, M425-T1556, G426-T1556, G427-T1556, A428-T1556, Y429-T1556, R430-T1556, C431-T1556, N432-T1556, Y433-T1556, T434-T1556, R435-T1556, K436-T1556, R437-T1556, F438-T1556, R439-T1556, T440-T1556, L441-T1556, Y442-T1556, H443-T1556, N444-T1556, L445-T1556, F446-T1556, G447-T1556, P448-T1556, K449-T1556, and/or R450-T1556 of SEQ ID NO:9. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these N-terminal LTRPC3e deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
In preferred embodiments, the following C-terminal LTRPC3e deletion polypeptides are encompassed by the present invention: M1-T1556, M1-H1555, M1-K1554, M1-S1553, M1-E1552, M1-F1551, M1-S1550, M1-Q1549, M1-F1548, M1-A1547, M1-S1546, M1-T1545, M1-R1544, M1-S1543, M1-L1542, M1-R1541, M1-R1540, M1-M1539, M1-S1538, M1-L1537, M1-S1536, M1-D1535, M1-G1534, M1-R1533, M1-G1532, M1-E1531, M1-P1530, M1-K1529, M1-S1528, M1-S1527, M1-K1526, M1-S1525, M1-R1524, M1-Q1523, M1-F1522, M1-P1521, M1-N1520, M1-Q1519, M1-L1518, M1-S1517, M1-A1516, M1-T1515, M1-N1514, M1-R1513, M1-Q1512, M1-R1511, M1-D1510, M1-L1509, M1-K1508, M1-D1507, M1-S1506, M1-I1505, M1-S1504, M1-F1503, M1-S1502, M1-K1501, M1-R1500, M1-T1499, M1-H1498, M1-A1497, M1-Y1496, M1-P1495, M1-A1494, M1-S1493, M1-P1492, M1-E1491, M1-E1490, M1-A1489, M1-S1488, M1-Y1487, M1-S1486, M1-N1485, M1-A1484, M1-R1483, M1-E1482, M1-I1481, M1-K1480, M1-P1479, M1-V1478, M1-T1477, M1-I1476, M1-N1475, M1-N1474, M1-S1473, M1-L1472, M1-T1471, M1-R1470, M1-E1469, M1-S1468, M1-N1467, M1-D1466, M1-G1465, M1-E1464, M1-Q1463, M1-S1462, M1-S1461, M1-I1460, M1-A1459, M1-I1458, M1-T1457, M1-A1456, M1-R1455, M1-R1454, M1-G1453, M1-K1452, M1-A1451, M1-E1450, M1-N1449, M1-E1448, M1-E1447, M1-S1446, M1-D1445, M1-S1444, M1-S1443, M1-P1442, M1-H1441, M1-S1440, M1-L1439, M1-E1438, M1-A1437, M1-E1436, M1-R1435, M1-E1434, M1-P1433, M1-H1432, M1-C1431, M1-C1430, M1-T1429, M1-L1428, M1-D1427, M1-E1426, M1-V1425, M1-K1424, M1-D1423, M1-G1422, M1-L1421, M1-G1420, M1-G1419, M1-P1418, M1-F1417, M1-A1416, M1-A1415, M1-R1414, M1-D1413, M1-A1412, M1-I1411, M1-A1410, M1-Q1409, M1-P1408, M1-A1407, M1-N1406, M1-V1405, M1-C1404, M1-R1403, M1-T1402, M1-D1401, M1-I1400, M1-C1399, M1-D1398, M1-T1397, M1-I1396, M1-S1395, M1-T1394, M1-Y1393, M1-E1392, M1-A1391, M1-T1390, M1-K1389, M1-V1388, M1-P1387, M1-V1386, M1-G1385, M1-F1384, M1-N1383, M1-A1382, M1-Y1381, M1-Y1380, M1-S1379, M1-R1378, M1-S1377, M1-P1376, M1-S1375, M1-F1374, M1-M1373, M1-F1372, M1-S1371, M1-H1370, M1-S1369, M1-K1368, M1-V1367, M1-I1366, M1-P1365, M1-A1364, M1-E1363, M1-E1362, M1-L1361, M1-L1360, M1-F1359, M1-P1358, M1-T1357, M1-T1356, M1-A1355, M1-L1354, M1-Y1353, M1-R1352, M1-S1351, M1-S1350, M1-K1349, M1-S1348, M1-R1347, M1-E1346, M1-I1345, M1-T1344, M1-H1343, M1-Y1342, M1-M1341, M1-P1340, M1-P1339, M1-E1338, M1-S1337, M1-D1336, M1-W1335, M1-P1334, M1-N1333, M1-Q1332, M1-C1331, M1-E1330, M1-P1329, M1-L1328, M1-H1327, M1-T1326, M1-Y1325, M1-D1324, M1-S1323, M1-S1322, M1-F1321, M1-S1320, M1-R1319, M1-T1318, M1-D1317, M1-M1316, M1-S1315, M1-T1314, M1-I1313, M1-D1312, M1-E1311, M1-F1310, M1-D1309, M1-I1308, M1-S1307, M1-R1306, M1-S1305, M1-P1304, M1-P1303, M1-R1302, M1-D1301, M1-T1300, M1-P1299, M1-A1298, M1-L1297, M1-T1296, M1-A1295, M1-Y1294, M1-A1293, M1-S1292, M1-S1291, M1-S1290, M1-P1289, M1-A1288, M1-T1287, M1-S1286, M1-P1285, M1-V1284, M1-P1283, M1-T1282, M1-S1281, M1-F1280, M1-S1279, M1-P1278, M1-E1277, M1-G1276, M1-L1275, M1-G1274, M1-L1273, M1-I1272, M1-N1271, M1-V1270, M1-S1269, M1-N1268, M1-D1267, M1-L1266, M1-P1265, M1-D1264, M1-I1263, M1-D1262, M1-C1261, M1-H1260, M1-L1259, M1-E1258, M1-D1257, M1-M1256, M1-A1255, M1-S1254, M1-V1253, M1-Y1252, M1-I1251, M1-D1250, M1-I1249, M1-C1248, M1-S1247, M1-S1246, M1-P1245, M1-R1244, M1-R1243, M1-S1242, M1-D1241, M1-P1240, M1-V1239, M1-I1238, M1-A1237, M1-L1236, M1-T1235, M1-N1234, M1-A1233, M1-P1232, M1-A1231, M1-A1230, M1-P1229, M1-A1228, M1-K1227, M1-P1226, M1-E1225, M1-K1224, M1-A1223, M1-V1222, M1-S1221, M1-H1220, M1-S1219, M1-S1218, M1-T1217, M1-A1216, M1-R1215, M1-H1214, M1-L1213, M1-S1212, M1-L1211, M1-S1210, M1-R1209, M1-E1208, M1-K1207, M1-F1206, M1-I1205, M1-S1204, M1-E1203, M1-L1202, M1-K1201, M1-E1200, M1-I1199, M1-G1198, M1-G1197, M1-K1196, M1-D1195, M1-K1194, M1-M1193, M1-N1192, M1-V1191, M1-S1190, M1-Y1189, M1-F1188, M1-S1187, M1-H1186, M1-S1185, M1-R1184, M1-M1183, M1-R1182, M1-P1181, M1-M1180, M1-L1179, M1-T1178, M1-P1177, M1-S1176, M1-T1175, M1-P1174, M1-S1173, M1-M1172, M1-T1171, M1-E1170, M1-E1169, M1-G1168, M1-A1167, M1-P1166, M1-D1165, M1-I1164, M1-S1163, M1-E1162, M1-Q161, M1-L1160, M1-K1159, M1-F1158, M1-T1157, M1-N1156, M1-G1155, M1-E1154, M1-Q1153, M1-S1152, M1-N1151, M1-F1150, M1-S1149, M1-S1148, M1-Q1147, M1-R1146, M1-V1145, M1-I1144, M1-Y1143, M1-A1142, M1-A1141, M1-D1140, M1-T1139, M1-C1138, M1-D1137, M1-S1136, M1-S1135, M1-T1134, M1-R1133, M1-S1132, M1-R1131, M1-I130, M1-K1129, M1-N1128, M1-S1127, M1-E1126, M1-A1125, M1-R1124, M1-E1123, M1-L1122, M1-G1121, M1-T1120, M1-L1119, M1-R1118, M1-E1117, M1-L1116, M1-A1115, M1-T1114, M1-A1113, M1-M1112, M1-R1111, M1-G1110, M1-I1109, M1-L1108, M1-D1107, M1-E1106, M1-L1105, M1-Q1104, M1-A1103, M1-L1102, M1-R1101, M1-I1100, M1-D1099, M1-V1098, M1-T1097, M1-Q1096, M1-L1095, M1-S1094, M1-A1093, M1-K1092, M1-M1091, M1-S1090, M1-H1089, M1-E1088, M1-R1087, M1-E1086, M1-N1085, M1-V1084, M1-E1083, M1-E1082, M1-L1081, M1-R1080, M1-M1079, M1-S1078, M1-M1077, M1-N1076, M1-E1075, M1-V1074, M1-R1073, M1-E1072, M1-S1071, M1-T1070, M1-V1069, M1-R1068, M1-I1067, M1-R1066, M1-E1065, M1-D1064, M1-N1063, M1-S1062, M1-S1061, M1-N1060, M1-F1059, M1-R1058, M1-D1057, M1-D1056, M1-K1055, M1-E1054, M1-R1053, M1-F1052, M1-Y1051, M1-E1050, M1-E1049, M1-I1048, M1-C1047, M1-Q1046, M1-E1045, M1-E1044, M1-F1043, M1-D1042, M1-H1041, M1-V1040, M1-K1039, M1-K1038, M1-L1037, M1-E1036, M1-D1035, M1-D1034, M1-T1033, M1-I1032, M1-F1031, M1-L1030, M1-K1029, M1-L1028, M1-G1027, M1-Y1026, M1-D1025, M1-R1024, M1-E1023, M1-D1022, M1-P1021, M1-D1020, M1-S1019, M1-E1018, M1-H1017, M1-K1016, M1-R1015, M1-W1014, M1-R1013, M1-C1012, M1-C1011, M1-L1010, M1-H1009, M1-Q1008, M1-F1007, M1-I1006, M1-M1005, M1-T1004, M1-M1003, M1-H1002, M1-S1001, M1-F1000, M1-I999, M1-I998, M1-L997, M1-P996, M1-P995, M1-P994, M1-L993, M1-V992, M1-P991, M1-R990, M1-E989, M1-H988, M1-F987, M1-T986, M1-M985, M1-I984, M1-L983, M1-Q982, M1-Y981, M1-R980, M1-Q979, M1-F978, M1-K977, M1-W976, M1-V975, M1-Q974, M1-N973, M1-S972, M1-I971, M1-S970, M1-K969, M1-V968, M1-E967, M1-F966, M1-F965, M1-T964, M1-N963, M1-N962, M1-F961, M1-V960, M1-A959, M1-I958, M1-L957, M1-L956, M1-N955, M1-V954, M1-L953, M1-L952, M1-I951, M1-N950, M1-A949, M1-V948, M1-L947, M1-L946, M1-Y945, M1-C944, M1-A943, M1-M942, M1-I941, M1-A940, M1-P939, M1-V938, M1-I937, M1-W936, M1-A935, M1-G934, M1-T933, M1-K932, M1-C931, M1-P930, M1-P929, M1-L928, M1-Q927, M1-I926, M1-I925, M1-K924, M1-G923, M1-D922, M1-E921, M1-R920, M1-T919, M1-E918, M1-N917, M1-Q916, M1-G915, M1-C914, M1-P913, M1-P912, M1-D911, M1-I910, M1-Q909, M1-D908, M1-A907, M1-F906, M1-V905, M1-E904, M1-G903, M1-Y902, M1-I901, M1-M900, M1-W899, M1-Y898, M1-P897, M1-M896, M1-Y895, M1-F894, M1-I893, M1-N892, M1-K891, M1-A890, M1-L889, M1-K888, M1-W887, M1-S886, M1-P885, M1-E884, M1-E883, M1-N882, M1-P881, M1-F880, M1-L879, M1-I878, M1-A877, M1-Q876, M1-R875, M1-A874, M1-V873, M1-G872, M1-F871, M1-S870, M1-M869, M1-L868, M1-V867, M1-V866, M1-L865, M1-M864, M1-I863, M1-I862, M1-V861, M1-F860, M1-Y859, M1-M858, M1-M857, M1-D856, M1-I855, M1-M854, M1-M853, M1-K852, M1-G851, M1-I850, M1-M849, M1-M848, M1-V847, M1-Y846, M1-P845, M1-G844, M1-L843, M1-Y842, M1-K841, M1-N840, M1-V839, M1-G838, M1-F837, M1-I836, M1-D835, M1-L834, M1-L833, M1-R832, M1-I831, M1-Y830, M1-W829, M1-Y828, M1-I827, M1-I826, M1-N825, M1-V824, M1-C823, M1-Y822, M1-I821, M1-V820, M1-R819, M1-G818, M1-D817, M1-S816, M1-R815, M1-F814, M1-P813, M1-Q812, M1-D811, M1-Q810, M1-L809, M1-R808, M1-L807, M1-I806, M1-M805, M1-G804, M1-V803, M1-S802, M1-F801, M1-L800, M1-L799, M1-I798, M1-A797, M1-I796, M1-L795, M1-D794, M1-T793, M1-V792, M1-N791, M1-W790, M1-Y789, M1-E788, M1-Q787, M1-L786, M1-W785, M1-V784, M1-K783, M1-V782, M1-K781, M1-Q780, M1-L779, M1-L778, M1-K777, M1-G776, M1-P775, M1-E774, M1-S773, M1-M772, M1-L771, M1-I770, M1-E769, M1-R768, M1-M767, M1-K766, M1-E765, M1-I764, M1-G763, M1-L762, M1-T761, M1-F760, M1-I759, M1-Y758, M1-S757, M1-I756, M1-V755, M1-I754, M1-W753, M1-E752, M1-Q751, M1-T750, M1-S749, M1-P748, M1-W747, M1-R746, M1-E745, M1-M744, M1-K743, M1-V742, M1-L741, M1-V740, M1-I739, M1-Y738, M1-N737, M1-F736, M1-L735, M1-M734, M1-L733, M1-Y732, M1-G731, M1-I730, M1-Y729, M1-A728, M1-L727, M1-T726, M1-Y725, M1-F724, M1-W723, M1-F722, M1-K721, M1-V720, M1-I719, M1-P718, M1-A717, M1-N716, M1-Y715, M1-F714, M1-E713, M1-Y712, M1-I711, M1-K710, M1-R709, M1-G708, M1-L707, M1-P706, M1-I705, M1-L704, M1-R703, M1-H702, M1-K701, M1-S700, M1-Q699, M1-V698, M1-E697, M1-E696, M1-E695, M1-D694, M1-K693, M1-K692, M1-R691, M1-S690, M1-S689, M1-E688, M1-G687, M1-N686, M1-N685, M1-R684, M1-G683, M1-L682, M1-M681, M1-A680, M1-T679, M1-L678, M1-E677, M1-M676, M1-D675, M1-E674, M1-E673, M1-E672, M1-K671, M1-E670, M1-K669, M1-T668, M1-P667, M1-K666, M1-E665, M1-P664, M1-E663, M1-E662, M1-A661, M1-E660, M1-K659, M1-E658, M1-Q657, M1-L656, M1-H655, M1-I654, M1-E653, M1-Q652, M1-A651, M1-Q650, M1-S649, M1-M648, M1-Y647, M1-P646, M1-M645, M1-D644, M1-D643, M1-K642, M1-N641, M1-K640, M1-F639, M1-E638, M1-L637, M1-S636, M1-L635, M1-I634, M1-S633, M1-P632, M1-P631, M1-L630, M1-L629, M1-I628, M1-G627, M1-L626, M1-I625, M1-V624, M1-K623, M1-L622, M1-G621, M1-S620, M1-N619, M1-K618, M1-R617, M1-M616, M1-R615, M1-L614, M1-R613, M1-G612, M1-M611, M1-W610, M1-M609, M1-D608, M1-T607, M1-L606, M1-L605, M1-M604, M1-Q603, M1-S602, M1-C601, M1-T600, M1-H599, M1-A598, M1-I597, M1-F596, M1-D595, M1-R594, M1-H593, M1-K592, M1-A591, M1-A590, M1-V589, M1-A588, M1-L587, M1-Q586, M1-L585, M1-C584, M1-T583, M1-A582, M1-N581, M1-S580, M1-W579, M1-N578, M1-K577, M1-L576, M1-E575, M1-Y574, M1-T573, M1-L572, M1-L571, M1-K570, M1-M569, M1-A568, M1-L567, M1-Q566, M1-E565, M1-D564, M1-Q563, M1-K562, M1-Y561, M1-S560, M1-Q559, M1-D558, M1-L557, M1-L556, M1-E555, M1-V554, M1-A553, M1-L552, M1-Q551, M1-G550, M1-F549, M1-D548, M1-R547, M1-S546, M1-N545, M1-H544, M1-N543, M1-L542, M1-E541, M1-Q540, M1-S539, M1-I538, M1-D537, M1-D536, M1-V535, M1-M534, M1-D533, M1-N532, M1-E531, M1-S530, M1-A529, M1-E528, M1-H527, M1-A526, M1-M525, M1-A524, M1-K523, M1-C522, M1-L521, M1-K520, M1-C519, M1-A518, M1-V517, M1-L516, M1-A515, M1-K514, M1-A513, M1-M512, M1-A511, M1-E510, M1-E509, M1-G508, M1-H507, M1-Q506, M1-W505, M1-F504, M1-F503, M1-L502, M1-A501, M1-M500, M1-K499, M1-Q498, M1-R497, M1-K496, M1-M495, M1-L494, M1-V493, M1-A492, M1-W491, M1-V490, M1-M489, M1-L488, M1-E487, M1-H486, M1-F485, M1-P484, M1-F483, M1-P482, M1-F481, M1-H480, M1-N479, M1-I478, M1-E477, M1-P476, M1-D475, M1-D474, M1-L473, M1-D472, M1-I471, M1-D470, M1-V469, M1-E468, M1-E467, M1-E466, M1-R465, M1-K464, M1-K463, M1-T462, M1-T461, M1-K460, M1-R459, M1-G458, M1-R457, M1-R456, M1-L455, M1-P454, M1-I453, M1-D452, and/or M1-D451 of SEQ ID NO:9. Polynucleotide sequences encoding these polypeptides are also provided. The present invention also encompasses the use of these C-terminal LTRPC3e deletion polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
Alternatively, preferred polypeptides of the present invention may comprise polypeptide sequences corresponding to, for example, internal regions of the LTRPC3e polypeptide (e.g., any combination of both N- and C-terminal LTRPC3e polypeptide deletions) of SEQ ID NO:9. For example, internal regions could be defined by the equation: amino acid NX to amino acid CX, wherein NX refers to any N-terminal deletion polypeptide amino acid of LTRPC3e (SEQ ID NO:9), and where CX refers to any C-terminal deletion polypeptide amino acid of LTRPC3e (SEQ ID NO:9). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these polypeptides as an immunogenic and/or antigenic epitope as described elsewhere herein.
The LTRPC3e polypeptides of the present invention were determined to comprise several phosphorylation sites based upon the Motif algorithm (Genetics Computer Group, Inc.). The phosphorylation of such sites may regulate some biological activity of the LTRPC3e polypeptide. For example, phosphorylation at specific sites may be involved in regulating the proteins ability to associate or bind to other molecules (e.g., proteins, ligands, substrates, DNA, etc.). In the present case, phosphorylation may modulate the ability of the LTRPC3e polypeptide to associate with other potassium channel alpha subunits, beta subunits, or its ability to modulate potassium channel function.
Specifically, the LTRPC3e polypeptide was predicted to comprise two tyrosine phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). Such sites are phosphorylated at the tyrosine amino acid residue. The consensus pattern for tyrosine phosphorylation sites are as follows: [RK]-x(2)-[DE]-x(3)-Y, or [RK]-x(3)-[DE]-x(2)-Y, where Y represents the phosphorylation site and ‘x’ represents an intervening amino acid residue. Additional information specific to tyrosine phosphorylation sites can be found in Patschinsky T., Hunter T., Esch F. S., Cooper J. A., Sefton B. M., Proc. Natl. Acad. Sci. U.S.A. 79:973-977 (1982); Hunter T., J. Biol. Chem. 257:4843-4848 (1982), and Cooper J. A., Esch F. S., Taylor S. S., Hunter T., J. Biol. Chem. 259:7835-7841(1984), which are hereby incorporated herein by reference.
In preferred embodiments, the following tyrosine phosphorylation site polypeptides are encompassed by the present invention: LSLEFKNKDDMPYMSQAQ (SEQ ID NO:209), and/or VMMIGKMMIDMMYFVIIM (SEQ ID NO:210). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these LTRPC3e tyrosine phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The LTRPC3e polypeptide was predicted to comprise twenty three PKC phosphorylation sites using the Motif algorithm (Genetics Computer Group, Inc.). In vivo, protein kinase C exhibits a preference for the phosphorylation of serine or threonine residues. The PKC phosphorylation sites have the following consensus pattern: [ST]-x-[RK], where S or T represents the site of phosphorylation and ‘x’ an intervening amino acid residue. Additional information regarding PKC phosphorylation sites can be found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem. 161:177-184 (1986), and Kishimoto A., Nishiyama K., Nakanishi H., Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem. 260:12492-12499 (1985); which are hereby incorporated by reference herein.
In preferred embodiments, the following PKC phosphorylation site polypeptides are encompassed by the present invention: ADNGTTGKYGAEV (SEQ ID NO:211), VVCDGSGRASDIL (SEQ ID NO:212), GLINESLRDQLLV (SEQ ID NO:213), YRCNYTRKRFRTL (SEQ ID NO:214), RRGRKTTKKREEE (SEQ ID NO:215), RGRKTTKKREEEV (SEQ ID NO:216), ELLDQSYKQDEQL (SEQ ID NO:217), RNNGESSRKKDEE (SEQ ID NO:218), NNGESSRKKDEEE (SEQ ID NO:219), PNEEPSWKLAKNI (SEQ ID NO:220), RIRVTSERVENMS (SEQ ID NO:221), RVENMSMRLEEVN (SEQ ID NO:222), NEREHSMKASLQT (SEQ ID NO:223), LERAESNKIRSRT (SEQ ID NO:224), SQEGNTFKLQESI (SEQ ID NO:225), AIVPDSRRPSSCI (SEQ ID NO:226), ATLAPTDRPPSRS (SEQ ID NO:227), IERSKSSRYLATT (SEQ ID NO:228), QEGDNSERTLSNN (SEQ ID NO:229), APYAHTRKSFSIS (SEQ ID NO:230), KSFSISDKLDRQR (SEQ ID NO:231), FQRSKSSKPEGRG (SEQ ID NO:232), and/or RGDSLSMRRLSRT (SEQ ID NO:233). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these LTRPC3e PKC phosphorylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The present invention also encompasses immunogenic and/or antigenic epitopes of the LTRPC3e polypeptide.
The LTRPC3e polypeptide has been shown to comprise twelve glycosylation sites according to the Motif algorithm (Genetics Computer Group, Inc.). As discussed more specifically herein, protein glycosylation is thought to serve a variety of functions including: augmentation of protein folding, inhibition of protein aggregation, regulation of intracellular trafficking to organelles, increasing resistance to proteolysis, modulation of protein antigenicity, and mediation of intercellular adhesion.
Asparagine phosphorylation sites have the following consensus pattern, N-{P}-[ST]-{P}, wherein N represents the glycosylation site. However, it is well known that that potential N-glycosylation sites are specific to the consensus sequence Asn-Xaa-Ser/Thr. However, the presence of the consensus tripeptide is not sufficient to conclude that an asparagine residue is glycosylated, due to the fact that the folding of the protein plays an important role in the regulation of N-glycosylation. It has been shown that the presence of proline between Asn and Ser/Thr will inhibit N-glycosylation; this has been confirmed by a recent statistical analysis of glycosylation sites, which also shows that about 50% of the sites that have a proline C-terminal to Ser/Thr are not glycosylated. Additional information relating to asparagine glycosylation may be found in reference to the following publications, which are hereby incorporated by reference herein: Marshall R. D., Annu. Rev. Biochem. 41:673-702 (1972); Pless D. D., Lennarz W. J., Proc. Natl. Acad. Sci. U.S.A. 74:134-138 (1977); Bause E., Biochem. J. 209:331-336 (1983); Gavel Y., von Heijne G., Protein Eng. 3:433-442 (1990); and Miletich J. P., Broze G. J. Jr., J. Biol. Chem. 265:11397-11404 (1990).
In preferred embodiments, the following asparagine glycosylation site polypeptides are encompassed by the present invention: FILADNGTTGKYGA (SEQ ID NO:234), EGGLINESLRDQLL (SEQ ID NO:235), LLKGANASAPDQLS (SEQ ID NO:236), GAYRCNYTRKRFRT (SEQ ID NO:237), TYELKNWSNATCLQ (SEQ ID NO:238), LKNWSNATCLQLAV (SEQ ID NO:239), LQEYWNVTDLIAIL (SEQ ID NO:240), PPCGQNETREDGKI (SEQ ID NO:241), LIAVFNNTFFEVKS (SEQ ID NO:242), KDDRFNSSNDERIR (SEQ ID NO:243), SERVENMSMRLEEV (SEQ ID NO:244), and/or RTLSNNITVPKIER (SEQ ID NO:245). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these LTRPC3e asparagine glycosylation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
The LTRPC3e polypeptide has been shown to comprise one RGD cell attachment site domain according to the Motif algorithm (Genetics Computer Group, Inc.). The sequence Arg-Gly-Asp, found in fibronectin, is crucial for its interaction with its cell surface receptor, an integrin. What has been called the ‘RGD’ tripeptide is also found in the sequences of a number of other proteins, where it has been shown to play a role in cell adhesion. Non-limiting examples of these proteins are the following: some forms of collagens, fibrinogen, vitronectin, von Willebrand factor (VWF), snake disintegrins, and slime mold discoidins. The ‘RGD’ tripeptide is also found in other proteins where it may serve the same purpose. A consensus pattern for RGD cell attachment sites is the following: R-G-D. Additional information relating to RGD cell attachment site domains may be found in reference to the following publications, which are hereby incorporated by reference herein: Ruoslahti E., Pierschbacher M. D., Cell 44:517-518 (1986); and d'Souza S. E., Ginsberg M. H., Plow E. F., Trends Biochem. Sci. 16:246-250 (1991).
In preferred embodiments, the following RGD cell attachment site domain polypeptide is encompassed by the present invention: SKPEGRGDSLSMR (SEQ ID NO:246). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this RGD cell attachment site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The LTRPC3e polypeptide has been shown to comprise one aminoacyl-transfer RNA synthetases class-II domain according to the Motif algorithm (Genetics Computer Group, Inc.). Aminoacyl-tRNA synthetases (EC 6.1.1.-) are a group of enzymes which activate amino acids and transfer them to specific tRNA molecules as the first step in protein biosynthesis. In prokaryotic organisms there are at least twenty different types of aminoacyl-tRNA synthetases, one for each different amino acid. In eukaryotes there are generally two aminoacyl-tRNA synthetases for each different amino acid: one cytosolic form and a mitochondrial form. While all these enzymes have a common function, they are widely diverse in terms of subunit size and of quaternary structure.
The synthetases specific for alanine, asparagine, aspartic acid, glycine, histidine, lysine, phenylalanine, proline, serine, and threonine are referred to as class-II synthetases and probably have a common folding pattern in their catalytic domain for the binding of ATP and amino acid which is different to the Rossmann fold observed for the class I synthetases.
Class-II tRNA synthetases do not share a high degree of similarity, however at least three conserved regions are present.
The consensus pattern for aminoacyl-transfer RNA synthetases class-II domains are as follows: [FYH]-R-x-[DE]-x(4,12)-[RH]-x(3)-F-x(3)-[DE]; and [GSTALVF]-{DENQHRKP}-[GSTA]-[LIVMF]-[DE]-R-[LIVMF]-x-[LIVMSTAG]-[LIVMFY], where ‘x’ represents an intervening amino acid residue.
Additional information specific to aminoacyl-transfer RNA synthetases class-II domains may be found in reference to the following publications, Schimmel P., Annu. Rev. Biochem. 56:125-158 (1987); Delarue M., Moras D., BioEssays 15:675-687 (1993); Schimmel P., Trends Biochem. Sci. 16:1-3 (1991); Nagel G. M., Doolittle R. F., Proc. Natl. Acad. Sci. U.S.A. 88:8121-8125 (1991); Cusack S., Haertlein M., Leberman R., Nucleic Acids Res. 19:3489-3498 (1991); Cusack S., Biochimie 75:1077-1081 (1993); Cusack S., Berthet-Colominas C., Haertlein M., Nassar N., Leberman R., Nature 347:249-255 (1990); and Leveque F., Plateau P., Dessen P., Blanquet S., Nucleic Acids Res. 18:305-312 (1990); which are hereby incorporated herein by reference in their entirety.
In preferred embodiments, the following aminoacyl-transfer RNA synthetases class-II domain polypeptide is encompassed by the present invention: LIGRMATALERLTGLERAES (SEQ ID NO:247). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of this RGD cell attachment site domain polypeptide as an immunogenic and/or antigenic epitope as described elsewhere herein.
The LTRPC3e polypeptide has been shown to comprise three amidation sites according to the Motif algorithm (Genetics Computer Group, Inc.). The precursor of hormones and other active peptides which are C-terminally amidated is always directly followed by a glycine residue which provides the amide group, and most often by at least two consecutive basic residues (Arg or Lys) which generally function as an active peptide precursor cleavage site. Although all amino acids can be amidated, neutral hydrophobic residues such as Val or Phe are good substrates, while charged residues such as Asp or Arg are much less reactive. A consensus pattern for amidation sites is the following: x-G-[RK]-[RK], wherein “X” represents the amidation site. Additional information relating to asparagine glycosylation may be found in reference to the following publications, which are hereby incorporated by reference herein: Kreil G., Meth. Enzymol. 106:218-223(1984); and Bradbury A. F., Smyth D. G., Biosci. Rep. 7:907-916 (1987).
In preferred embodiments, the following amidation site polypeptides are encompassed by the present invention: DIPLRRGRKTTKKR (SEQ ID NO:248), HRLIPLGRKIYEFY (SEQ ID NO:249), and/or EENEAKGRRATIAI (SEQ ID NO:250). Polynucleotides encoding these polypeptides are also provided. The present invention also encompasses the use of these LTRPC3e amidation site polypeptides as immunogenic and/or antigenic epitopes as described elsewhere herein.
Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:8 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides consisting of a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 4657 of SEQ ID NO:8, b is an integer between 15 to 4671, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:8, and where b is greater than or equal to a+14.
















TABLE I







ATCC

NT
Total
5′ NT







Deposit

SEQ
NT Seq
of Start
3′ NT

Total


Gene
CDNA
No. Z and

ID.
of
Codon
of
AA Seq
AA of


No.
CloneID
Date
Vector
No. X
Clone
of ORF
ORF
ID No. Y
ORF







1.
LTRPC3
PTA-4175
PBSII-
1
4665
1
4662
2
1554



(AL358786,
Mar. 21, 2002
SKSΔP-



NT_008306,

XB



clone



86,



TRPM3,



and/or



LTRPC6)


2
LTRPC3b
N/A
N/A
3
4701
1
4698
4
1566



(AL358786,



NT_008306,



clone



86,



TRPM3,



and/or



LTRPC6



splice



variant)


3.
LTRPC3c
N/A
N/A
5
4701
1
4698
6
1566



(AL358786,



NT_008306,



clone



86,



TRPM3,



and/or



LTRPC6



splice



variant)


4
LTRPC3e
N/A
N/A
8
4671
1
4668
9
1556



(AL358786,



NT_008306,



clone



86,



TRPM3,



and/or



LTRPC6



splice



variant)









Table 1 summarizes the information corresponding to each “Gene No.” described above. The nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous (“overlapping”) sequences obtained from the “cDNA clone ID” identified in Table 1 and, in some cases, from additional related DNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually several overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:X.
The cDNA Clone ID was deposited on the date and given the corresponding deposit number listed in “ATCC Deposit No:Z and Date.” “Vector” refers to the type of vector contained in the cDNA Clone ID.
“Total NT Seq. Of Clone” refers to the total number of nucleotides in the clone contig identified by “Gene No.” The deposited clone may contain all or most of the sequence of SEQ ID NO:X. The nucleotide position of SEQ ID NO:X of the putative start codon (methionine) is identified as “5′ NT of Start Codon of ORF.”
The translated amino acid sequence, beginning with the methionine, is identified as “AA SEQ ID NO:Y,” although other reading frames can also be easily translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.
The total number of amino acids within the open reading frame of SEQ ID NO:Y is identified as “Total AA of ORF”.
SEQ ID NO:X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further herein. For instance, SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the proteins encoded by the cDNA clones identified in Table 1.
Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides may cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).
Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:1, 3, 5, 8, 317, and/or 318 and the predicted translated amino acid sequence identified as SEQ ID NO:2, 4, 6, 7, 9, and/or 10. The nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell, collecting the protein, and determining its sequence.
The present invention also relates to the genes corresponding to SEQ ID NO:1, 3, 5, 8, 317, and/or 318, SEQ ID NO:2, 4, 6, 7, 9, and/or 10. The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
Also provided in the present invention are species homologs, allelic variants, and/or orthologs. The skilled artisan could, using procedures well-known in the art, obtain the polynucleotide sequence corresponding to full-length genes (including, but not limited to the full-length coding region), allelic variants, splice variants, orthologs, and/or species homologues of genes corresponding to SEQ ID NO:1, 3, 5, 8, 317, and/or 318, SEQ ID NO:2, 4, 6, 7, 9, and/or 10, or a deposited clone, relying on the sequence from the sequences disclosed herein. For example, allelic variants and/or species homologues may be isolated and identified by making suitable probes or primers which correspond to the 5′, 3′, or internal regions of the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
The polypeptides may be in the form of the protein, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.
The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using protocols described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the full-length form of the protein.
The present invention provides a polynucleotide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:1, 3, 5, 8, 317, and/or 318. The present invention also provides a polypeptide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:2, 4, 6, 7, 9, and/or 10. The present invention also provides polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:2, 4, 6, 7, 9, and/or 10.
Preferably, the present invention is directed to a polynucleotide comprising, or alternatively consisting of, the sequence identified as SEQ ID NO:1, 3, 5, 8, 317, and/or 318 that is less than, or equal to, a polynucleotide sequence that is 5 mega basepairs, 1 mega basepairs, 0.5 mega basepairs, 0.1 mega basepairs, 50,000 basepairs, 20,000 basepairs, or 10,000 basepairs in length.
The present invention also encompasses polynucleotides capable of hybridizing, preferably under reduced stringency conditions, more preferably under stringent conditions, and most preferably under highly stringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in Table 2 below: highly stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.











TABLE 2








Hyridization
Wash


Stringency
Polynucleotide
Hybrid Length
Temperature
Temperature


Condition
Hybrid±
(bp)‡
and Buffer†
and Buffer†







A
DNA:DNA
> or equal to 50
65° C.; 1xSSC -or-
65° C.;





42° C.; 1xSSC,
0.3xSSC





50% formamide


B
DNA:DNA
<50
Tb*; 1xSSC
Tb*; 1xSSC


C
DNA:RNA
> or equal to 50
67° C.; 1xSSC -or-
67° C.;





45° C.; 1xSSC,
0.3xSSC





50% formamide


D
DNA:RNA
<50
Td*; 1xSSC
Td*; 1xSSC


E
RNA:RNA
> or equal to 50
70° C.; 1xSSC -or-
70° C.;





50° C.; 1xSSC,
0.3xSSC





50% formamide


F
RNA:RNA
<50
Tf*; 1xSSC
Tf*; 1xSSC


G
DNA:DNA
> or equal to 50
65° C.; 4xSSC -or-
65° C.; 1xSSC





45° C.; 4xSSC,





50% formamide


H
DNA:DNA
<50
Th*; 4xSSC
Th*; 4xSSC


I
DNA:RNA
> or equal to 50
67° C.; 4xSSC -or-
67° C.; 1xSSC





45° C.; 4xSSC,





50% formamide


J
DNA:RNA
<50
Tj*; 4xSSC
Tj*; 4xSSC


K
RNA:RNA
> or equal to 50
70° C.; 4xSSC -or-
67° C.; 1xSSC





40° C.; 6xSSC,





50% formamide


L
RNA:RNA
<50
Tl*; 2xSSC
Tl*; 2xSSC


M
DNA:DNA
> or equal to 50
50° C.; 4xSSC -or-
50° C.; 2xSSC





40° C. 6xSSC,





50% formamide


N
DNA:DNA
<50
Tn*; 6xSSC
Tn*; 6xSSC


O
DNA:RNA
> or equal to 50
55° C.; 4xSSC -or-
55° C.; 2xSSC





42° C.; 6xSSC,





50% formamide


P
DNA:RNA
<50
Tp*; 6xSSC
Tp*; 6xSSC


Q
RNA:RNA
> or equal to 50
60° C.; 4xSSC -or-
60° C.; 2xSSC





45° C.; 6xSSC,





50% formamide


R
RNA:RNA
<50
Tr*; 4xSSC
Tr*; 4xSSC





‡The “hybrid length” is the anticipated length for the hybridized region(s) of the hybridizing polynucleotides. When hybridizing a polynucletotide of unknown sequence, the hybrid is assumed to be that of the hybridizing polynucleotide of the present invention. When polynucleotides of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity. Methods of aligning two or more polynucleotide sequences and/or determining the percent identity between two polynucleotide sequences are well known in the art (e.g., MegAlign program of the DNA*Star suite of programs, etc).


†SSPE (1xSSPE is 0.15 M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1xSSC is 0.15 M NaCl anmd 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete. The hydridizations and washes may additionally include 5X Denhardt's reagent, .5-1.0% SDS, 100 ug/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, and up to 50% formamide.


*Tb − Tr: The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature Tm of the hybrids there Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(° C.) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49 base pairs in length, Tm(° C.) = 81.5 +16.6(log10[Na+]) + 0.41 (% G + C) − (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([NA+] for 1xSSC = .165 M).


±The present invention encompasses the substitution of any one, or more DNA or RNA hybrid partners with either a PNA, or a modified polynucleotide. Such modified polynucleotides are known in the art and are more particularly described elsewhere herein.






Additional examples of stringency conditions for polynucleotide hybridization are provided, for example, in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M., Ausubel et al., eds, John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4, which are hereby incorporated by reference herein.
Preferably, such hybridizing polynucleotides have at least 70% sequence identity (more preferably, at least 80% identity; and most preferably at least 90% or 95% identity) with the polynucleotide of the present invention to which they hybridize, where sequence identity is determined by comparing the sequences of the hybridizing polynucleotides when aligned so as to maximize overlap and identity while minimizing sequence gaps. The determination of identity is well known in the art, and discussed more specifically elsewhere herein.
The invention encompasses the application of PCR methodology to the polynucleotide sequences of the present invention, and/or the cDNA encoding the polypeptides of the present invention. PCR techniques for the amplification of nucleic acids are described in U.S. Pat. No. 4,683,195 and Saiki et al., Science, 239:487-491 (1988). PCR, for example, may include the following steps, of denaturation of template nucleic acid (if double-stranded), annealing of primer to target, and polymerization. The nucleic acid probed or used as a template in the amplification reaction may be genomic DNA, cDNA, RNA, or a PNA. PCR may be used to amplify specific sequences from genomic DNA, specific RNA sequence, and/or cDNA transcribed from mRNA. References for the general use of PCR techniques, including specific method parameters, include Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR Technology, Stockton Press, NY, 1989; Ehrlich et al., Science, 252:1643-1650, (1991); and “PCR Protocols, A Guide to Methods and Applications”, Eds., Innis et al., Academic Press, New York, (1990).
Polynucleotide and Polypeptide Variants
The present invention also encompasses variants (e.g., allelic variants, orthologs, etc.) of the polynucleotide sequence disclosed herein in SEQ ID NO:1, 3, 5, 8, 317, and/or 318, the complementary strand thereto, and/or the cDNA sequence contained in the deposited clone.
The present invention also encompasses variants of the polypeptide sequence, and/or fragments therein, disclosed in SEQ ID NO:2, 4, 6, 7, 9, and/or 10, a polypeptide encoded by the polynucleotide sequence in SEQ ID NO:1, 3, 5, 8, 317, and/or 318, and/or a polypeptide encoded by a cDNA in the deposited clone.
“Variant” refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.

    
    
        Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a LTRPC3 related polypeptide having an amino acid.- sequence as shown in the sequence listing and described in SEQ ID NO:1, 3, 5, 8, 317, and/or 318; (b) a nucleotide sequence encoding a mature LTRPC3 related polypeptide having the amino acid sequence as shown in the sequence listing and described in SEQ ID NO:1, 3, 5, 8, 317, and/or 318; (c) a nucleotide sequence encoding a biologically active fragment of a LTRPC3 related polypeptide having an amino acid sequence shown in the sequence listing and described in SEQ ID NO:1, 3, 5, 8, 317, and/or 318; (d) a nucleotide sequence encoding an antigenic fragment of a LTRPC3 related polypeptide having an amino acid sequence shown in the sequence listing and described in SEQ ID NO:1, 3, 5, 8, 317, and/or 318; (e) a nucleotide sequence encoding a LTRPC3 related polypeptide comprising the complete amino acid sequence encoded by a human cDNA plasmid containined in SEQ ID NO:1, 3, 5, 8, 317, and/or 318; (f) a nucleotide sequence encoding a mature LTRPC3 realted polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1, 3, 5, 8, 317, and/or 318; (g) a nucleotide sequence encoding a biologically active fragement of a LTRPC3 related polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1, 3, 5, 8, 317, and/or 318; (h) a nucleotide sequence encoding an antigenic fragment of a LTRPC3 related polypeptide having an amino acid sequence encoded by a human cDNA plasmid contained in SEQ ID NO:1, 3, 5, 8, 317, and/or 318; (I) a nucleotide sequence complimentary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above.
    
    


The present invention is also directed to polynucleotide sequences which comprise, or alternatively consist of, a polynucleotide sequence which is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides encoded by these nucleic acid molecules are also encompassed by the invention. In another embodiment, the invention encompasses nucleic acid molecule which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polypeptides.
Another aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively, consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a LTRPC3 related polypeptide having an amino acid sequence as shown in the sequence listing and described in Table 1; (b) a nucleotide sequence encoding a mature LTRPC3 related polypeptide having the amino acid sequence as shown in the sequence listing and described in Table 1; (c) a nucleotide sequence encoding a biologically active fragment of a LTRPC3 related polypeptide having an amino acid sequence as shown in the sequence listing and described in Table 1; (d) a nucleotide sequence encoding an antigenic fragment of a LTRPC3 related polypeptide having an amino acid sequence as shown in the sequence listing and described in Table 1; (e) a nucleotide sequence encoding a biologically active fragment of a LTRPC3 related polypeptide having an amino acid sequence encoded by a human cDNA (f) a nucleotide sequence encoding an antigenic fragment of a LTRPC3 related polypeptide.
The present invention is also directed to nucleic acid molecules which comprise, or alternatively, consist of, a nucleotide sequence which is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above.
The present invention encompasses polypeptide sequences which comprise, or alternatively consist of, an amino acid sequence which is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, the following non-limited examples, the polypeptide sequence identified as SEQ ID NO:2, 4, 6, 7, 9, and/or 10, the polypeptide sequence encoded by a cDNA provided in the deposited clone, and/or polypeptide fragments of any of the polypeptides provided herein. Polynucleotides encoded by these nucleic acid molecules are also encompassed by the invention. In another embodiment, the invention encompasses nucleic acid molecule which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polypeptides.
The present invention is also directed to polypeptides which comprise, or alternatively consist of, an amino acid sequence which is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for example, the polypeptide sequence shown in SEQ ID NO:2, 4, 6, 7, 9, and/or 10, a polypeptide sequence encoded by the nucleotide sequence in SEQ ID NO:1, 3, 5, 8, 317, and/or 318,and/or polypeptide fragments of any of these polypeptides (e.g., those fragments described herein). Polynucleotides which hybridize to the complement of the nucleic acid molecules encoding these polypeptides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompasses by the present invention, as are the polypeptides encoded by these polynucleotides.
By a nucleic acid having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence referenced in Table 1, the ORF (open reading frame), or any fragment specified as described herein.
As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the CLUSTALW computer program (Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based on the algorithm of Higgins, D. G., et al., Computer Applications in the Biosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. However, the CLUSTALW algorithm automatically converts U's to T's when comparing RNA sequences to DNA sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a CLUSTALW alignment of DNA sequences to calculate percent identity via pairwise alignments are: Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent, Window Size=5 or the length of the subject nucleotide sequence, whichever is shorter. For multiple alignments, the following CLUSTALW parameters are preferred: Gap Opening Penalty=10; Gap Extension Parameter=0.05; Gap Separation Penalty Range=8; End Gap Separation Penalty=Off; % Identity for Alignment Delay=40%; Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. The pairwise and multiple alignment parameters provided for CLUSTALW above represent the default parameters as provided with the AlignX software program (Vector NTI suite of programs, version 6.0).
The present invention encompasses the application of a manual correction to the percent identity results, in the instance where the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions. If only the local pairwise percent identity is required, no manual correction is needed. However, a manual correction may be applied to determine the global percent identity from a global polynucleotide alignment. Percent identity calculations based upon global polynucleotide alignments are often preferred since they reflect the percent identity between the polynucleotide molecules as a whole (i.e., including any polynucleotide overhangs, not just overlapping regions), as opposed to, only local matching polynucleotides. Manual corrections for global percent identity determinations are required since the CLUSTALW program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the CLUSTALW sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above CLUSTALW program using the specified parameters, to arrive at a final percent identity score. This corrected score may be used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALW alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the CLUSTALW alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the CLUSTALW program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by CLUSTALW is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are required for the purposes of the present invention.
By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino- or carboxy-terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to, for instance, an amino acid sequence referenced in Table 1 (SEQ ID NO:2), can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the CLUSTALW computer program (Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based on the algorithm of Higgins, D. G., et al., Computer Applications in the Biosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment the query and subject sequences are both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a CLUSTALW alignment of DNA sequences to calculate percent identity via pairwise alignments are: Matrix=BLOSUM, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, Gap Open Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent, Window Size=5 or the length of the subject nucleotide sequence, whichever is shorter. For multiple alignments, the following CLUSTALW parameters are preferred: Gap Opening Penalty=10; Gap Extension Parameter=0.05; Gap Separation Penalty Range=8; End Gap Separation Penalty=Off; % Identity for Alignment Delay=40%; Residue Specific Gaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. The pairwise and multiple alignment parameters provided for CLUSTALW above represent the default parameters as provided with the AlignX software program (Vector NTI suite of programs, version 6.0).
The present invention encompasses the application of a manual correction to the percent identity results, in the instance where the subject sequence is shorter than the query sequence because of N- or C-terminal deletions, not because of internal deletions. If only the local pairwise percent identity is required, no manual correction is needed. However, a manual correction may be applied to determine the global percent identity from a global polypeptide alignment. Percent identity calculations based upon global polypeptide alignments are often preferred since they reflect the percent identity between the polypeptide molecules as a whole (i.e., including any polypeptide overhangs, not just overlapping regions), as opposed to, only local matching polypeptides. Manual corrections for global percent identity determinations are required since the CLUSTALW program does not account for N- and C-terminal truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the CLUSTALW sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above CLUSTALW program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what may be used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.
For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the CLUSTALW alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the CLUSTALW program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence, which are not matched/aligned with the query. In this case the percent identity calculated by CLUSTALW is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the CLUSTALW alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are required for the purposes of the present invention.
In addition to the above method of aligning two or more polynucleotide or polypeptide sequences to arrive at a percent identity value for the aligned sequences, it may be desirable in some circumstances to use a modified version of the CLUSTALW algorithm which takes into account known structural features of the sequences to be aligned, such as for example, the SWISS-PROT designations for each sequence. The result of such a modified CLUSTALW algorithm may provide a more accurate value of the percent identity for two polynucleotide or polypeptide sequences. Support for such a modified version of CLUSTALW is provided within the CLUSTALW algorithm and would be readily appreciated to one of skill in the art of bioinformatics.
The variants may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the mRNA to those preferred by a bacterial host such as E. coli).
Naturally occurring variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).) These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the protein without substantial loss of biological function. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biotechnology 7:199-216 (1988)).
Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that “[m]ost of the molecule could be altered with little effect on either [binding or biological activity].” In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.
Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the protein will likely be retained when less than the majority of the residues of the protein are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.
Alternatively, such N-terminus or C-terminus deletions of a polypeptide of the present invention may, in fact, result in a significant increase in one or more of the biological activities of the polypeptide(s). For example, biological activity of many polypeptides are governed by the presence of regulatory domains at either one or both termini. Such regulatory domains effectively inhibit the biological activity of such polypeptides in lieu of an activation event (e.g., binding to a cognate ligand or receptor, phosphorylation, proteolytic processing, etc.). Thus, by eliminating the regulatory domain of a polypeptide, the polypeptide may effectively be rendered biologically active in the absence of an activation event.
The invention further includes polypeptide variants that show substantial biological activity. Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity. For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can then be tested for biological activity.
As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved.
The invention encompasses polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by the polypeptide of the present invention. Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics (e.g., chemical properties). According to Cunningham et al above, such conservative substitutions are likely to be phenotypically silent. Additional guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).
Tolerated conservative amino acid substitutions of the present invention involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
In addition, the present invention also encompasses the conservative substitutions provided in Table III below.









TABLE III





For Amino Acid
Code
Replace with any of:







Alanine
A
D-Ala, Gly, beta-Ala, L-Cys, D-Cys


Arginine
R
D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-




Met, D-Ile, Orn, D-Orn


Asparagine
N
D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln


Aspartic Acid
D
D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln


Cysteine
C
D-Cys, S—Me-Cys, Met, D-Met, Thr, D-Thr


Glutamine
Q
D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp


Glutamic Acid
E
D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln


Glycine
G
Ala, D-Ala, Pro, D-Pro, β-Ala, Acp


Isoleucine
I
D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met


Leucine
L
D-Leu, Val, D-Val, Met, D-Met


Lysine
K
D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met,




Ile, D-Ile, Orn, D-Orn


Methionine
M
D-Met, S—Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val


Phenylalanine
F
D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-




3,4, or 5-phenylproline, cis-3,4, or 5-phenylproline


Proline
P
D-Pro, L-1-thioazolidine-4-carboxylic acid, D- or L-1-




oxazolidine-4-carboxylic acid


Serine
S
D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(O),




L-Cys, D-Cys


Threonine
T
D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O),




Val, D-Val


Tyrosine
Y
D-Tyr, Phe, D-Phe, L-Dopa, His, D-His


Valine
V
D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met









Aside from the uses described above, such amino acid substitutions may also increase protein or peptide stability. The invention encompasses amino acid substitutions that contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the protein or peptide sequence. Also included are substitutions that include amino acid residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., β or γ amino acids.
Both identity and similarity can be readily calculated by reference to the following publications: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Informatics Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.
In addition, the present invention also encompasses substitution of amino acids based upon the probability of an amino acid substitution resulting in conservation of function. Such probabilities are determined by aligning multiple genes with related function and assessing the relative penalty of each substitution to proper gene function. Such probabilities are often described in a matrix and are used by some algorithms (e.g., BLAST, CLUSTALW, GAP, etc.) in calculating percent similarity wherein similarity refers to the degree by which one amino acid may substitute for another amino acid without lose of function. An example of such a matrix is the PAM250 or BLOSUM62 matrix.
Aside from the canonical chemically conservative substitutions referenced above, the invention also encompasses substitutions which are typically not classified as conservative, but that may be chemically conservative under certain circumstances. Analysis of enzymatic catalysis for proteases, for example, has shown that certain amino acids within the active site of some enzymes may have highly perturbed pKa's due to the unique microenvironment of the active site. Such perturbed pKa's could enable some amino acids to substitute for other amino acids while conserving enzymatic structure and function. Examples of amino acids that are known to have amino acids with perturbed pKa's are the Glu-35 residue of Lysozyme, the Ile-16 residue of Chymotrypsin, the His-159 residue of Papain, etc. The conservation of function relates to either anomalous protonation or anomalous deprotonation of such amino acids, relative to their canonical, non-perturbed pKa. The pKa perturbation may enable these amino acids to actively participate in general acid-base catalysis due to the unique ionization environment within the enzyme active site. Thus, substituting an amino acid capable of serving as either a general acid or general base within the microenvironment of an enzyme active site or cavity, as may be the case, in the same or similar capacity as the wild-type amino acid, would effectively serve as a conservative amino substitution.
Besides conservative amino acid substitution, variants of the present invention include, but are not limited to, the following: (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification. Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.
For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).)
A further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of the present invention having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions. Of course, in order of ever-increasing preference, it is highly preferable for a peptide or polypeptide to have an amino acid sequence which comprises the amino acid sequence of the present invention, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions. In specific embodiments, the number of additions, substitutions, and/or deletions in the amino acid sequence of the present invention or fragments thereof (e.g., the mature form and/or other fragments described herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions are preferable.
Polynucleotide and Polypeptide Fragments
The present invention is directed to polynucleotide fragments of the polynucleotides of the invention, in addition to polypeptides encoded therein by said polynucleotides and/or fragments.
In the present invention, a “polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence which: is a portion of that contained in a deposited clone, or encoding the polypeptide encoded by the cDNA in a deposited clone; is a portion of that shown in SEQ ID NO:1, 3, 5, 8, 317, and/or 318 or the complementary strand thereto, or is a portion of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2, 4, 6, 7, 9, and/or 10. The nucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length. A fragment “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in a deposited clone or the nucleotide sequence shown in SEQ ID NO:1, 3, 5, 8, 317, and/or 318. In this context “about” includes the particularly recited value, a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus, or at both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600, 2000 nucleotides) are preferred.
Moreover, representative examples of polynucleotide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:1, 3, 5, 8, 317, and/or 318. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has biological activity. More preferably, these polynucleotides can be used as probes or primers as discussed herein. Also encompassed by the present invention are polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions, as are the polypeptides encoded by these polynucleotides.
In the present invention, a “polypeptide fragment” refers to an amino acid sequence which is a portion of that contained in SEQ ID NO:2, 4, 6, 7, 9, and/or 10. Protein (polypeptide) fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context “about” includes the particularly recited ranges or values, and ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptides are also encompassed by the invention.
Preferred polypeptide fragments include the full-length protein. Further preferred polypeptide fragments include the full-length protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of the full-length polypeptide. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the full-length protein. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.
Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Polypeptide fragments of SEQ ID NO:2, 4, 6, 7, 9, and/or 10 falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotides encoding these domains are also contemplated.
Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
In a preferred embodiment, the functional activity displayed by a polypeptide encoded by a polynucleotide fragment of the invention may be one or more biological activities typically associated with the full-length polypeptide of the invention. Illustrative of these biological activities includes the fragments ability to bind to at least one of the same antibodies which bind to the full-length protein, the fragments ability to interact with at least one of the same proteins which bind to the full-length, the fragments ability to elicit at least one of the same immune responses as the full-length protein (i.e., to cause the immune system to create antibodies specific to the same epitope, etc.), the fragments ability to bind to at least one of the same polynucleotides as the full-length protein, the fragments ability to bind to a receptor of the full-length protein, the fragments ability to bind to a ligand of the full-length protein, and the fragments ability to multimerize with the full-length protein. However, the skilled artisan would appreciate that some fragments may have biological activities which are desirable and directly inapposite to the biological activity of the full-length protein. The functional activity of polypeptides of the invention, including fragments, variants, derivatives, and analogs thereof can be determined by numerous methods available to the skilled artisan, some of which are described elsewhere herein.
The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:2, 4, 6, 7, 9, and/or 10 or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO:1, 3, 5, 8, 317, and/or 318 under stringent hybridization conditions or lower stringency hybridization conditions as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:1), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
The term “epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Pat. No. 4,631,211).
In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length, or longer. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μg of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences. For example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides. Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion disulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (“HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:1, 3, 5, 8, 317, and/or 318 and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
Fusion Proteins
Any polypeptide of the present invention can be used to generate fusion proteins. For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide. Moreover, because certain proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.
Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.
Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. Similarly, peptide cleavage sites can be introduced in-between such peptide moieties, which could additionally be subjected to protease activity to remove said peptide(s) from the protein of the present invention. The addition of peptide moieties, including peptide cleavage sites, to facilitate handling of polypeptides are familiar and routine techniques in the art.
Moreover, polypeptides of the present invention, including fragments, and specifically epitopes, can be combined with parts of the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86 (1988).) Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)
Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of the constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).)
Moreover, the polypeptides of the present invention can be fused to marker sequences (also referred to as “tags”). Due to the availability of antibodies specific to such “tags”, purification of the fused polypeptide of the invention, and/or its identification is significantly facilitated since antibodies specific to the polypeptides of the invention are not required. Such purification may be in the form of an affinity purification whereby an anti-tag antibody or another type of affinity matrix (e.g., anti-tag antibody attached to the matrix of a flow-thru column) that binds to the epitope tag is present. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767 (1984)).
The skilled artisan would acknowledge the existence of other “tags” which could be readily substituted for the tags referred to supra for purification and/or identification of polypeptides of the present invention (Jones C., et al., J Chromatogr A. 707(1):3-22 (1995)). For example, the c-myc tag and the 8F9, 3 C7, 6E10, G4m B7 and 9E10 antibodies thereto (Evan et al., Molecular and Cellular Biology 5:3610-3616 (1985)); the Herpes Simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering, 3(6):547-553 (1990), the Flag-peptide—i.e., the octapeptide sequence DYKDDDDK (SEQ ID NO:251), (Hopp et al., Biotech. 6:1204-1210 (1988); the KT3 epitope peptide (Martin et al., Science, 255:192-194 (1992)); a-tubulin epitope peptide (Skinner et al., J. Biol. Chem., 266:15136-15166, (1991)); the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Sci. USA, 87:6363-6397 (1990)), the FITC epitope (Zymed, Inc.), the GFP epitope (Zymed, Inc.), and the Rhodamine epitope (Zymed, Inc.).
The present invention also encompasses the attachment of up to nine codons encoding a repeating series of up to nine arginine amino acids to the coding region of a polynucleotide of the present invention. The invention also encompasses chemically derivitizing a polypeptide of the present invention with a repeating series of up to nine arginine amino acids. Such a tag, when attached to a polypeptide, has recently been shown to serve as a universal pass, allowing compounds access to the interior of cells without additional derivitization or manipulation (Wender, P., et al., unpublished data).
Protein fusions involving polypeptides of the present invention, including fragments and/or variants thereof, can be used for the following, non-limiting examples, subcellular localization of proteins, determination of protein-protein interactions via immunoprecipitation, purification of proteins via affinity chromatography, functional and/or structural characterization of protein. The present invention also encompasses the application of hapten specific antibodies for any of the uses referenced above for epitope fusion proteins. For example, the polypeptides of the present invention could be chemically derivatized to attach hapten molecules (e.g., DNP, (Zymed, Inc.)). Due to the availability of monoclonal antibodies specific to such haptens, the protein could be readily purified using immunoprecipation, for example.
Polypeptides of the present invention, including fragments and/or variants thereof, in addition to, antibodies directed against such polypeptides, fragments, and/or variants, may be fused to any of a number of known, and yet to be determined, toxins, such as ricin, saporin (Mashiba H, et al., Ann. N.Y. Acad. Sci. 1999; 886:233-5), or HC toxin (Tonukari N J, et al., Plant Cell. 2000 February; 12(2):237-248), for example. Such fusions could be used to deliver the toxins to desired tissues for which a ligand or a protein capable of binding to the polypeptides of the invention exists.
The invention encompasses the fusion of antibodies directed against polypeptides of the present invention, including variants and fragments thereof, to said toxins for delivering the toxin to specific locations in a cell, to specific tissues, and/or to specific species. Such bifunctional antibodies are known in the art, though a review describing additional advantageous fusions, including citations for methods of production, can be found in P. J. Hudson, Curr. Opp. In. Imm. 11:548-557, (1999); this publication, in addition to the references cited therein, are hereby incorporated by reference in their entirety herein. In this context, the term “toxin” may be expanded to include any heterologous protein, a small molecule, radionucleotides, cytotoxic drugs, liposomes, adhesion molecules, glycoproteins, ligands, cell or tissue-specific ligands, enzymes, of bioactive agents, biological response modifiers, anti-fungal agents, hormones, steroids, vitamins, peptides, peptide analogs, anti-allergenic agents, anti-tubercular agents, anti-viral agents, antibiotics, anti-protozoan agents, chelates, radioactive particles, radioactive ions, X-ray contrast agents, monoclonal antibodies, polyclonal antibodies and genetic material. In view of the present disclosure, one skilled in the art could determine whether any particular “toxin” could be used in the compounds of the present invention. Examples of suitable “toxins” listed above are exemplary only and are not intended to limit the “toxins” that may be used in the present invention.
Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.
Vectors, Host Cells, and Protein Production
The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlsbad, Calif.). Other suitable vectors will be readily apparent to the skilled artisan.
Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.
A polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
Polypeptides of the present invention, and preferably the secreted form, can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
In one embodiment, the yeast Pichia pastoris is used to express the polypeptide of the present invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O2. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O2. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See, Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.
In one example, the plasmid vector pPIC9K is used to express DNA encoding a polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in “Pichia Protocols: Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. This expression vector allows expression and secretion of a protein of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.
Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG, as required.
In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.
In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination, resulting in the formation of a new transcription unit (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No. 5,733,761, issued Mar. 31, 1998; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).
In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide sequence of the invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).
The invention encompasses polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein, the addition of epitope tagged peptide fragments (e.g., FLAG, HA, GST, thioredoxin, maltose binding protein, etc.), attachment of affinity tags such as biotin and/or streptavidin, the covalent attachment of chemical moieties to the amino acid backbone, N- or C-terminal processing of the polypeptides ends (e.g., proteolytic processing), deletion of the N-terminal methionine residue, etc.
Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
The invention further encompasses chemical derivitization of the polypeptides of the present invention, preferably where the chemical is a hydrophilic polymer residue. Exemplary hydrophilic polymers, including derivatives, may be those that include polymers in which the repeating units contain one or more hydroxy groups (polyhydroxy polymers), including, for example, poly(vinyl alcohol); polymers in which the repeating units contain one or more amino groups (polyamine polymers), including, for example, peptides, polypeptides, proteins and lipoproteins, such as albumin and natural lipoproteins; polymers in which the repeating units contain one or more carboxy groups (polycarboxy polymers), including, for example, carboxymethylcellulose, alginic acid and salts thereof, such as sodium and calcium alginate, glycosaminoglycans and salts thereof, including salts of hyaluronic acid, phosphorylated and sulfonated derivatives of carbohydrates, genetic material, such as interleukin-2 and interferon, and phosphorothioate oligomers; and polymers in which the repeating units contain one or more saccharide moieties (polysaccharide polymers), including, for example, carbohydrates.
The molecular weight of the hydrophilic polymers may vary, and is generally about 50 to about 5,000,000, with polymers having a molecular weight of about 100 to about 50,000 being preferred. The polymers may be branched or unbranched. More preferred polymers have a molecular weight of about 150 to about 10,000, with molecular weights of 200 to about 8,000 being even more preferred.
For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
Additional preferred polymers which may be used to derivatize polypeptides of the invention, include, for example, poly(ethylene glycol) (PEG), poly(vinylpyrrolidine), polyoxomers, polysorbate and poly(vinyl alcohol), with PEG polymers being particularly preferred. Preferred among the PEG polymers are PEG polymers having a molecular weight of from about 100 to about 10,000. More preferably, the PEG polymers have a molecular weight of from about 200 to about 8,000, with PEG 2,000, PEG 5,000 and PEG 8,000, which have molecular weights of 2,000, 5,000 and 8,000, respectively, being even more preferred. Other suitable hydrophilic polymers, in addition to those exemplified above, will be readily apparent to one skilled in the art based on the present disclosure. Generally, the polymers used may include polymers that can be attached to the polypeptides of the invention via alkylation or acylation reactions.
The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminus) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
As with the various polymers exemplified above, it is contemplated that the polymeric residues may contain functional groups in addition, for example, to those typically involved in linking the polymeric residues to the polypeptides of the present invention. Such functionalities include, for example, carboxyl, amine, hydroxy and thiol groups. These functional groups on the polymeric residues can be further reacted, if desired, with materials that are generally reactive with such functional groups and which can assist in targeting specific tissues in the body including, for example, diseased tissue. Exemplary materials which can be reacted with the additional functional groups include, for example, proteins, including antibodies, carbohydrates, peptides, glycopeptides, glycolipids, lectins, and nucleosides.
In addition to residues of hydrophilic polymers, the chemical used to derivatize the polypeptides of the present invention can be a saccharide residue. Exemplary saccharides which can be derived include, for example, monosaccharides or sugar alcohols, such as erythrose, threose, ribose, arabinose, xylose, lyxose, fructose, sorbitol, mannitol and sedoheptulose, with preferred monosaccharides being fructose, mannose, xylose, arabinose, mannitol and sorbitol; and disaccharides, such as lactose, sucrose, maltose and cellobiose. Other saccharides include, for example, inositol and ganglioside head groups. Other suitable saccharides, in addition to those exemplified above, will be readily apparent to one skilled in the art based on the present disclosure. Generally, saccharides which may be used for derivitization include saccharides that can be attached to the polypeptides of the invention via alkylation or acylation reactions.
Moreover, the invention also encompasses derivitization of the polypeptides of the present invention, for example, with lipids (including cationic, anionic, polymerized, charged, synthetic, saturated, unsaturated, and any combination of the above, etc.). stabilizing agents.
The invention encompasses derivitization of the polypeptides of the present invention, for example, with compounds that may serve a stabilizing function (e.g., to increase the polypeptides half-life in solution, to make the polypeptides more water soluble, to increase the polypeptides hydrophilic or hydrophobic character, etc.). Polymers useful as stabilizing materials may be of natural, semi-synthetic (modified natural) or synthetic origin. Exemplary natural polymers include naturally occurring polysaccharides, such as, for example, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans (such as, for example, inulin), levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectins, including amylose, pullulan, glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, glucose, polyglucose, polydextrose, pustulan, chitin, agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum, starch and various other natural homopolymer or heteropolymers, such as those containing one or more of the following aldoses, ketoses, acids or amines: erythose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid, and naturally occurring derivatives thereof. Accordingly, suitable polymers include, for example, proteins, such as albumin, polyalginates, and polylactide-coglycolide polymers. Exemplary semi-synthetic polymers include carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose. Exemplary synthetic polymers include polyphosphazenes, hydroxyapatites, fluoroapatite polymers, polyethylenes (such as, for example, polyethylene glycol (including for example, the class of compounds referred to as Pluronics®, commercially available from BASF, Parsippany, N.J.), polyoxyethylene, and polyethylene terephthlate), polypropylenes (such as, for example, polypropylene glycol), polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinyl chloride and polyvinylpyrrolidone), polyamides including nylon, polystyrene, polylactic acids, fluorinated hydrocarbon polymers, fluorinated carbon polymers (such as, for example, polytetrafluoroethylene), acrylate, methacrylate, and polymethylmethacrylate, and derivatives thereof. Methods for the preparation of derivatized polypeptides of the invention which employ polymers as stabilizing compounds will be readily apparent to one skilled in the art, in view of the present disclosure, when coupled with information known in the art, such as that described and referred to in Unger, U.S. Pat. No. 5,205,290, the disclosure of which is hereby incorporated by reference herein in its entirety.
Moreover, the invention encompasses additional modifications of the polypeptides of the present invention. Such additional modifications are known in the art, and are specifically provided, in addition to methods of derivitization, etc., in U.S. Pat. No. 6,028,066, which is hereby incorporated in its entirety herein.
The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.
Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer, refers to a multimer containing only polypeptides corresponding to the amino acid sequence of SEQ ID NO:2, 4, 6, 7, 9, and/or 10 or encoded by the cDNA contained in a deposited clone (including fragments, variants, splice variants, and fusion proteins, corresponding to these polypeptides as described herein). These homomers may contain polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in the sequence listing, or contained in the polypeptide encoded by a deposited clone). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein of the invention.
In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in an Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, osteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.
Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.
Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference. Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention.
In another example, proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide sequence. In a further embodiment, associations proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti-Flag® antibody.
The multimers of the invention may be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).
Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hydrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).
In addition, the polynucleotide insert of the present invention could be operatively linked to “artificial” or chimeric promoters and transcription factors. Specifically, the artificial promoter could comprise, or alternatively consist, of any combination of cis-acting DNA sequence elements that are recognized by trans-acting transcription factors. Preferably, the cis acting DNA sequence elements and trans-acting transcription factors are operable in mammals. Further, the trans-acting transcription factors of such “artificial” promoters could also be “artificial” or chimeric in design themselves and could act as activators or repressors to said “artificial” promoter.
Uses of the Polynucleotides
Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.
The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each polynucleotide of the present invention can be used as a chromosome marker.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the sequences shown in SEQ ID NO:1, 3, 5, 8, 317, and/or 318. Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the SEQ ID NO:1, 3, 5, 8, 317, and/or 318 will yield an amplified fragment.
Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries.
Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. For a review of this technique, see Verma et al., “Human Chromosomes: a Manual of Basic Techniques,” Pergamon Press, New York (1988).
For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes). Preferred polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross hybridization during chromosomal mapping.
Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. Disease mapping data are known in the art. Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.
Thus, once coinheritance is established, differences in the polynucleotide and the corresponding gene between affected and unaffected organisms can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected organisms, but not in normal organisms, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene from several normal organisms is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.
Furthermore, increased or decreased expression of the gene in affected organisms as compared to unaffected organisms can be assessed using polynucleotides of the present invention. Any of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker.
Thus, the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an organism and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder.
By “measuring the expression level of a polynucleotide of the present invention” is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the present invention or the level of the mRNA encoding the polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of organisms not having a disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
By “biological sample” is intended any biological sample obtained from an organism, body fluids, cell line, tissue culture, or other source which contains the polypeptide of the present invention or mRNA. As indicated, biological samples include body fluids (such as the following non-limiting examples, sputum, amniotic fluid, urine, saliva, breast milk, secretions, interstitial fluid, blood, serum, spinal fluid, etc.) which contain the polypeptide of the present invention, and other tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from organisms are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
The method(s) provided above may Preferably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides are attached to a solid support. In one exemplary method, the support may be a “gene chip” or a “biological chip” as described in U.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip with polynucleotides of the present invention attached may be used to identify polymorphisms between the polynucleotide sequences, with polynucleotides isolated from a test subject. The knowledge of such polymorphisms (i.e. their location, as well as, their existence) would be beneficial in identifying disease loci for many disorders, including proliferative diseases and conditions. Such a method is described in U.S. Pat. Nos. 5,858,659 and 5,856,104. The US patents referenced supra are hereby incorporated by reference in their entirety herein.
The present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art. The use of PNAs would serve as the preferred form if the polynucleotides are incorporated onto a solid support, or gene chip. For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M. Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M. Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the stronger binding characteristics of PNA:DNA hybrids. In addition, it is more likely that single base mismatches can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by 8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.
In addition to the foregoing, a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991). Both methods rely on binding of the polynucleotide to a complementary DNA or RNA. For these techniques, preferred polynucleotides are usually oligonucleotides 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat or prevent disease.
The present invention encompasses the addition of a nuclear localization signal, operably linked to the 5′ end, 3′ end, or any location therein, to any of the oligonucleotides, antisense oligonucleotides, triple helix oligonucleotides, ribozymes, PNA oligonucleotides, and/or polynucleotides, of the present invention. See, for example, G. Cutrona, et al., Nat. Biotech., 18:300-303, (2000); which is hereby incorporated herein by reference.
Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. In one example, polynucleotide sequences of the present invention may be used to construct chimeric RNA/DNA oligonucleotides corresponding to said sequences, specifically designed to induce host cell mismatch repair mechanisms in an organism upon systemic injection, for example (Bartlett, R. J., et al., Nat. Biotech, 18:615-622 (2000), which is hereby incorporated by reference herein in its entirety). Such RNA/DNA oligonucleotides could be designed to correct genetic defects in certain host strains, and/or to introduce desired phenotypes in the host (e.g., introduction of a specific polymorphism within an endogenous gene corresponding to a polynucleotide of the present invention that may ameliorate and/or prevent a disease symptom and/or disorder, etc.). Alternatively, the polynucleotide sequence of the present invention may be used to construct duplex oligonucleotides corresponding to said sequence, specifically designed to correct genetic defects in certain host strains, and/or to introduce desired phenotypes into the host (e.g., introduction of a specific polymorphism within an endogenous gene corresponding to a polynucleotide of the present invention that may ameliorate and/or prevent a disease symptom and/or disorder, etc). Such methods of using duplex oligonucleotides are known in the art and are encompassed by the present invention (see EP1007712, which is hereby incorporated by reference herein in its entirety).
The polynucleotides are also useful for identifying organisms from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel. This method does not suffer from the current limitations of “Dog Tags” which can be lost, switched, or stolen, making positive identification difficult. The polynucleotides of the present invention can be used as additional DNA markers for RFLP.
The polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an organisms genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, organisms can be identified because each organism will have a unique set of DNA sequences. Once an unique ID database is established for an organism, positive identification of that organism, living or dead, can be made from extremely small tissue samples. Similarly, polynucleotides of the present invention can be used as polymorphic markers, in addition to, the identification of transformed or non-transformed cells and/or tissues.
There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA probes or primers specific to particular tissue prepared from the sequences of the present invention. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination. Moreover, as mentioned above, such reagents can be used to screen and/or identify transformed and non-transformed cells and/or tissues.
In the very least, the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to “subtract-out” known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a “gene chip” or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.
Uses of the Polypeptides
Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.
A polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques. For example, protein expression in tissues can be studied with classical immunohistological methods. (Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987).) Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
In addition to assaying protein levels in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, 131I, 112In, 99mTc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously, or intraperitoneally) into the mammal. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)
Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.
Moreover, polypeptides of the present invention can be used to treat, prevent, and/or diagnose disease. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor suppressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).
Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat, prevent, and/or diagnose disease. For example, administration of an antibody directed to a polypeptide of the present invention can bind and reduce overproduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).
At the very least, the polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the following biological activities.
Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.
REFERENCES


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EXAMPLES
Description of the Preferred Embodiments
Example 1
Method Used to Identify the Novel LTRPC3 Polynucleotide of the Present Invention—Bioinformatics Analysis
Ion channel sequences (mouse TRP1a, gi|1911245, SEQ ID NO:27; mouse TRP2, gi|4324938, SEQ ID NO:26; mouse TRP4, gi|4200415, SEQ ID NO:25; mouse TRP5, gi|6048344, SEQ ID NO:24; human TRP7, gi|13928756, SEQ ID NO:23; mouse TRP3, gi|6014703, SEQ ID NO:22; mouse TRP6, gi|2979524, SEQ ID NO:21; mouse TRP8, gi|5326854, SEQ ID NO:20; Drosophila NOMPC, gi|7328583, SEQ ID NO:19; C. elegans Y71A12B.4, gi|1065673, SEQ ID NO:18) in the TRP/NOMPC family were used as probes to search the human genomic sequence database (see FIGS. 13A-C). The search program used was the gapped BLAST program TBLASTN (Altschul et al., 1997). A multiple sequence alignment of TRP/NOMPC family members was generated using the Align program in software Vector NTI 5.5, using the ClustalW algorithm. A Hidden Markov Model (HMM) specific for NOMPC family was constructed using the HMMERBUILD program in the Genewise/Wise2 package from the above multiple sequence alignment (Bateman et al., 2000). This HMM model was then used to search the human genomic sequence database using the software program GENEWISEDB in the Genewise/Wise2 package (http://www.sanger.ac.uk/Software/Wise2/index.shtml). Results from the TBLASTN and GENEWISEDB searches were pooled and a potential TRP family member was identified in human BAC AL358786 (Genbank Accession No. gi|AL358786). The high scoring hit segments from the genomic sequence hits from BAC AL358786 were extracted and searched back against non-redundant protein and patent sequence databases. The most similar protein sequence for each genomic sequence hit was used as a template to predict putative exons from the BAC AL358786 genomic sequence using the GENEWISEDB program in the Genewise/Wise2 package (trpc7; SEQ ID NO:23). From this analysis, exons encoding the potential novel ion channel, LTRPC3, was identified based upon sequence homology. To extend the 5′ and 3′ sequences of putative novel ion channel molecules, the genomic regions surrounding the matching exons in genomic sequence NT—008306 (SEQ ID NO:17) using GENSCAN and FGENESH programs to generate de novo exons. Based on these analysis, partial sequences of novel human ion channel related genes were identified (shown in FIG. 10; SEQ ID NO:12, 13, and 14). The full-length clone of one novel ion channel gene LTRPC3 was experimentally obtained by using the putative exon sequences as templates to design PCR cloning primers as described below.
Example 2
Cloning the Novel Transient Receptor Potential Channel Member, LTRPC3
Probe Design
Using the predicted exon genomic sequence from BAC AL358786 (FIG. 10; SEQ ID NO:12, 13, and 14), oligonucleotides with the following sequences was used to amplify fragments from the human kidney Marathon-Ready cDNA library (Clontech). The reaction mixture in 50 ul containing 5 ul cDNA library, 0.5 mM each primer, 5 mM dNTPs (1.25 mM each), 5 ul of 10×PCR and 0.5 unit of TaqPlus Precision polymerase (Stratagene). The reaction was repeated for 30 cycles (94° C. for 45 seconds, 55° C. for 45 seconds, and 72° C. for 4 minutes). The amplified fragments were cloned into the sequencing vector pCR4 Blunt-TOPO (Invitrogen) for sequence analysis. For functional studies the cDNA was fused in-frame with an HA epitope at its C terminus and subcloned into the mammalian expression vector pcDNA3.1/Hygro (Invitrogen).











Primer Name
Primer Sequence


















86-N2-C
ATGTATGTGCGAGTATCTTTTGATACAAAACCT
(SEQ ID NO:253)



 


86-1-NC
AGCCAAATCAATGTCCTGGTGTCC
(SEQ ID NO:254)


 


86-1-C
GAAGGACACCAGGACATTGATTTG
(SEQ ID NO:255)


 


86-2-NC
GTCACTCCTGAAGGGCTGGTCTTG
(SEQ ID NO:256)


 


86-2-C
CAAGACCAGCCCTTCAGGAGTGAC
(SEQ ID NO:257)


 


86-3-NC
CGCCCGATAAGGTCTTCCAGCTG
(SEQ ID NO:258)


 


86-3-C
CAGCTGGAAGACCTTATCGGGCG
(SEQ ID NO:259)


 


86-END-NC
TTAGGTGTGCTTGCTTTCAAAGCT
(SEQ ID NO:260)









The resulting full-length encoding polynucleotide sequence for LTRPC3 is shown in FIGS. 1A-F (SEQ ID NO:1).
Additional clones corresponding to the LTRPC3 splice variants of the present invention were isolated according to the above methods. The full-length polynucleotide sequence of each of the LTRPC3 splice variants are provided in FIGS. 2A-F (SEQ ID NO:3), FIGS. 3A-F (SEQ ID NO:5), FIGS. 4A-F (SEQ ID NO:8), for LTRPC3b, LTRPC3c, and LTRPC3e, respectively. Additional splice variants of LTRPC3 were also isolated corresponding to LTRPC3d and LTRPC3f. The amino acid sequences of these splice variants are provided in FIGS. 9A-E (SEQ ID NO:7 and 10, respectively), while the polynucleotide sequences of these splice variants are provided in FIG. 14 (LTRPC3d, SEQ ID NO:281) and FIG. 15 (LTRPC3f, SEQ ID NO:282).
Example 3
Expression Profiling of Novel Human Immunoglobulin Protein, LTRPC3
RT-PCR
A PCR primer pair was designed to measure the steady state levels of the LTRPC3 mRNA by quantitative RT-PCR.










LTRP6.tp1s
CGCAGCTGGAAGACCTTATC
(SEQ ID NO:261)



 


LTRP6.tp1a
AAGCTGCTCTGACGGACAAT
(SEQ ID NO:262)






Briefly, first strand cDNA was made from commercially available mRNA. The relative amount of cDNA used in each assay was determined by performing a parallel experiment using a primer pair for a gene expressed in equal amounts in all tissues, cyclophilin. The cyclophilin primer pair detected small variations in the amount of cDNA in each sample and these data were used for normalization of the data obtained with the primer pair for the LTRPC3 transcript. The PCR data was converted into a relative assessment of the difference in transcript abundance amongst the tissues tested and the data is presented in FIG. 8. Transcripts corresponding to the LTRPC3 transcript were found to be highly expressed in the kidney; and significantly in spinal cord, testis, and brain.
Northern Blot Analysis
Human tissue Northern blots (Clontech) were probed with an RNA probe derived from a 645-bp DNA fragment amplified from the primer pair 86-1-C (SEQ ID NO:255) and 86-5-NC (5′-AGGGAAGGGGAAGTGGTTGATCTC-3′, SEQ ID NO:263). Hybridization of the blot was performed at 68° C. in ExpressHyb (Clontech) for 6 hours, with 1×106 cpm/ml of 32P-labeled probe. Autoradiography was performed for 1 week at −70° C.
The results of the Northern hybridization are shown in FIG. 9. As shown, Transcripts corresponding to the LTRPC3 transcript were found to be highly expressed in kidney, and to a lesser extent in brain, and testis.
Example 4
Method of Assessing the Expression Profile of the Novel LTRPC3 Polypeptides of the Present Invention Using Expanded mRNA Tissue and Cell Sources
Total RNA from tissues was isolated using the TriZol protocol (Invitrogen) and quantified by determining its absorbance at 260 nM. An assessment of the 18s and 28s ribosomal RNA bands was made by denaturing gel electrophoresis to determine RNA integrity.
The specific sequence to be measured was aligned with related genes found in GenBank to identity regions of significant sequence divergence to maximize primer and probe specificity. Gene-specific primers and probes were designed using the ABI primer express software to amplify small amplicons (150 base pairs or less) to maximize the likelihood that the primers function at 100% efficiency. All primer/probe sequences were searched against Public Genbank databases to ensure target specificity. Primers and probes were obtained from ABI. For LTRPC3, the primer probe sequences were as follows








Forward Primer



5′-TCAGAGAATGGGCCAACAAGA-3′
(SEQ ID NO:270)


 


Reverse Primer


5′-CGAAAACGCTCGAGGAATGA-3′
(SEQ ID NO:271)


 


TaqMan Probe


5′-CAGGCCTAGGTTCCTCCTCTCGGAAA-3′
(SEQ ID NO:272)






DNA Contamination

To access the level of contaminating genomic DNA in the RNA, the RNA was divided into 2 aliquots and one half was treated with Rnase-free Dnase (Invitrogen). Samples from both the Dnase-treated and non-treated were then subjected to reverse transcription reactions with (RT+) and without (RT−) the presence of reverse transcriptase. TaqMan assays were carried out with gene-specific primers (see above) and the contribution of genomic DNA to the signal detected was evaluated by comparing the threshold cycles obtained with the RT+/RT− non-Dnase treated RNA to that on the RT+/RT− Dnase treated RNA. The amount of signal contributed by genomic DNA in the Dnased RT− RNA must be less that 10% of that obtained with Dnased RT+RNA. If not the RNA was not used in actual experiments.
Reverse Transcription Reaction and Sequence Detection
100 ng of Dnase-treated total RNA was annealed to 2.5 μM of the respective gene-specific reverse primer in the presence of 5.5 mM Magnesium Chloride by heating the sample to 72° C. for 2 min and then cooling to 55° C. for 30 min. 1.25 U/μl of MuLv reverse transcriptase and 500 μM of each dNTP was added to the reaction and the tube was incubated at 37° C. for 30 min. The sample was then heated to 90° C. for 5 min to denature enzyme.
Quantitative sequence detection was carried out on an ABI PRISM 7700 by adding to the reverse transcribed reaction 2.5 μM forward and reverse primers, 500 μM of each dNTP, buffer and 5 U AmpliTaq GoldTM. The PCR reaction was then held at 94° C. for 12 min, followed by 40 cycles of 94° C. for 15 sec and 60° C. for 30 sec.
Data Handling
The threshold cycle (Ct) of the lowest expressing tissue (the highest Ct value) was used as the baseline of expression and all other tissues were expressed as the relative abundance to that tissue by calculating the difference in Ct value between the baseline and the other tissues and using it as the exponent in 2(ΔCt) 
The expanded expression profile of the LTRPC3 polypeptide is provided in FIGS. 12 and 13 and described elsewhere herein.
Example 5
Complementary Oligonucleotides to the LTRPC3 Polynucleotide
Antisense molecules or nucleic acid sequences complementary to the LTRPC3 protein-encoding sequence, or any part thereof, was used to decrease or to inhibit the expression of naturally occurring LTRPC3. Although the use of antisense or complementary oligonucleotides comprising about 15 to 35 base-pairs is described, essentially the same procedure is used with smaller or larger nucleic acid sequence fragments. An oligonucleotide based on the coding sequence of LTRPC3 protein, as shown in FIGS. 1A-F, or as depicted in SEQ ID NO:1, for example, is used to inhibit expression of naturally occurring LTRPC3. The complementary oligonucleotide is typically designed from the most unique 5′ sequence and is used either to inhibit transcription by preventing promoter binding to the coding sequence, or to inhibit translation by preventing the ribosome from binding to the LTRPC3 protein-encoding transcript. However, other regions may also be targeted.
Using an appropriate portion of a 5′ sequence of SEQ ID NO:1, an effective antisense oligonucleotide includes any of about 15-35 nucleotides spanning the region which translates into the signal or 5′ coding sequence, among other regions, of the polypeptide as shown in FIGS. 1A-F (SEQ ID NO:2). Appropriate oligonucleotides are designed using OLIGO 4.06 software and the LTRPC3 protein coding sequence (SEQ ID NO:1). Preferred oligonucleotides are deoxynucleotide, or chimeric deoxynucleotide/ribonucleotide based and are provided below. The oligonucleotides were synthesized using chemistry essentially as described in U.S. Pat. No. 5,849,902; which is hereby incorporated herein by reference in its entirety.











ID#
Sequence

















15737
CCAUGGACAGAGAUGAGAAGCUUGGU
(SEQ ID NO:276)


 


15738
AGUGGUCCCGUUGUCAGCCAGAAUGU
(SEQ ID NO:277)


 


15739
CCUUCCACUAUGAGUGCCACCACAGU
(SEQ ID NO:278)


 


15740
GUGUCCUUCUGAUCCCAUCCGAAAUU
(SEQ ID NO:279)


 


15741
UGGUAUGGCCGGACAACAUCUCUUCU
(SEQ ID NO:280)









The LTRPC3 polypeptide has been shown to be involved in the regulation of mammalian base-excision repair. Subjecting cells with an effective amount of a pool of all five of the above antisense oligonucleotides resulted in a significant increase in FEN1 expression/activity providing convincing evidence that LTRPC3 at least regulates the activity and/or expression of FEN1 either directly, or indirectly. Moreover, the results suggest that LTRPC3 is involved in the negative regulation of FEN1 activity and/or expression, either directly or indirectly. The FEN1 assay used is described below and was based upon the analysis of FEN1 activity as a downstream marker for proliferative signal transduction events.
Transfection of Post-Quiescent A549 Cells with AntiSense Oligonucleotides.
Materials needed:


    
    
        A549 cells maintained in DMEM with high glucose (Gibco-BRL) supplemented with 10% Fetal Bovine Serum, 2 mM L-Glutamine, and 1× penicillin/streptomycin.
        Opti-MEM (Gibco-BRL)
        Lipofectamine 2000 (Invitrogen)
        Antisense oligomers (Sequitur)
        Polystyrene tubes.
        Tissue culture treated plates.
    
    


Quiescent cells were prepared as follows:

    Day 0: 300,000 A549 cells were seeded in a T75 tissue culture flask in 10 ml of A549 media (as specified above), and incubated in at 37° C., 5% CO2 in a humidified incubator for 48 hours.
    Day 2: The T75 flasks were rocked to remove any loosely adherent cells, and the A549 growth media removed and replenished with 10 ml of fresh A549 media. The cells were cultured for six days without changing the media to create a quiescent cell population.
    Day 8: Quiescent cells were plated in multi-well format and transfected with antisense oligonucleotides.


A549 cells were transfected according to the following:

    
    
        1. Trypsinize T75 flask containing quiescent population of A549 cells.
        2. Count the cells and seed 24-well plates with 60K quiescent A549 cells per well.
        3. Allow the cells to adhere to the tissue culture plate (approximately 4 hours).
        4. Transfect the cells with antisense and control oligonucleotides according to the following:
        
            a. A 10× stock of lipofectamine 2000 (10 ug/ml is 10×) was prepared, and diluted lipid was allowed to stand at RT for 15 minutes.
            
                Stock solution of lipofectamine 2000 was 1 mg/ml.
                10× solution for transfection was 10 ug/ml.
                To prepare 10× solution, dilute 10 ul of lipofectamine 2000 stock per 1 ml of Opti-MEM (serum free media).
            
            
            b. A 10× stock of each oligomer was prepared to be used in the transfection.
            
                Stock solutions of oligomers were at 100 uM in 20 mM HEPES, pH 7.5.
                10× concentration of oligomer was 0.25 uM.
                To prepare the 10× solutions, dilute 2.5 ul of oligomer per 1 ml of Opti-MEM.
            
            
            c. Equal volumes of the 10× lipofectamine 2000 stock and the 10× oligomer solutions were mixed well, and incubated for 15 minutes at RT to allow complexation of the oligomer and lipid. The resulting mixture was 5×.
            d. After the 15 minute complexation, 4 volumes of full growth media was added to the oligomer/lipid complexes (solution was 1×).
            e. The media was aspirated from the cells, and 0.5 ml of the 1× oligomer/lipid complexes added to each well.
            f. The cells were incubated for 16-24 hours at 37° C. in a humidified CO2 incubator.
            g. Cell pellets were harvested for RNA isolation and TaqMan analysis of downstream marker genes.

TaqMan Reactions

        
        
    
    


Quantitative RT-PCR analysis was performed on total RNA preps that had been treated with DNaseI or poly A selected RNA. The Dnase treatment may be performed using methods known in the art, though preferably using a Qiagen Rneasy kit to purify the RNA samples, wherein DNAse I treatment is performed on the column.
Briefly, a master mix of reagents was prepared according to the following table:










Dnase I Treatment










Reagent
Per r'xn (in uL)














10 × Buffer
2.5



Dnase I (1 unit/ul @1 unit per ug
2



sample)



DEPC H2O
0.5



RNA sample @ 0.1 ug/ul
20



(2-3 ug total)




Total
25










Next, 5 ul of master mix was aliquoted per well of a 96-well PCR reaction plate (PE part #N801-0560). RNA samples were adjusted to 0.1 ug/ul with DEPC treated H2O (if necessary), and 20 ul was added to the aliquoted master mix for a final reaction volume of 25 ul.
The wells were capped using strip well caps (PE part #N801-0935), placed in a plate, and briefly spun in a plate centrifuge (Beckman) to collect all volume in the bottom of the tubes. Generally, a short spin up to 500 rpm in a Sorvall RT is sufficient
The plates were incubated at 37° C. for 30 mins. Then, an equal volume of 0.1 mM EDTA in 10 mM Tris was added to each well, and heat inactivated at 70° C. for 5 min. The plates were stored at −80° C. upon completion.
RT Reaction
A master mix of reagents was prepared according to the following table:










RT reaction










RT
No RT


Reagent
Per Rx'n (in ul)
Per Rx'n (in ul)














10 × RT buffer
5

2.5



MgCl2
11

5.5


DNTP mixture
10

5


Random Hexamers
2.5

1.25


Rnase inhibitors
1.25

0.625










RT enzyme
1.25

—











Total RNA 500 ng (100 ng no RT)
19.0
max
10.125
max









DEPC H2O
—
—











Total
50
uL
25
uL









Samples were adjusted to a concentration so that 500 ng of RNA was added to each RT rx'n (100 ng for the no RT). A maximum of 19 ul can be added to the RT rx'n mixture (10.125 ul for the no RT.) Any remaining volume up to the maximum values was filled with DEPC treated H2O, so that the total reaction volume was 50 ul (RT) or 25 ul (no RT).
On a 96-well PCR reaction plate (PE part #N801-0560), 37.5 ul of master mix was aliquoted (22.5 ul of no RT master mix), and the RNA sample added for a total reaction volume of 50 ul (25 ul, no RT). Control samples were loaded into two or even three different wells in order to have enough template for generation of a standard curve.
The wells were capped using strip well caps (PE part #N801-0935), placed in a plate, and spin briefly in a plate centrifuge (Beckman) to collect all volume in the bottom of the tubes. Generally, a short spin up to 500 rpm in a Sorvall RT is sufficient.
For the RT-PCR reaction, the following thermal profile was used:

    
    
        25° C. for 10 min
        48° C. for 30 min
        95° C. for 5 min
        4° C. hold (for 1 hour)
        Store plate @-20° C. or lower upon completion.

TaqMan Reaction (Template Comes from RT Plate.)

    
    


A master mix was prepared according to the following table:










TaqMan reaction (per well)










Reagent
Per Rx'n (in ul)














TaqMan Master Mix
4.17



100 uM Probe
.025



(SEQ ID NO: 311)



100 uM Forward
.05



primer (SEQ ID



NO: 309)



100 uM Reverse
.05



primer (SEQ ID



NO: 310)



Template
—



DEPC H2O
18.21



Total
22.5










The primers used for the RT-PCR reaction is as follows:











FEN1 primer and probes:




Forward Primer:



CCACCTGATGGGCATGTTCT
(SEQ ID NO:273)



 



Reverse Primer:



CGGCTTGCCATCAAAGACATA
(SEQ ID NO:274)



 



TaqMan Probe:



CCGCACCATTCGCATGATGGAG
(SEQ ID NO:275)






Using a Gilson P-10 repeat pipetter, 22.5 ul of master mix was aliquouted per well of a 96-well optical plate. Then, using P-10 pipetter, 2.5 ul of sample was added to individual wells. Generally, RT samples are run in triplicate with each primer/probe set used, and no RT samples are run once and only with one primer/probe set, often gapdh (or other internal control).
A standard curve is then constructed and loaded onto the plate. The curve has five points plus one no template control (NTC, =DEPC treated H2O). The curve was made with a high point of 50 ng of sample (twice the amount of RNA in unknowns), and successive samples of 25, 10, 5, and 1 ng. The curve was made from a control sample(s) (see above).
The wells were capped using optical strip well caps (PE part #N801-0935), placed in a plate, and spun in a centrifuge to collect all volume in the bottom of the tubes. Generally, a short spin up to 500 rpm in a Sorvall RT is sufficient.
Plates were loaded onto a PE 5700 sequence detector making sure the plate is aligned properly with the notch in the upper right hand corner. The lid was tightened down and run using the 5700 and 5700 quantitation programs and the SYBR probe using the following thermal profile:

    
    
        50° C. for 2 min
        95° C. for 10 min
        and the following for 40 cycles:
        
            95° C. for 15 sec
            60° C. for 1 min
        
        
        Change the reaction volume to 25 ul.
    
    


Once the reaction was complete, a manual threshold of around 0.1 was set to minimize the background signal. Additional information relative to operation of the GeneAmp 5700 machine may be found in reference to the following manuals: “GeneAmp 5700 Sequence Detection System Operator Training CD”; and the “User's Manual for 5700 Sequence Detection System”; available from Perkin-Elmer and hereby incorporated by reference herein in their entirety.
Example 6
Method of Assessing the Cellular Localization of the LTRPC3 Polypeptide
HEK 293 cells were cultured in Dulbecco's modified medium containing 10% heat-inactivated fetal bovine serum and grown on the poly-D-lysine-coated glass coverslips. The cells were transiently transfected with the pcDNA3.1/Hygro-LTRPC3-HA vector with Fugene (Roche). Forty-eight hours later, cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100, blocked in PBS containing 5% FBS and 5% normal goat serum, and stained with 10 μg/ml Fluorescein-conjugated anti-HA High Affinity antibody (3F10, Roche) and DAPI (0.5 μg/ml; Molecular Probes). Immunostained cell cultures were examined using a laser scanning confocal microscope (ZEISS LSM510). A 63× oil immersion objective was used with appropriate filter sets. Images were reconstructed from confocal stacks of Z-series scans of 10-20 optical sections through a depth of 5-15 μM.
The cellular localization of HA-tagged LTRPC3 was exclusively detected at the subplasma membrane region of the transfected cells (see FIG. 18), which is consistent with LTRPC3 being an integral membrane protein. Under these conditions approximately  ̃70% of cells were expressing LTRPC3.
Example 7
Method of Assessing the Ion Channel Activity of the LTRPC3 Polypeptide
The cytoplasmic Ca2+ indicator Fluo-4-AM (Molecular Probe) and the Fluorometric Imaging Plate Reader (FLIPRTM, Molecular Devices) instrument were used to detect changes in intracellular Ca2+. The pcDNA3.1/Hygro-LTRPC3-HA transfected cells were seeded on PDL-coated 96-well plates at a density of 70,000 cells/well 24 hours after transfection and used 24 hours after plating. Transfected cells were loaded with 4 μM Fluo-4-AM at 37° C. for 30 min in a nominally Ca2+-free or 1 mM CaCl2 buffer containing 140 mM NaCl, 4.7 mM KCl, 1 mM MgCl2, 10M HEPES, 10 mM Glucose, and 2.5 mM Probenecid (Sigma), pH 7.4. Extracellular Fluo-4-AM was removed and cells were maintained in either Ca2+-free buffer or buffer containing 1 mM Ca2+ at room temperature prior to the experiments, which were conducted within 30 min after dye removal. Fluo-4 was excited at 488 nm using an argon laser and emitted light was selected using a 510-570 nm bandpass filter. Baseline intracellular fluorescence was established during the initial 50 seconds of the FLIPR read, then 1, 3 or 10 mM Ca2+ was added to each well and subsequent changes in the intracellular Ca2+ were monitored for 8 minutes. For store-depletion studies, 2 μM thapsigargin or 100 μM carbachol was added to Fluo-4-loaded cells in Ca2+-free buffer before adding Ca2+ on FLIPR. For pharmacology studies, 100 μM LaCl3 or 100 μM GdCl3 were added to Fluo-4-loaded cells in 1 mM Ca2+ buffer before adding Ca2+ on FLIPR. Experiments were carried out at room temperature. The results of these physiometric studies are shown in FIGS. 18 and 19.
The addition of Ca2+ to the media resulted in a concentration-dependent influx of Ca2+ into LTRPC3-expressing cells (as shown in FIG. 18, right panels), indicating that LTRPC3 is a functional Ca2+ channel. In contrast, vector-transfected cells showed minimal Ca2+ influx under the same experimental conditions (as shown in FIG. 18, left panels). The non-transfected cells were indistinguishable from the vector-transfected cells (data not shown). Therefore, LTRPC3 is a constitutively active channel capable of mediating Ca2+ influx.
To further address the mechanism of LTRPC3-mediated Ca2+ entry, similar Ca2+ addition experiments were performed on transfected cells incubated ( ̃30 min) in a nominally Ca2+-free solution. Previous studies have shown that lowering extracellular Ca2+ concentration below physiological levels can deplete intracellular Ca2+ stores in many cell types including HEK 293 (EMBO J. 17, 4274-4282 (1998)). Incubating vector-transfected HEK 293 cells in a nominally Ca2+-free solution gave rise to Ca2+ entry that was dependent on the concentration of Ca2+ added to the buffers, indicating Ca2+ influx was mediated through endogenous SOCs in HEK293 cells (as shown in FIG. 19; left panel). In LTRPC3 cells, the Ca2+ transients triggered by similar Ca2+ treatment were much larger (as shown in FIG. 19, right panel). This Ca2+ entry observed in LTRPC3 cells incubated in Ca2+-free media were greater than in 1 mM Ca2+ media, indicating that LTRPC3-mediated Ca2+ entry can be potentiated by the store-depletion.
Example 8
Method of Assessing the Expression Profile of the Ion Channel Activity of the LTRPC3 Polypeptide Using In Situ Hybridization
Human Kidney was collected and received from the National Disease Research Interchange (Philadelphia, Pa.) according to IRB approved protocol. Tissue sections were embedded in OCT compound (Miles) and snap-frozen by immersion in 2-methylbutane cooled in dry ice and subsequently stored at −70° C.
Templates for LTRPC3 cRNA probes were derived from a 678-bp LTRPC3 fragment, cloned in a pCR-BluntII-TOPO vector (Invitrogen) utilizing the primer pair: (forward: 5′-CAGCTGGAAGACCTTATCGGG-3′ (SEQ ID NO:285); reverse: 5′-TGGGAGGTGGGTGTAGTCTGAAGA-3′ (SEQ ID NO:286)). The template for positive control cRNA human lysozyme probe was derived from a 638 bp EST (Incyte Genomics, GenBank Accession No:AA588081). 35S-labeled riboprobes were synthesized via in vitro transcription utilizing the Riboprobe®Combination System (Promega) where T7 and Sp6 RNA polymerase yielded sense and antisense probes respectively for LTRPC3 while T7 and T3 RNA polymerases yielded antisense and sense probes respectively for human lysozyme. Cryostat tissue sections cut at 10 μm and fixed in 4.0% formalin were used for in situ hybridization as previously described (Dambach, D. M., et al., (2002) Hepatology 35, 1093-1103.): Briefly, tissue sections were acetylated; dehydrated in a graded ethanol series; immersed in chloroform; alcohol rinsed; air dried and then hybridized with sense and antisense 35S RNA probes (1.5×106 cpm/slide) for 16-20 hours at 60° C. Following hybridization, slides were rinsed in 4×SSC/50% formamide and 4×SSC; treated with RNAse A (20 μg/ml; Invitrogen) at 37° C.; washed through increasing stringent solutions to final high stringency wash in 0.1×SSC at 60° C.; dehydrated; air dried and then coated with NTB-2 emulsion (Eastman Kodak). Slides were placed in a dark box with desiccant at 4° C. and developed after one and four weeks exposure. Sections were stained with hematoxylin and eosin, and coverslipped. Expression signals were detected by dark phase microscopy. Cellular phenotype identification was by bright field microscopy.
The In situ hybridization results of the LTRPC3 polypeptide is provided in FIG. 20 and described elsewhere herein.
Example 9
Method of Further Assessing Cellular Localization of the LTRPC3 Polypeptide
HEK 293 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% heat-inactivated fetal bovine serum and grown on poly-D-lysine-coated (PDL) glass coverslips. The cells were transiently transfected with hLTRPC3-HA with FuGENE 6 (Roche Molecular Biochemicals). Forty-eight hours later, cells were stained in culture media with the membrane probe VybrantTM CM-DiI (5 μl/ml; Molecular Probes) at 37° C. for 5 min and 4° C. for 15 min. After washing with PBS, cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100, blocked in PBS containing 5% FBS and 5% normal goat serum, and stained with 10 μg/ml Fluorescein-conjugated anti-HA High Affinity antibody (3F10; Roche Molecular Biochemicals) and DAPI (0.5 μg/ml; Molecular Probes). Immunostained cell cultures were examined using a laser scanning confocal microscope (ZEISS LSM510), a 63× oil immersion objective, and appropriate filter sets. Images shown are of a single optical section approximately 1 μm thick.
The expanded confocal microscopy results of the LTRPC3 polypeptide is provided in FIG. 21 and described elsewhere herein.
Example 10
Method of Further Assessing the Ion Channel Activity of the LTRPC3 Polypeptide
The cytoplasmic Ca2+ indicator Fluo-4-AM (Molecular Probes) and a Fluorometric Imaging Plate Reader (FLIPRTM; Molecular Devices) instrument were used to detect changes in intracellular Ca2+ concentration. The hLTRPC3-transfected cells were seeded on PDL-coated 96-well plates at a density of 70,000 cells/well 24 hours after transfection and used 24 hours after plating. Cells were loaded with 4 μM Fluo-4-AM at 37° C. for 30 min in a nominally Ca2+-free or 1 mM CaCl2 buffer containing (in mM): 140 NaCl, 4.7 KCl, 1 MgCl2, 10 HEPES, 10 Glucose, and 2.5 Probenecid (Sigma), pH 7.4. Extracellular Fluo-4-AM was removed and cells were maintained in either Ca2+-free buffer or buffer containing 1 mM Ca2+ at room temperature prior to the experiments, which were conducted within 30 min after dye removal. Fluo-4 was excited at 488 nm using an argon laser and emitted light was selected using a 510-570 nm bandpass filter. Baseline intracellular fluorescence was established during the initial 50 seconds of the FLIPR read, then 1, 3, or 10 mM Ca2+ was added to each well and subsequent changes in the intracellular Ca2+ were monitored for 8 minutes. For store-depletion or receptor activation studies, 2 μM thapsigargin or 50 μM carbachol, respectively, was added to Fluo-4-loaded cells in Ca2+-free buffer before adding 2 mM Ca2+ on FLIPR. For pharmacology studies, 100 μM GdCl3 was added to Fluo-4-loaded cells in 0 or 1 mM Ca2+ buffer, as described in herein, prior to the start of the FLIPR recordings. Experiments were carried out at room temperature.
The results of the expanded physiometric experiments are provided in FIG. 22 and described herein.
Example 11
Method of Assessing the Putative Kinase Activity of the LTRPC3 Polypeptide
A number of methods may be employed to assess the potential kinase activity of the LTRPC3 polypeptides. One preferred method is described below. A fusion construct is made whereby the LTRPC3 encoding polynucleotide is operably linked to the coding region of the HA protein. CHO-K1 or HEK-293 cells grown on 100-mm dishes are transiently transfected with 8 μg of novel LTRPC3-HA cDNA construct in the pTracer-CMV2 (Invitrogen) vector with LipofectAMINE 2000 (Gibco). Cells are harvested after 48 hours with 3 ml of RIPA buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% IGEPAL CA-630, 0.5% (w/v) deoxycholate, 0.1% (w/v) SDS, and 10 mM iodoacetamide]. LTRPC3-HA is immunoprecipitated with mouse monoclonal immunoglobulin G2a (IgG2a) HA probe (F-7) coupled to agarose (Santa Cruz Biotechnology). The agarose is sedimented and washed three times with RIPA buffer, and 2×SDS sample buffer is added. The samples may be resolved by SDS-PAGE and Western blotting following standard methods. HA probe Y-11 antibody could be the primary antibody (Santa Cruz Biotechnology), and horseradish peroxidase-linked antibody to rabbit Ig (Amersham Pharmacia Biotech) could be the secondary antibody. The SuperSignal West Dura substrate may be used for chemiluminescent detection (Pierce)
For phosphorylation experiments, purified GST-kinase fusion proteins and mutants are incubated at 37° C. for 30 min in the presence or absence of MBP as a test substrate in a 50-μl reaction. These reactions are performed in KIN buffer {50 mM Mops (pH 7.2), 100 mM NaCl, 20 mM MgCl2, 0.5 mM ATP, and 2 μCi of [-32P]ATP}. Immunokinase reactions containing immunopurified LTRPC3—HA are incubated at 37° C. for 30 min in a 50-μl reaction containing KIN buffer with 75 mM n-octyl—D-glucopyranoside. The reactions are terminated by the addition of 2×SDS sample buffer, and the proteins were resolved by SDS-PAGE and Coomassie staining for the GST-kinase experiment or by SDS-PAGE and Western blotting for the immunokinase assay. The gels are dried, and 32P incorporation is visualized by autoradiography for the GST-kinase experiment. For the immunokinase experiment, 32P incorporation may be visualized by autoradiography of the transferred proteins on polyvinylidene difluoride membrane (Bio-Rad) before Western blotting.
Example 12
Bacterial Expression of a Polypeptide
A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 15, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites, such as BamHI and XbaI, at the 5′ end of the primers in order to clone the amplified product into the expression vector. For example, BamHI and XbaI correspond to the restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.
The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture is then used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4, that expresses the lacI repressor and also confers kanamycin resistance (Kanr). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.
Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.600) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration of 1 mM. IPTG induces by inactivating the lacI repressor, clearing the P/O leading to increased gene expression.
Cells are grown for an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 mins at 6000×g). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at 4 degree C. The cell debris is removed by centrifugation, and the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind to the Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist (1995) QIAGEN, Inc., supra).
Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.
The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on the Ni-NTA column. The recommended conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM imidazole. Imidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein is stored at 4 degree C. or frozen at −80 degree C.
Example 13
Purification of a Polypeptide from an Inclusion Body
The following alternative method can be used to purify a polypeptide expressed in E coli when it is present in the form of inclusion bodies. Unless otherwise specified, all of the following steps are conducted at 4-10 degree C.
Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10 degree C. and the cells harvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste, by weight, is suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a high shear mixer.
The cells are then lysed by passing the solution through a microfluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000×g for 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
The resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×g centrifugation for 15 min., the pellet is discarded and the polypeptide containing supernatant is incubated at 4 degree C. overnight to allow further GuHCl extraction.
Following high speed centrifugation (30,000×g) to remove insoluble particles, the GuHCl solubilized protein is refolded by quickly mixing the GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded diluted protein solution is kept at 4 degree C. without mixing for 12 hours prior to further purification steps.
To clarify the refolded polypeptide solution, a previously prepared tangential filtration unit equipped with 0.16 um membrane filter with appropriate surface area (e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loaded onto a cation exchange resin (e.g., Poros HS-50, Perceptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. The absorbance at 280 nm of the effluent is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.
Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample is then loaded onto a previously prepared set of tandem columns of strong anion (Poros HQ-50, Perceptive Biosystems) and weak anion (Poros CM-20, Perceptive Biosystems) exchange resins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10 column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant A280 monitoring of the effluent. Fractions containing the polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.
The resultant polypeptide should exhibit greater than 95% purity after the above refolding and purification steps. No major contaminant bands should be observed from Coomassie blue stained 16% SDS-PAGE gel when 5 ug of purified protein is loaded. The purified protein can also be tested for endotoxin/LPS contamination, and typically the LPS content is less than 0.1 ng/ml according to LAL assays.
Example 14
Cloning and Expression of a Polypeptide in a Baculovirus Expression System
In this example, the plasmid shuttle vector pAc373 is used to insert a polynucleotide into a baculovirus to express a polypeptide. A typical baculovirus expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites, which may include, for example BamHI, Xba I and Asp718. The polyadenylation site of the simian virus 40 (“SV40”) is often used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate a viable virus that express the cloned polynucleotide.
Many other baculovirus vectors can be used in place of the vector above, such as pVL941 and pAcIM1, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectors are described, for instance, in Luckow et al., Virology 170:31-39 (1989).
A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 15, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites at the 5′ end of the primers in order to clone the amplified product into the expression vector. Specifically, the cDNA sequence contained in the deposited clone, including the AUG initiation codon and the naturally associated leader sequence identified elsewhere herein (if applicable), is amplified using the PCR protocol described in Example 15. If the naturally occurring signal sequence is used to produce the protein, the vector used does not need a second signal peptide. Alternatively, the vector can be modified to include a baculovirus leader sequence, using the standard methods described in Summers et al., “A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures,” Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.
The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).
The fragment and the dephosphorylated plasmid are ligated together with T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing the plasmid are identified by digesting DNA from individual colonies and analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.
Five ug of a plasmid containing the polynucleotide is co-transformed with 1.0 ug of a commercially available linearized baculovirus DNA (“BaculoGoldTM baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofection method described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One ug of BaculoGoldTM virus DNA and 5 ug of the plasmid are mixed in a sterile well of a microtiter plate containing 50 ul of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27 degrees C. The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27 degrees C. for four days.
After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a “plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10.) After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 ul of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4 degree C.
To verify the expression of the polypeptide, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus containing the polynucleotide at a multiplicity of infection (“MOI”) of about 2. If radiolabeled proteins are desired, 6 hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of 35S-methionine and 5 uCi 35S-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then are harvested by centrifugation. The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).
Microsequencing of the amino acid sequence of the amino terminus of purified protein may be used to determine the amino terminal sequence of the produced protein.
Example 15
Expression of a Polypeptide in Mammalian Cells
The polypeptide of the present invention can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).
Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC 67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
Alternatively, the polypeptide can be expressed in stable cell lines containing the polynucleotide integrated into a chromosome. The co-transformation with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transformed cells.
The transformed gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.
A polynucleotide of the present invention is amplified according to the protocol outlined in herein. If the naturally occurring signal sequence is used to produce the protein, the vector does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891.) The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.
The amplified fragment is then digested with the same restriction enzyme and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.
Chinese hamster ovary cells lacking an active DHFR gene is used for transformation. Five μg of an expression plasmid is cotransformed with 0.5 ug of the plasmid pSVneo using lipofectin (Felgner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100-200 uM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.
Example 16
Protein Fusions
The polypeptides of the present invention are preferably fused to other proteins. These fusion proteins can be used for a variety of applications. For example, fusion of the present polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See Example described herein; see also EP A 394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusion to IgG-1, IgG-3, and albumin increases the half-life time in vivo. Nuclear localization signals fused to the polypeptides of the present invention can target the protein to a specific subcellular localization, while covalent heterodimer or homodimers can increase or decrease the activity of a fusion protein. Fusion proteins can also create chimeric molecules having more than one function. Finally, fusion proteins can increase solubility and/or stability of the fused protein compared to the non-fused protein. All of the types of fusion proteins described above can be made by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG molecule.
Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5′ and 3′ ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian expression vector. Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protein will not be produced.
The naturally occurring signal sequence may be used to produce the protein (if applicable). Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891 and/or U.S. Pat. No. 6,066,781, supra.)








Human IgG Fc region:









(SEQ ID NO:252)









GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGC



 


CCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAA


 


ACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGG


 


TGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG


 


GACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTA


 


CAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACT


 


GGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCA


 


ACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACC


 


ACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG


 


TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTG


 


GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCC


 


CGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG


 


ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT


 


GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGG


 


TAAATGAGTGCGACGGCCGCGACTCTAGAGGAT






Example 17
Method of Creating N- and C-Terminal Deletion Mutants Corresponding to the LTRPC3 Polypeptide of the Present Invention
As described elsewhere herein, the present invention encompasses the creation of N- and C-terminal deletion mutants, in addition to any combination of N- and C-terminal deletions thereof, corresponding to the LTRPC3 polypeptide of the present invention. A number of methods are available to one skilled in the art for creating such mutants. Such methods may include a combination of PCR amplification and gene cloning methodology. Although one of skill in the art of molecular biology, through the use of the teachings provided or referenced herein, and/or otherwise known in the art as standard methods, could readily create each deletion mutant of the present invention, exemplary methods are described below.
Briefly, using the isolated cDNA clone encoding the full-length LTRPC3 polypeptide sequence (as described in Example 15, for example), appropriate primers of about 15-25 nucleotides derived from the desired 5′ and 3′ positions of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:281, or SEQ ID NO:282 may be designed to PCR amplify, and subsequently clone, the intended N- and/or C-terminal deletion mutant. Such primers could comprise, for example, an initiation and stop codon for the 5′ and 3′ primer, respectively. Such primers may also comprise restriction sites to facilitate cloning of the deletion mutant post amplification. Moreover, the primers may comprise additional sequences, such as, for example, flag-tag sequences, kozac sequences, or other sequences discussed and/or referenced herein.
For example, in the case of the G620 to T1554 LTRPC3 N-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant:










5′ Primer
5′-GCAGCA GCGGCCGC CTCAAGGTAATTCTGGGAATTCTAC-3′
(SEQ ID NO:378)




            NotI


 


3′ Primer
5′-GCAGCA GTCGAC GGTGTGCTTGCTTTCAAAGCTTTGG-3′
(SEQ ID NO:379)



   SalI






For example, in the case of the M1 to N960 LTRPC3 C-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant:










5′ Primer
5′-GCAGCA GCGGCCGC ATGTATGTGCGAGTATCTTTTG-3′
(SEQ ID NO:380)




          NotI


 


3′ Primer
5′-GCAGCA GTCGAC GTTAAAGACAGCAATGAGGAGGTTG-3′
(SEQ ID NO:381)



           SalI






The resulting C-terminal deletion mutant could be used as a potential, membrane bound, LTRPC3 decoy receptor.
For example, in the case of the M1 to N1061 LTRPC3 C-terminal deletion mutant, the following primers could be used to amplify a cDNA fragment corresponding to this deletion mutant:










5′ Primer
5′-GCAGCA GCGGCCGC ATGTATGTGCGAGTATCTTTTG-3′
(SEQ ID NO:382)




          NotI


 


3′ Primer
5′-GCAGCA GTCGAC ATTAGATGAGTTGAACCGATCATCC-3′
(SEQ ID NO:383)



          SalI






The resulting C-terminal deletion mutant could be used as a potential, membrane bound, LTRPC3 decoy receptor.
Representative PCR amplification conditions are provided below, although the skilled artisan would appreciate that other conditions may be required for efficient amplification. A 100 ul PCR reaction mixture may be prepared using 10 ng of the template DNA (cDNA clone of LTRPC3, LTRPC3b, LTRPC3c, LTRPC3d, LTRPC3e, or LTRPC3f), 200 uM 4dNTPs, 1 uM primers, 0.25 U Taq DNA polymerase (PE), and standard Taq DNA polymerase buffer. Typical PCR cycling condition are as follows:
20-25 cycles: 45 sec, 93 degrees

    
    
        2 min, 50 degrees
        2 min, 72 degrees
    
    


1 cycle: 10 min, 72 degrees
After the final extension step of PCR, 5 U Klenow Fragment may be added and incubated for 15 min at 30 degrees.
Upon digestion of the fragment with the NotI and SalI restriction enzymes, the fragment could be cloned into an appropriate expression and/or cloning vector which has been similarly digested (e.g., pSport1, among others). The skilled artisan would appreciate that other plasmids could be equally substituted, and may be desirable in certain circumstances. The digested fragment and vector are then ligated using a DNA ligase, and then used to transform competent E. coli cells using methods provided herein and/or otherwise known in the art.
The 5′ primer sequence for amplifying any additional N-terminal deletion mutants may be determined by reference to the following formula:

(S+(X*3)) to ((S+(X*3))+25),

wherein ‘S’ is equal to the nucleotide position of the initiating start codon of the LTRPC3 gene (SEQ ID NO:1), LTRPC3b gene (SEQ ID NO:3), LTRPC3c gene (SEQ ID NO:5), LTRPC3d gene (SEQ ID NO:281), LTRPC3e gene (SEQ ID NO:8), LTRPC3f gene (SEQ ID NO:282) and ‘X’ is equal to the most N-terminal amino acid of the intended N-terminal deletion mutant. The first term will provide the start 5′ nucleotide position of the 5′ primer, while the second term will provide the end 3′ nucleotide position of the 5′ primer corresponding to sense strand of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:281, or SEQ ID NO:282. Once the corresponding nucleotide positions of the primer are determined, the final nucleotide sequence may be created by the addition of applicable restriction site sequences to the 5′ end of the sequence, for example. As referenced herein, the addition of other sequences to the 5′ primer may be desired in certain circumstances (e.g., kozac sequences, etc.).

The 3′ primer sequence for amplifying any additional N-terminal deletion mutants may be determined by reference to the following formula:

(S+(X*3)) to ((S+(X*3))−25),

wherein ‘S’ is equal to the nucleotide position of the initiating start codon of the LTRPC3 gene (SEQ ID NO:1), LTRPC3b gene (SEQ ID NO:3), LTRPC3c gene (SEQ ID NO:5), LTRPC3d gene (SEQ ID NO:281), LTRPC3e gene (SEQ ID NO:8), LTRPC3f gene (SEQ ID NO:282) and ‘X’ is equal to the most C-terminal amino acid of the intended N-terminal deletion mutant. The first term will provide the start 5′ nucleotide position of the 3′ primer, while the second term will provide the end 3′ nucleotide position of the 3′ primer corresponding to the anti-sense strand of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:281, or SEQ ID NO:282. Once the corresponding nucleotide positions of the primer are determined, the final nucleotide sequence may be created by the addition of applicable restriction site sequences to the 5′ end of the sequence, for example. As referenced herein, the addition of other sequences to the 3′ primer may be desired in certain circumstances (e.g., stop codon sequences, etc.). The skilled artisan would appreciate that modifications of the above nucleotide positions may be necessary for optimizing PCR amplification.

The same general formulas provided above may be used in identifying the 5′ and 3′ primer sequences for amplifying any C-terminal deletion mutant of the present invention. Moreover, the same general formulas provided above may be used in identifying the 5′ and 3′ primer sequences for amplifying any combination of N-terminal and C-terminal deletion mutant of the present invention. The skilled artisan would appreciate that modifications of the above nucleotide positions may be necessary for optimizing PCR amplification.
One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides of the invention (e.g., gene therapy), agonists, and/or antagonists of polynucleotides or polypeptides of the invention.
It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.
The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background of the Invention, Detailed Description, and Examples is hereby incorporated herein by reference. Further, the hard copy of the sequence listing submitted herewith and the corresponding computer readable form are both incorporated herein by reference in their entireties.

## Claims

1. An isolated polynucleotide comprising a sequence selected from the group consisting of:
(a) an isolated polynucleotide comprising the sequence encoding amino acids 1 to 1556 of SEQ ID NO: 9; and
(b) an isolated polynucleotide comprising the sequence encoding amino acids 2 to 1556 of SEQ ID NO: 9.

2. The isolated polynucleotide of claim 1, wherein said polynucleotide is (a).

3. The isolated polynucleotide of claim 1, wherein said polynucleotide comprises nucleotides 1 to 4668 of SEQ ID NO:8.

4. The isolated nucleic acid molecule of claim 1, wherein said polynucleotide is (b).

5. The isolated nucleic acid molecule of claim 4, wherein said polynucleotide comprises nucleotides 4 to 4668 of SEQ ID NO:8.

6. The isolated polynucleotide of claim 1 wherein said nucleic acid sequence further comprises a heterologous nucleic acid sequence.

7. The isolated polynucleotide of claim 6 wherein said heterologous nucleic acid sequence encodes a heterologous polypeptide.

8. The isolated polynucleotide of claim 7 wherein said heterologous polypeptide is the Fc domain of immunoglobulin.

9. An isolated polynucleotide comprising a polynucleotide that encodes a polypeptide sequence having at least 99.0% identity to amino acids 1 to 1556 of SEQ ID NO:9, wherein percent identity is calculated using a CLUSTLAW global sequence alignment, and wherein said polynucleotide is a transient receptor potential channel having Ca++ flux activity.

10. The isolated polynucleotide of claim 9 further comprising a heterologous polynucleotide.

11. The isolated polynucleotide of claim 10 wherein said heterologous polynucleotide is the Fc domain of immunoglobulin.

12. An isolated polynucleotide comprising a polynucleotide sequence that encodes at least 1116 contiguous amino acids of SEQ ID NO:9, wherein said polynucleotide is a transient receptor potential channel having Ca++ flux activity.

13. The isolated polynucleotide of claim 12, wherein said polynucleotide comprises at least 3348 contiguous nucleotides of SEQ ID NO:8.

14. A recombinant vector comprising the isolated nucleic acid molecule of claim 1.

15. An isolated recombinant host cell comprising the recombinant vector of claim 14.

16. A method of making an isolated polypeptide comprising:
(a) culturing the isolated recombinant host cell of claim 15 under conditions such that a polypeptide comprising either amino acids 1 to 1556 of SEQ ID NO:9 or amino acids 2 to 1556 of SEQ ID NO:9 is expressed; and
(b) recovering said polypeptide.