# Patent

## Title
PKD ligands and polynucleotides encoding PKD ligands

## Abstract

The invention relates to kinase ligands and polyligands. In particular, the invention relates to ligands, homopolyligands, and heteropolyligands that modulate protein kinase D (PKD) activity. The ligands and polyligands are utilized as research tools or as therapeutics. The invention includes linkage of the ligands and polyligands to cellular localization signals, epitope tags and/or reporters. The invention also includes polynucleotides encoding the ligands and polyligands.

## Background
This application is the U.S. national stage of International Application No. PCT/US2006/060062, filed Oct. 18, 2006, which claims the benefit of U.S. provisional Application No. 60/728,259, filed Oct. 19, 2005.


Reference To Sequence Listing Submitted Electronically Via EFS-Web
This application includes a “sequencelisting ascii.txt”, 159,159 bytes, created on Jul. 20, 2011, and submitted electronically via EFS-Web, which is hereby incorporated by reference in its entirety.
FIELD OF INVENTION
The invention relates to mammalian kinase ligands, substrates and modulators. In particular, the invention relates to polypeptides, polypeptide compositions and polynucleotides that encode polypeptides that are ligands, substrates, and/or modulators of PKD. The invention also relates to polyligands that are homopolyligands or heteropolyligands that modulate PKD activity. The invention also relates to ligands and polyligands tethered to a subcellular location.
This application has subject matter related to application Ser. No. 10/724,532 (now U.S. Pat. No. 7,071,295), Ser. No. 10/682,764 (US2004/0185556, PCT/US2004/013517, WO2005/040336), Ser. No. 11/233,246, and US20040572011P (WO2005116231). Each of these patents and applications is hereby incorporated by reference.
BACKGROUND AND PRIOR ART
Kinases are enzymes that catalyze the addition of phosphate to a molecule. The addition of phosphate by a kinase is called phosphorylation. When the kinase substrate is a protein molecule, the amino acids commonly phosphorylated are serine, threonine and tyrosine. Phosphatases are enzymes that remove phosphate from a molecule. The removal of phosphate is called dephosphorylation. Kinases and phosphatases often represent competing forces within a cell to transmit, attenuate, or otherwise modulate cellular signals and cellular control mechanisms. Kinases and phosphatases have both overlapping and unique natural substrates. Cellular signals and control mechanisms, as regulated by kinases, phosphatases, and their natural substrates are a target of research tool design and drug design.
Mammalian Protein Kinase D is also known as PKD. The enzymatic activity, activation and autoregulation of PKD have been studied. Several cellular substrates of PKD have been identified. Substrates and modified substrates are generically referred to herein as ligands. Natural and synthetic peptide ligands have been studied to examine PKD substrate specificity. While peptide ligands and variants thereof have been studied as individuals PKD ligands, mixed ligands linked together as polyligands have not been demonstrated before this invention.
Design and synthesis of polypeptide ligands that modulate calcium/calmodulin-dependent protein kinase and that localize to the cardiac sarco(endo)plasmic reticulum was performed by Ji et al. (J Biol Chem (2003) 278:25063-71). Ji et al. accomplished this by generating expression constructs that localized calcium/calmodulin-dependent protein kinase inhibitory polypeptide ligands to the sarcoplasmic reticulum by fusing a sarcoplasmic reticulum localization signal derived from phospholamban to a polypeptide ligand. See also U.S. Pat. No. 7,071,295.
DETAILED DESCRIPTION OF POLYPEPTIDE AND POLYNUCLEOTIDE SEQUENCES
SEQ ID NOS:1-104 represent examples of monomeric peptide ligand sequences.
Specifically, SEQ ID NOS:1-52 are fragments of natural substrates of mammlain PKD, wherein each fragment contains at least one amino acid residue phosphorylatable by PKD. SEQ ID NOS:53-104 are fragments of natural PKD substrates, where the location of the PKD phosphorylatable serine or threonine in the natural polypeptide is designated as Xaa.
SEQ ID NOS:105-116 are example polyligands and polynucleotides encoding them.
Specifically, SEQ ID NO:105 is encoded by SEQ ID NO:106, SEQ ID NO:107 and by SEQ ID NO:108, wherein the the codons of SEQ ID NO:107 and SEQ ID NO:108 have been optimized for vector insertion. SEQ ID NO:108 includes flanking restriction sites. SEQ ID NO:105 is an embodiment of a polyligand of the structure X-S1-Y-S2-Z, wherein X is SEQ ID NO:53, Y is SEQ ID NO:55, Z is SEQ ID NO:58, wherein Xaa is alanine, and wherein S1 is a four amino acid spacer of the amino acid sequence AGAG and S2 is a four amino acid spacer of amino acid sequence GAGA. A polyligand of structure A-S1-B-S2-C is called herein a heteropolyligand, shown generically in FIG. 4B.
SEQ ID NO:109 is an embodiment of a polyligand of the structure X-S1-Y-S2-Z, wherein X is SEQ ID NO:61, Y is SEQ ID NO:65, Z is SEQ ID NO:66, wherein Xaa is alanine, wherein S1 is a four amino acid spacer of amino acid sequence AGAG and S2 is a four amino acid spacer of amino acid sequence GAGA. The PKD polyligand of SEQ ID NO:109 is encoded by SEQ ID NO:110, SEQ ID NO:111 and by SEQ ID NO:112, wherein the the codons of SEQ ID NO:111 and SEQ ID NO:112 have been optimized for vector insertion. SEQ ID NO:112 includes flanking restriction sites. A polyligand of structure X-S1-Y-S2-Z is also called herein a heteropolyligand, shown generically in FIG. 4B.
SEQ ID NO:113 is an embodiment of a polyligand of the structure X-Y-S3-Y-X, wherein X is SEQ ID NO:53, Y is SEQ ID NO:54, wherein Xaa is alanine, and wherein S3 is a five amino acid spacer with the sequence PAGAG. The PKD polyligand of SEQ ID NO :113 is encoded by SEQ ID NO:114, SEQ ID NO:115, and by SEQ ID NO:116, wherein the the codons of SEQ ID NO:115 and SEQ ID NO:116 have been optimized for vector insertion. SEQ ID NO:116 includes flanking restriction sites. A polyligand of structure X-Y-S3-Y-X is also called herein a heteropolyligand, shown generically in FIG. 4C.
SEQ ID NOS:117-132 are full length PKD substrates. These sequences have the following public database accession numbers: AAH64840, NP000354, CAA79356, NP001015053, NP009112, NP001531, NP065789, AAH00029, AAB48596, NP—536728, AAH47282, NP004283, NP005219,CAB95729,NP—057541, NP—849231. Each of the protein sequences represented by these accession numbers is incorporated by reference herein. In SEQ ID NOS:117-132, the positions of the amino acid(s) phosphorylatable by PKD are represented by Xaa. In wild-type proteins, Xaa is serine or threonine. In the ligands of the invention, Xaa is any amino acid.
Amino acid sequences containing Xaa encompass polypeptides where Xaa is any amino acid.



DETAILED DESCRIPTION OF DRAWINGS
FIGS. 1A-1C show examples of homopolymeric ligands without spacers.
FIGS. 2A-2C show examples of homopolymeric ligands with spacers.
FIGS. 3A-3C show examples of heteropolymeric ligands without spacers.
FIGS. 4A-4C show examples of heteropolymeric ligands with spacers.
FIGS. 5A-5G show examples of ligands and polymeric ligands linked to an optional epitope tag.
FIGS. 6A-6G show examples of ligands and polymeric ligands linked to an optional reporter.
FIGS. 7A-7G show examples of ligands and polymeric ligands linked to an optional localization signal.
FIGS. 8A-8G show examples of ligands and polymeric ligands linked to an optional localization signal and an optional epitope tag.
FIGS. 9A-9G show examples of gene constructs where ligands and polyligands are linked to an optional localization signal, an optional epitope tag, and an optional reporter.
FIGS. 10A-10D show examples of vectors containing ligand gene constructs.
FIG. 11 shows an example of a sequential cloning process useful for combinatorial synthesis of polyligands.



BRIEF DESCRIPTION OF THE INVENTION
The invention relates to polypeptide ligands and polyligands for PKD. Various embodiments of the PKD ligands and polyligands are represented in SEQ ID NOS:1-132. More specifically, the invention relates to ligands, homopolyligands, and heteropolyligands that comprise any one or more of SEQ ID NOS:1-104. Additionally, the invention relates to ligands and polyligands comprising one or more subsequences of SEQ ID NOS:117-132 or any portion thereof. Furthermore, the invention relates to polyligands with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% sequence identity to a polyligand comprising one or more of SEQ ID NOS:1-104 or any portion thereof. Furthermore, the invention relates to polyligands with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% sequence identity to a polyligand comprising one or more subsequences of SEQ ID NOS:117-132 or portions thereof.
Polyligands, which can be homopolyligands or heteropolyligands, are chimeric ligands composed of two or more monomeric polypeptide ligands. An example of a monomeric ligand is the polypeptide represented by SEQ ID NO:61, wherein Xaa is any amino acid. An example of a homopolyligand is a polypeptide comprising a dimer or multimer of SEQ ID NO:61, wherein Xaa is any amino acid. An example of a heteropolyligand is a polypeptide comprising SEQ ID NO:1 and one or more of SEQ ID NOS:2-104, wherein Xaa is any amino acid. There are numerous ways to combine SEQ ID NOS:1-104 into homopolymeric or heteropolymeric ligands. Furthermore, there are numerous ways to combine portions or subsequences of SEQ ID NOS:117-132 with each other and with SEQ ID NOS:1-104 to make polymeric ligands that modulate PKD.
The polyligands of the invention optionally comprise spacer amino acids before, after, or between monomers. SEQ ID NO:105 is an embodiment of a polyligand of the structure X-S1-Y-S2-Z, wherein X is SEQ ID NO:53, Y is SEQ ID NO:55, Z is SEQ ID NO:58, wherein Xaa is alanine, and wherein S1 and S2 are four amino acid spacers. This invention intends to capture all combinations of homopolyligands and heteropolyligands without limitation to the examples given above or below. In this description, use of the term “ligand(s)” encompasses monomeric ligands, polymeric ligands, homopolymeric ligands and/or heteropolymeric ligands.
A monomeric ligand is a polypeptide where at least a portion of the polypeptide is capable of being recognized by PKD. The portion of the polypeptide capable of recognition is termed the recognition motif. In the present invention, recognition motifs can be natural or synthetic. Examples of recognition motifs are well known in the art and include, but are not limited to, naturally occurring PKD substrates and pseudosubstrate motifs.
A polymeric ligand comprises two or more monomeric ligands.
A homopolymeric ligand is a polymeric ligand where each of the monomeric ligands is identical in amino acid sequence, except that a phosphorylatable residue may be substituted or modified in one or more of the monomeric ligands.
A heteropolymeric ligand is a polymeric ligand where some of the monomeric ligands do not have an identical amino acid sequence.
The ligands of the invention are optionally linked to additional molecules or amino acids that provide an epitope tag, a reporter, and/or a cellular localization signal. The cellular localization signal targets the ligands to a region of a cell. The epitope tag and/or reporter and/or localization signal may be the same molecule. The epitope tag and/or reporter and/or localization signal may also be different molecules.
The invention also encompasses polynucleotides comprising a nucleotide sequence encoding ligands, homopolyligands, and heteropolyligands. The nucleic acids of the invention are optionally linked to additional nucleotide sequences encoding polypeptides with additional features, such as an epitope tag, a reporter, and/or a cellular localization signal. The polynucleotides are optionally flanked by nucleotide sequences comprising restriction endonuclease sites and other nucleotides needed for restriction endonuclese activity. The flanking sequences optionally provide unique cloning sites within a vector and optionally provide directionality of subsequence cloning. Further, the nucleic acids of the invention are optionally incorporated into vector polynucleotides. The ligands, polyligands, and polynucleotides of this invention have utility as research tools and/or therapeutics.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to ligands and polyligands that are PKD modulators. Various embodiments of ligands and polyligands are represented in SEQ ID NOS:1-132. Polyligands are chimeric ligands comprising two or more monomeric polypeptide ligands. An example of a monomeric ligand is the polypeptide represented by SEQ ID NO:54, wherein Xaa is any amino acid. Another example of a monomeric ligand is the polypeptide represented by SEQ ID NO:1. Each of SEQ ID NOS:1-104 represents an individual polypeptide ligand in monomeric form, wherein Xaa is any amino acid. Additional examples of monomeric ligands are subsequence portions of SEQ ID NOS:117-132 containing a PKD recognition motif. Monomeric ligand subsequences of SEQ ID NOS:117-132 may be wild-type subsequences. Additionally, monomeric ligand subsequences of SEQ ID NOS:117-132 may have the PKD phosphorylatable amino acids replaced by other amino acids. Furthermore, monomeric ligands and polyligands may have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a ligand comprising an amino acid sequence in one or more of SEQ ID NOS:1-104. Furthermore, monomeric ligands and polyligands may have at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% sequence identity to a subsequence of SEQ ID NOS:117-132.
An example of a homopolyligand is a polypeptide comprising a dimer or multimer of SEQ ID NO:66, wherein Xaa is any amino acid. An example of a heteropolyligand is a polypeptide comprising SEQ ID NO:104 and one or more of SEQ ID NOS:1-103, wherein Xaa is any amino acid. There are numerous ways to combine SEQ ID NOS:1-104 into homopolymeric or heteropolymeric ligands. Furthermore, there are numerous ways to combine PKD recogntition motif-containing portions of SEQ ID NOS:117-132 with each other and with SEQ ID NOS:1-104 to make polymeric ligands.
Polyligands may comprise any two or more of a sequence selected from SEQ ID NOS:1-104 and recognition motif-containing portions of SEQ ID NOS:117-132, wherein Xaa is any amino acid. A dimer or multimer of SEQ ID NO:91 is an example of a homopolyligand. An example of a heteropolyligand is a polypeptide comprising SEQ ID NO:5 and one or more of a recognition motif-containing portion of SEQ ID NOS:117-132. There are numerous ways to combine SEQ ID NOS:1-104 and portions of SEQ ID NOS:117-132 into homopolymeric or heteropolymeric ligands. The instant invention is directed to all possible combinations of homopolyligands and heteropolyligands without limitation.
SEQ ID NOS:53-104 and SEQ ID NOS:117-132 show polypeptides that contain at least one serine or threonine residue phosphorylatable by PKD, the positions of which are represented by Xaa. In nature, Xaa is, generally speaking, serine or threonine. In one embodiment of the instant invention, Xaa can be any amino acid. Ligands where Xaa is serine or threonine can be used as part of a polyligand, however in one embodiment, at least one phosphorylatable serine or threonine is replaced with another amino acid, such as one of the naturally occurring amino acids including, alanine, aspartate, asparagine, cysteine, glutamate, glutamine, phenylalanine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, arginine, valine, tryptophan, or tyrosine. The Xaa may also be a non-naturally occurring amino acid. In another embodiment, the PKD phosphorylatable serine(s) or threonine(s) are replaced by alanine. The ligands and polyligands of the invention are designed to modulate the endogenous effects of PKD.
In general, ligand monomers based on natural PKD substrates are built by isolating a putative PKD phosphorylation recognition motif in a PKD substrate. Sometimes it is desirable to modify the phosphorylatable residue to an amino acid other than serine or threonine. Additional monomers include the PKD recognition motif as well as amino acids adjacent and contiguous on either side of the PKD recognition motif. Monomeric ligands may therefore be any length provided the monomer includes the PKD recognition motif. For example, the monomer may comprise a PKD recognition motif and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30-100 or more amino acids adjacent to the recognition motif.
For example, in one embodiment, the invention comprises an inhibitor of PKD comprising at least one copy of a peptide selected from the group consisting of:

    a) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 194-201 of SEQ ID NO:117, wherein the amino acid residue corresponding to amino acid residue 199 of SEQ ID NO:117 is an amino acid residue other than serine or threonine;
    b) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 191-203 of SEQ ID NO:117, wherein the amino acid residue corresponding to amino acid residue 199 of SEQ ID NO:117 is an amino acid residue other than serine or threonine;
    c) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 189-205 of SEQ ID NO:117, wherein the amino acid residue corresponding to amino acid residue 199 of SEQ ID NO:117 is an amino acid residue other than serine or threonine; and
    d) a peptide at least 80% identical to a peptide comprising amino acid residues corresponding to amino acid residues 184-207 of SEQ ID NO:117, wherein the amino acid residue corresponding to amino acid residue 199 of SEQ ID NO:117 is an amino acid residue other than serine or threonine.


As used herein, the terms “correspond(s) to” and “corresponding to,” as they relate to sequence alignment, are intended to mean enumerated positions within a reference protein, e.g., HDAC5 (SEQ ID NO:119), and those positions that align with the positions on the reference protein. Thus, when the amino acid sequence of a subject peptide is aligned with the amino acid sequence of a reference peptide, e.g., SEQ ID NO:119, the amino acids in the subject peptide sequence that “correspond to” certain enumerated positions of the reference peptide sequence are those that align with these positions of the reference peptide sequence, but are not necessarily in these exact numerical positions of the reference sequence. Methods for aligning sequences for determining corresponding amino acids between sequences are described below.
Additional embodiments of the invention include monomers (as described above) based on any putative or real substrate for PKD, such as substrates identified by SEQ ID NOS:1-104 and SEQ ID NOS:117-132. Furthermore, if the substrate has more than one recognition motif, then more than one monomer may be identified therein.
Another embodiment of the invention is a nucleic acid molecule comprising a polynucleotide sequence encoding at least one copy of a ligand peptide.
Another embodiment of the invention is a nucleic acid molecule wherein the polynucleotide sequence encodes one or more copies of one or more peptide ligands.
Another embodiment of the invention is a nucleic acid molecule wherein the polynucleotide sequence encodes at least a number of copies of the peptide selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9 or 10.
Another embodiment of the invention is a vector comprising a nucleic acid molecule encoding at least one copy of a ligand or polyligand.
Another embodiment of the invention is a recombinant host cell comprising a vector comprising a nucleic acid molecule encoding at least one copy of a ligand or polyligand.
Another embodiment of the invention is a method of inhibiting PKD in a cell comprising transfecting a vector comprising a nucleic acid molecule encoding at least one copy of a ligand or polyligand into a host cell and culturing the transfected host cell under conditions suitable to produce at least one copy of the ligand or polyligand.
The invention also relates to modified inhibitors that are at least about 80%, 85%, 90% 95%, 96%, 97%, 98% or 99% identical to a reference inhibitor. A “modified inhibitor” is used to mean a peptide that can be created by addition, deletion or substitution of one or more amino acids in the primary structure (amino acid sequence) of a inhibitor protein or polypeptide. A “modified recognition motif” is a naturally occurring PKD recognition motif that has been modified by addition, deletion, or substitution of one or more amino acids in the primary structure (amino acid sequence) of the motif. For example, a modified PKD recognition motif may be a motif where the phosphorylatable amino acid has been modified to a non-phosphorylatable amino acid. The terms “protein” and “polypeptide” are used interchangeably herein. The reference inhibitor is not necessarily a wild-type protein or a portion thereof. Thus, the reference inhibitor may be a protein or peptide whose sequence was previously modified over a wild-type protein. The reference inhibitor may or may not be the wild-type protein from a particular organism.
A polypeptide having an amino acid sequence at least, for example, about 95% “identical” to a reference an amino acid sequence is understood to mean that the amino acid sequence of the polypeptide is identical to the reference sequence except that the amino acid sequence may include up to about five modifications per each 100 amino acids of the reference amino acid sequence encoding the reference peptide. In other words, to obtain a peptide having an amino acid sequence at least about 95% identical to a reference amino acid sequence, up to about 5% of the amino acid residues of the reference sequence may be deleted or substituted with another amino acid or a number of amino acids up to about 5% of the total amino acids in the reference sequence may be inserted into the reference sequence. These modifications of the reference sequence may occur at the N-terminus or C-terminus positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
As used herein, “identity” is a measure of the identity of nucleotide sequences or amino acid sequences compared to a reference nucleotide or amino acid sequence. In general, the sequences are aligned so that the highest order match is obtained. “Identity” per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g., 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); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); von Heinje, G., Sequence Analysis In Molecular Biology, Academic Press (1987); and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York (1991)). While there exist several methods to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to skilled artisans (Carillo, H. & Lipton, D., Siam J Applied Math 48:1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego (1994) and Carillo, H. & Lipton, D., Siam J Applied Math 48:1073 (1988). Computer programs may also contain methods and algorithms that calculate identity and similarity. Examples of computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 12(i):387 (1984)), BLASTP, ExPASy, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)) and FASTDB. Examples of methods to determine identity and similarity are discussed in Michaels, G. and Garian, R., Current Protocols in Protein Science, Vol 1, John Wiley & Sons, Inc. (2000), which is incorporated by reference. In one embodiment of the present invention, the algorithm used to determine identity between two or more polypeptides is BLASTP.
In another embodiment of the present invention, the algorithm used to determine identity between two or more polypeptides is FASTDB, which is based upon the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990), incorporated by reference). In a FASTDB sequence alignment, the query and subject sequences are amino sequences. The result of sequence alignment is in percent identity. Parameters that may be used in a FASTDB alignment of amino acid sequences to calculate percent identity include, but are not limited to: Matrix=PAM, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject amino sequence, whichever is shorter.
If the subject sequence is shorter or longer than the query sequence because of N-terminus or C-terminus additions or deletions, not because of internal additions or deletions, a manual correction can be made, because the FASTDB program does not account for N-terminus and C-terminus truncations or additions of the subject sequence when calculating percent identity. For subject sequences truncated at both ends, relative to the query sequence, the percent identity is corrected by calculating the number of amino acids of the query sequence that are N-and C-terminus to the reference sequence that are not matched/aligned, as a percent of the total bases of the query sequence. The results of the FASTDB sequence alignment determine matching/alignment. The alignment percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score can be used for the purposes of determining how alignments “correspond” to each other, as well as percentage identity. Residues of the query (subject) sequences or the reference sequence that extend past the N-or C-termini of the reference or subject sequence, respectively, may be considered for the purposes of manually adjusting the percent identity score. That is, residues that are not matched/aligned with the N-or C-termini of the comparison sequence may be counted when manually adjusting the percent identity score or alignment numbering.
For example, a 90 amino acid residue subject sequence is aligned with a 100 residue reference sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a match/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 FASTDB 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 reference 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 FASTDB is not manually corrected.
The polyligands of the invention optionally comprise spacer amino acids before, after, or between monomers. The length and composition of the spacer may vary. An example of a spacer is glycine, alanine, polyglycine, or polyalanine. Specific examples of spacers used between monomers in SEQ ID NO:105 are the four amino acids AGAG, and the four amino acids GAGA. Spacer amino acids may be any amino acid and are not limited to alanine and glycine. The instant invention is directed to all combinations of homopolyligands and heteropolyligands, with or without spacers, and without limitation to the examples given above or below.
The ligands and polyligands of the invention are optionally linked to additional molecules or amino acids that provide an epitope tag, a reporter, and/or localize the ligand to a region of a cell (See FIGS. 5A-5G, FIGS. 6A-6G, FIGS. 7A-7G, and FIGS. 8A-8G). Non-limiting examples of epitope tags are FLAG (Kodak; Rochester, N.Y.), HA (hemagluttinin), c-Myc and His6. Additional examples of epitope tags are given in Jarvik & Telmer 1998 Annual Reviw of Genetics 32:601-18. Non-limiting examples of reporters are alkaline phosphatase, galactosidase, peroxidase, luciferase and green fluorescent protein (GFP). Non-limiting examples of cellular localizations are sarcoplamic reticulum, endoplasmic reticulum, mitochondria, golgi apparatus, nucleus, plasma membrane, apical membrane, and basolateral membrane. The epitopes, reporters and localization signals are given by way of example and without limitation. The epitope tag, reporter and/or localization signal may be the same molecule. The epitope tag, reporter and/or localization signal may also be different molecules.
Ligands and polyligands and optional amino acids linked thereto can be synthesized chemically or recombinantly using techniques known in the art. Chemical synthesis techniques include but are not limited to peptide synthesis which is often performed using an automated peptide synthesizer. Pepetides can also be synthesized utilizing non-automated peptide sythesis methods known in the art. Recombinant techniques include insertion of ligand-encoding nucleic acids into expression vectors, wherein nucleic acid expression products are synthesized using cellular factors and processes.
Linkage of a cellular localization signal, epitope tag, or reporter to a ligand or polyligand can include covalent or enzymatic linkage to the ligand. When the localization signal comprises material other than a polypeptide, such as a lipid or carbohydrate, a chemical reaction to link molecules may be utilized. Additionally, non-standard amino acids and amino acids modified with lipids, carbohydrates, phosphate or other molecules may be used as precursors to peptide synthesis. The ligands of the invention have therapeutic utility with or without localization signals. However, ligands linked to localization signals have utility as subcellular tools or therapeutics. For example, ligands depicted generically in FIGS. 7A-7G and FIGS. 8A-8G represent ligands with utility as subcellular tools or therapeutics. PKD ligand-containing gene constructs are also delivered via gene therapy. FIGS. 10B and 10C depict embodiments of gene therapy vectors for delivering and controlling polypeptide expression in vivo. Polynucleotide sequences linked to the gene construct in FIGS. 10B and 10C include genome integration domains to facilitate integration of the transgene into a viral genome and/or host genome.
FIG. 10A shows a vector containing a PKD ligand gene construct, wherein the ligand gene construct is releasable from the vector as a unit useful for generating transgenic animals. For example, the ligand gene construct, or transgene, is released from the vector backbone by restriction endonuclease digestion. The released transgene is then injected into pronuclei of fertilized mouse eggs; or the transgene is used to transform embryonic stem cells. The vector containing a ligand gene construct of FIG. 10A is also useful for transient transfection of the trangene, wherein the promoter and codons of the transgene are optimized for the host organism. The vector containing a ligand gene construct of FIG. 10A is also useful for recombinant expression of polypeptides in fermentable organisms adaptable for small or large scale production, wherein the promoter and codons of the transgene are optimized for the fermentation host organism.
FIG. 10D shows a vector containing a PKD ligand gene construct useful for generating stable cell lines.
The invention also encompasses polynucleotides comprising nucleotide sequences encoding ligands, homopolyligands, and heteropolyligands. The polynucleotides of the invention are optionally linked to additional nucleotide sequences encoding epitopes, reporters and/or localization signals. Further, the nucleic acids of the invention are optionally incorporated into vector polynucleotides. The polynucleotides are optionally flanked by nucleotide sequences comprising restriction endonuclease sites and other nucleotides needed for restriction endonuclese activity. The flanking sequences optionally provide cloning sites within a vector. The restriction sites can include, but are not limited to, any of the commonly used sites in most commercially available cloning vectors. Examples of such sites are those recognized by BamHI, ClaI, EcoRI, EcoRV, SpeI, AflII, NdeI, NheI, XbaI, XhoI, SphI, NaeI, SexAI, HindIII, HpaI, and PstI restriction endonucleases. Sites for cleavage by other restriction enzymes, including homing endonucleases, are also used for this purpose. The polynucleotide flanking sequences also optionally provide directionality of subsequence cloning. It is preferred that 5′ and 3′ restriction endonuclease sites differ from each other so that double-stranded DNA can be directionally cloned into corresponding complementary sites of a cloning vector.
Ligands and polyligands with or without localization signals, epitopes or reporters are alternatively synthesized by recombinant techniques. Polynucleotide expression constructs are made containing desired components and inserted into an expression vector. The expression vector is then transfected into cells and the polypeptide products are expressed and isolated. Ligands made according to recombinant DNA techniques have utility as research tools and/or therapeutics.
An example of how nucleotide sequences encoding ligands, homopolyligands, and heteropolyligands are produced is as follows. Complimentary oligonucleotides encoding the ligands and flanking sequences are synthesized and annealled. The resulting double-stranded DNA molecule is inserted into a cloning vector using techniques known in the art. When the ligands and polyligands are placed in-frame adjacent to sequences within a transgenic gene construct that is translated into a protein product, they form part of a fusion protein when expressed in cells or transgenic animals.
Another embodiment of the invention relates to selective control of transgene expression in a desired cell or organism. The promotor portion of the recombinant gene can be a constitutive promotor, a non-constitutive promotor, a tissue-specific promotor (constitutive or non-constitutive) or a selectively controlled promotor. Different selectively controlled promotors are controlled by different mechanisms. RheoSwitchR is an inducible promotor system available from RheoGene. Temperature sensitive promotors can also be used to increase or decrease gene expression. An embodiment of the invention comprises a ligand or polyligand gene construct whose expression is controlled by an inducible promotor.
Polyligands are modular in nature. An aspect of the instant invention is the combinatorial modularity of the disclosed polyligands. Another aspect of the invention are methods of making these modular polyligands easily and conveniently. In this regard, an embodiment of the invention comprises methods of modular subsequence cloning of genetic expression components. When the ligands, homopolyligands, heteropolyligands and optional amino acid expression components are synthesized recombinantly, one can consider each clonable element as a module. For speed and convenience of cloning, it is desirable to make modular elements that are compatible at cohesive ends and are easy to insert and clone sequentially. This is accomplished by exploiting the natural properties of restriction endonuclease site recognition and cleavage. One aspect of the invention encompasses module flanking sequences that, at one end of the module, are utilized for restriction enzyme digestion once, and at the other end, utilized for restriction enzyme digestion as many times as desired. In other words, a restriction site in one part of the module is utilized and destroyed in order to effect sequential cloning of modular elements. An example of restriction sites flanking a coding region of interest are sequences recognized by the restriction enzymes NgoM IV and Cla I; or Xma I and Cla I. Cutting a first circular DNA with NgoM IV and Cla I to yield linear DNA with a 5′ NgoM IV overhang and a 3′ Cla I overhang; and cutting a second circular DNA with Xma I and Cla I to yield linear DNA with a 5′ Cla I overhang and a 3′ Xma I overhang generates first and second DNA fragments with compatible cohesive ends. When these first and second DNA fragments are mixed together, annealed, and ligated to form a third circular DNA fragment, the NgoM IV site that was in the first DNA and the Xma I site that was in the second DNA are destroyed in the third circular DNA. Now this vestigial region of DNA is protected from further Xma I or NgoM IV digestion, but flanking sequences remaining in the third circular DNA still contain intact 5′ NgoM IV and 3′ Cla I and Xma I sites. This process can be repeated numerous times to achieve directional, sequential, modular cloning events. Restriction sites recognized by NgoM IV, Xma I, and Cla I endonucleases represent a group of sites that permit sequential cloning when used as flanking sequences.
Another way to assemble coding region modules directionally and sequentially employs linear DNA in addition to circular DNA. For example, like the sequential cloning process described above, restriction sites flanking a coding region module are sequences recognized by the restriction enzymes NgoM IV and Cla I; or Xma I and Cla I. Referring to FIG. 11, a first circular DNA is cut with Xma I and Cla I to yield linear DNA with a 5′ Cla I overhang and a 3′ Xma I overhang. A second linear double-stranded DNA is generated by PCR amplification followed by digestion, or by synthesizing and annealing complimentary oligonucleotides. The second linear DNA has 5′ NgoM IV overhang and a 3′ Cla I overhang, which are compatible cohesive ends with the first DNA linearized. When these first and second DNA fragments are mixed together, annealed, and ligated to form a third circular DNA fragment, the Xma I site that was in the first DNA and the NgoM IV site that was in the second DNA are destroyed in the third circular DNA. Flanking sequences remaining in the third circular DNA still contain intact 5′ N NgoM IV and 3′ Cla I and Xma I sites. This process can be repeated numerous times to achieve directional, sequential, modular cloning events. Restriction sites recognized by NgoM IV, Xma I, and Cla I endonucleases represent a group of sites that permit sequential cloning when used as flanking sequences. This process is depicted in FIG. 11.
One of ordinary skill in the art recognizes that other restriction site groups can accomplish sequential, directional cloning as described herein. Preferred criteria for restriction endonuclease selection are selecting a pair of endonucleases that generate compatible cohesive ends but whose sites are destroyed upon ligation with each other. Another criteria is to select a third endouclease site that does not generate sticky ends compatible with either of the first two. When such criteria are utilized as a system for sequential, directional cloning, ligands, polyligands and other coding regions or expression components can be combinatorially assembled as desired. The same sequential process can be utilized for epitope, reporter, and/or localization signals.
Polyligands and methods of making polyligands that modulate PKD activity are disclosed. Therapeutics include delivery of purified ligand or polyligand with or without a localization signal to a cell. Alternatively, ligands and polyligands with or without a localization signals are delivered via adenovirus, lentivirus, adeno-associated virus, or other viral constructs that express protein product in a cell.
EXAMPLE 1
A polypeptide comprising a heteropolyligand, an endoplasmic reticulum cellular localization signal, and a His6 epitope is synthesized. Examples of such polypeptides are generically represented by FIGS. 8B, 8D, 8E and 8F. The polypeptide is synthesized on an automated peptide synthesizer or is recombinantly expressed and purified. Purified polypeptide is solubilized in media and added to cells. The polypeptide is endocytosed by the cells, and transported to the endoplasmic reticulum. Verification is performed by immunohistochemical staining using an anti-His6 antibody.
EXAMPLE 2
A transgene is constructed using a human cytomegalovirus (CMV) promoter to direct expression of a fusion protein comprising SEQ ID NO:113 (POLYLIGAND), green fluorescent protein (REPORTER), and a nuclear localization signal (LOCALIZATION SIGNAL). Such a transgene is generically represented by FIG. 9A-9D. The transgene is transfected into cells for transient expression. Verification of expression and location is performed by visualization of green fluorescent protein (GFP) by confocal microscopy.
EXAMPLE 3
A transgene construct is built to produce a protein product with expression driven by a tissue-specific promoter. The transgene comprises a synthetic gene expression unit engineered to encode three domains. Each of these three domains is synthesized as a pair of complimentary polynucleotides that are annealed in solution, ligated and inserted into a vector. Starting at the amino-terminus, the three domains in the expression unit are nucleotide sequences that encode a PKD ligand, a FLAGTM epitope, and an endoplasmic reticulum localization signal. The PKD ligand is a monomeric ligand, homopolymeric ligand or heteropolymeric ligand as described herein. Nucleotide sequences encoding a FLAGTM epitope are placed downstream of nucleotide sequences encoding the PKD ligand. Finally, nucleotide sequences encoding the localization signal are placed downstream of those encoding the FLAGTM epitope. The assembled gene expression unit is subsequently subcloned into an expression vector, such as that shown in FIG. 10A, and used to transiently transfect cells. Verification is performed by immunohistochemical staining using an anti-FLAGTM antibody.
EXAMPLE 4
Ligand function and localization is demonstrated in vivo by making a transgene construct used to generate mice expressing a ligand fusion protein targeted to the endoplasmic reticulum. The transgene construct is shown generically in FIG. 10B. The expression unit contains nucleotides that encode a tetramer of SEQ ID NO:33, a hemagluttinin epitope, and a mitochondrial localization signal. This expression unit is subsequently subcloned into a pBluscript-based vector (Stratagene; La Jolla, Calif.) between nucleotide sequences including a promoter and an SV40 polyadenylation signal. The completed transgene is then injected into pronuclei of fertilized mouse oocytes. The resultant pups are screened for the presence of the transgene by PCR. Transgenic founder mice are bred with wild-type mice. Heterozygous transgenic animals from at least the third generation are used for the following tests, with their non-transgenic littermates serving as controls.
Test 1: Southern blotting analysis is performed to determine the copy number. Southern blots are hybridized with a radio-labeled probe generated from a fragment of the transgene. The probe detects bands containing DNA from transgenic mice, but does not detect bands containing DNA from non-transgenic mice. Intensities of the transgenic mice bands are measured and compared with the transgene plasmid control bands to estimate copy number. This demonstrates that mice in Example 4 harbor the transgene in their genomes.
Test 2: Tissue homogenates are prepared for Western blot analysis. This experiment demonstrates the transgene is expressed in tissues of transgenic mice because hemagluttinin epitope is detected in transgenic homogenates but not in non-transgenic homogenates.
These examples demonstrate delivery of ligands to a localized region of a cell for therapeutic or experimental purposes. The purified polypeptide ligands can be formulated for oral or parenteral administration, topical administration, or in tablet, capsule, or liquid form, intranasal or inhaled aerosol, subcutaneous, intramuscular, intraperitoneal, or other injection; intravenous instillation; or any other routes of administration. Furthermore, the nucleotide sequences encoding the ligands permit incorporation into a vector designed to deliver and express a gene product in a cell. Such vectors include plasmids, cosmids, artificial chromosomes, and modified viruses. Delivery to eukaryotic cells can be accomplished in vivo or ex vivo. Ex vivo delivery methods include isolation of the intended recipient's cells or donor cells and delivery of the vector to those cells, followed by treatment of the recipient with the cells.
Disclosed are ligands and polyligands that modulate PKD activity and methods of making and using these ligands. The ligands and polyligands are synthesized chemically or recombinantly and are utilized as research tools or as therapeutics. The invention includes linking the ligands and polyligands to cellular localization signals for subcellular therapeutics.

## Claims

1. An isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 105, 109 or 113.

2. The polypeptide of claim 1 linked to a subcellular localization signal.

3. The polypeptide of claim 1 linked to an epitope.

4. The polypeptide of claim 1 linked to a reporter.

5. A composition comprising the polypeptide of claim 1.

6. An isolated polynucleotide comprising a polynucleotide encoding the polypeptide of claim 1.

7. A vector comprising the polynucleotide of claim 6.

8. A recombinant host cell comprising the vector of claim 7.

9. The polynucleotide of claim 6, wherein the polynucleotide is flanked on one end by a sequence cleavable by a first restriction endonuclease, and wherein the polynucleotide is flanked on the other end by a sequence cleavable by a second restriction endonuclease, and wherein the first and second restriction endonucleases generate noncompatible cohesive ends.

10. A method of inhibiting PKD in a cell comprising transfecting the vector of claim 7 into a host cell and culturing the transfected host cell under conditions suitable to produce at least one copy of the polypeptide, thereby inhibiting PKD.

11. The isolated polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 105.

12. The isolated polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 109.

13. The isolated polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 113.

14. The isolated polynucleotide of claim 6, wherein the polynucleotide comprises the polynucleotide of SEQ ID NO: 105.

15. The isolated polynucleotide of claim 6, wherein the polynucleotide comprises the polynucleotide of SEQ ID NO: 109.

16. The isolated polynucleotide of claim 6, wherein the polynucleotide comprises the polynucleotide of SEQ ID NO: 113.

17. The method of claim 10, wherein the vector comprises the polynucleotide of SEQ ID NO: 105.

18. The method of claim 10, wherein the vector comprises the polynucleotide of SEQ ID NO: 109.

19. The method of claim 10, wherein the vector comprises the polynucleotide of SEQ ID NO: 113.