Patent Document

[0001]    This application is a regular application filed under 35 USC § 111 (a), claiming priority from U.S. Provisional Patent Application No. 60/039,204 filed Feb. 28, 1997. 
     
    
     
       TECHNICAL FIELD  
         [0002]    The present invention relates to novel human calcium channel compositions, and to the expression of these compositions in cell lines for use in evaluating calcium channel function.  
         BACKGROUND OF THE INVENTION  
         [0003]    The rapid entry of calcium into cells is mediated by a class of proteins called voltage-gated calcium channels. Calcium channels are a heterogeneous class of molecules that respond to depolarization by opening a calcium-selective pore through the plasma membrane. The entry of calcium into cells mediates a wide variety of cellular and physiological responses including excitation-contraction coupling, hormone secretion and gene expression. In neurons, calcium entry directly affects membrane potential and contributes to electrical properties such as excitability, repetitive firing patterns and pacemaker activity. Miller, R. J. (1987) Multiple calcium channels and neuronal function. Science 235:46-52. Calcium entry further affects neuronal functions by directly regulating calcium-dependent ion channels and modulating the activity of calcium-dependent enzymes such as protein kinase C and calmodulin-dependent protein kinase II. An increase in calcium concentration at the presynaptic nerve terminal triggers the release of neurotransmitter. Calcium entry also plays a role in neurite outgrowth and growth cone migration in developing neurons and has been implicated in long-term changes in neuronal activity. In addition to the variety of normal physiological functions mediated by calcium channels, they are also implicated in a number of human disorders. Recently, mutations identified in human and mouse calcium channel genes have been found to account for several disorders including, familial hemiplegic migraine, episodic ataxia type 2, cerebellar ataxia, absence epilepsy and seizures. Fletcher, C. F., Lutz, C. M., O&#39;Sullivan, T. N., Shaughnessy, Jr., J. D., Hawkes, R., Frankel, W. N., Copeland, N. G. and Jenkins, N. A. (1996) Absence epilepsy in tottering mutant mice is associated with calcium channel defects. Cell 87:607-617; Burgess, D. L., Jones, J. M., Meiser, M. H. and Noebels, J. L. (1997) Mutation of the Ca2+channel β subunit gene Cchb4 is associated with ataxia and seizures in the lethargic (lh) mouse. Cell 88:385-392; Ophoff, R. A., Terwindt, G. M., Vergouwe, M. N., van Eijk, R., Oefner, P. J., Hoffman, S. M. G., Lamerdin, J. E., Mohrenweiser, H. W., Bulman, D. E., Ferrari, M., Haan, J., Lindhout, D., van Ommen, G. -J. B., Hofker, M. H., Ferrari, M. D. and Frants, R. R. (1996) Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+channel gene CACNL1A4. cell 87:543-552; Zhuchenko, O., Bailey, J., Bonnen, P., Ashizawa, T., Stockton, D. W., Amos, C., Dobyns, W. B., Subramony, S. H., Zogbhbi, H. Y. and Lee, C. C. (1997) Autosomal dominat cerebellar ataxia (SCA6) associated with the small polyglutamine expansions in the α1A-voltage-dependent calcium channel. Nature Genetics 15:62-69. The clinical treatment of some disorders has been aided by the development of therapeutic calcium channel antagonists. Janis, R. J and Triggle, D. J. (1991) In Calcium Channels: Their Properties, Functions, Regulation and Clinical Relevance. CRC Press, London.  
           [0004]    Native calcium channels have been classified by their electrophysiological and pharmacological properties as T, L, N, P and Q types (for reviews see McCleskey, E. W. and Schroeder, J. E. (1991) Functional properties of voltage-dependent calcium chnanels. Curr. Topics Membr. 39: 295-326, and Dunlap, K., Luebke, J. I. and Turner, T. J. (1995) Exocytotic Ca 2+  channels in mammalian central neurons. Trends Neurosci. 18:89-98.). T-type (or low voltage-activated) channels describe a broad class of molecules that transiently activate at negative potentials and are highly sensitive to changes in resting potential. The L, N, P and Q-type channels activate at more positive potentials and display diverse kinetics and voltage-dependent properties. There is some overlap in biophysical properties of the high voltage-activated channels, consequently pharmacological profiles are useful to further distinguish them. L-type channels are sensitive to dihydropyridine (DHP) agonists and antagonists, N-type channels are blocked by the  Conus geographus  peptide toxin, ω-conotoxin GVIA, and P-type channels are blocked by the peptide ω-agatoxin IVA from the venom of the funnel web spider,  Agelenopsis aperta . A fourth type of high voltage-activated Ca channel (Q-type) has been described, although whether the Q- and P-type channels are distinct molecular entities is controversial (Sather, W. A., Tanabe, T., Zhang, J. -F., Mori, Y., Adams, M. E., and Tsien, R. W. (1993) Distinctive biophysical and pharmacological properties of class A (B 1) calcium channel α1 subunits. Neuron 11: 291-303; Stea, A., Tomlinson, W. J., Soong, T. W., Bourinet, E., Dubel, S. J., Vincent, S. R and Snutch, T. P. (1994) Localization and functional properties of a rat brain α1A calcium channel reflect similarities to neuronal Q- and P-type channels. PNAS 91: 10576-10580.). Several types of calcium conductances do not fall neatly into any of the above categories and there is variability of properties even within a category suggesting that additional calcium channels subtypes remain to be classified.  
           [0005]    Biochemical analyses show that neuronal calcium channels are heterooligomeric complexes consisting of three distinct subunits (α 1 , α 2 δ and β) (reveiwed by De Waard, M., Gurnett, C. A. and Campbell, K. P. (1997) In Ion Channels, Volume 4, edited by Narahashi, T. Plenum Press, New York). The α 1  subunit is the major pore-forming subunit and contains the voltage sensor and binding sites for calcium channel antagonists. The mainly extracellular α 2  is disulphide-linked to the transmembrane δ subunit and both are derived from the same gene and are proteolytically cleaved in vivo. The β subunit is a non-glycosylated, hydrophilic protein with a high affinity of binding to a cytoplasmic region of the α 1  subunit. A fourth subunit, γ, is unique to L-type Ca channels expressed in skeletal muscle T-tubules. The isolation and characterization of γ-subunit-encoding cDNAs is described in U.S. Pat. No. 5,386,025 which is incorporated herein by reference.  
           [0006]    Molecular cloning has revealed the cDNA and corresponding amino acid sequences of six different types of α 1  subunits (α 1A , α 1B , α 1C , α 1D , α 1E  and α 1S ) and four types of β subunits (β 1 , β 2 , β 3  and β 4 ) (reviewed in Stea, A., Soong, T. W. and Snutch, T. P. (1994) Voltage-gated calcium channels. PCT Patent Publication WO 95/04144, which is incorporated herein by reference, discloses the sequence and expression of α 1E  calcium channel subunits. In Handbook of Receptors and Channels. Edited by R. A. North, CRC Press.).  
           [0007]    The different classes of (α1 and β subunits have been identified in different animals including, rat, rabbit and human and share a significant degree of amino acid conservation across species (for examples see: Castellano, A., Wei, X., Bimbaumer, L., and Perez-Reyes, E. (1993) Cloning and expression of a third calcium channel β subunit. J. Biol. Chem. 268: 3450-3455; Castellano, A., Wei, X., Bimbaumer, L., and Perez-Reyes, E. (1993) Cloning and expression of a neuronal calcium channel β subunit. J. Biol. Chem. 268: 12359-12366; Dubel, S. J., Starr, T. V. B., Hell, J., Ahlijanian, M. K., Enyeart, J. J., Catterall, W. A., and Snutch, T. P. (1992). Molecular cloning of the α 1  subunit of an co-conotoxin-sensitive calcium channel. Proc. Natl. Acad. Sci. USA 89: 5058-5062; Fujita, Y., Mynlieff, M., Dirksen, R. T., Kim, M., Niidome, T., Nakai, J., Friedrich, T., Iwabe, N., Miyata, T., Furuichi, T., Furutama, D., Mikoshiba, K., Mori, Y., and Beam, K. G. (1993) Primary structure and functional expression of the ω-conotoxin-sensitive N-type calcium channel from rabbit brain. Neuron 10: 585-598; Mikami, A., Imoto, K., Tanabe, T., Niidome, T., Mori, Y., Takeshima, H., Narumiya, S., and Numa, S. (1989). Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel. Nature 340: 230-233; Mori, Y., Friedrich, T., Kim, M. -S., Mikami, A., Nakai, J., Ruth, P., Bosse, E., Hofmann, F., Flockerzi, V., Furuichi, T., Mikoshiba, K., Imoto, K., Tanabe, T., and Numa, S. (1991) Primary structure and functional expression from complementary DNA of a brain calcium channel. Nature 350: 398-402; Perez-Reyes, E., Castellano, A., Kim, H. S., Bertrand, P., Baggstrom, E., Lacerda, A. E., Wei, X., and Bimbaumer, L. (1992). Cloning and expression of a cardiac/brain β subunit of the L-type calcium channel. S. Biol. Chem. 267: 1792-1797; Pragnell, M., Sakamoto, J., Jay, S. D., and Campbell, K. P. (1991). Cloning and tissue-specific expression of the brain calcium channel β-subunit. FEBS Lett. 291: 253-258; Snutch, T. P., Tomlinson, W. J., Leonard, J. P., and Gilbert, M. M. (1991) Distinct calcium channels are generated by alternative splicing and are differentially expressed in the mammalian CNS. Neuron 7: 45-57; Soong, T. W., Stea, A., Hodson, C. D., Dubel, S. J., Vincent, S. R., and Snutch, T. P. (1993) Structure and functional expression of a member of the low voltage-activated calcium channel family. Science 260: 1133-1136; Tomlinson, W. J., Stea, A., Bourinet, E., Charnet, P., Nargeot, J., and Snutch, T. P. (1993) Functional properties of a neuronal class C L-type channel. Neuropharmacology 32: 1117-1126; Williams, M. E., Feldman, D. H., McCue, A. F., Brenner, R., Velicelebi, G., Ellis, S. B., and Harpold, M. M. (1992) Structure and functional expression of α1, α2, and β subunits of a novel human neuronal calcium channel subtype. Neuron 8: 71-84; Williams, M. E., Brust, P. F., Feldman, D. H., Patthi, S., Simerson, S., Maroufi, A., McCue, A. F., Velicelebi, G., Ellis, S. B., and Harpold, M. (1992) Structure and functional expression of an Co-conotoxin-sensitive human N-type calcium channel. Science 257: 389-395.  
           [0008]    In some expression systems the α 1  subunits alone can form functional calcium channels although their electrophysiological and pharmacological properties can be differentially modulated by coexpression with any of the four β subunits. Until recently, the reported modulatory affects of β subunit coexpression were to mainly alter kinetic and voltage-dependent properties. More recently it has been shown that β subunits also play crucial roles in modulating channel activity by protein kinase A, protein kinase C and direct G-protein interaction. (Bourinet, E., Charnet, P., Tomlinson, W. J., Stea, A., Snutch, T. P. and Nargeot, J. (1994) Voltage-dependent facilitation of a neuronal α1C L-type calcium channel. EMBO J. 13: 5032-5039; Stea, A., Soong, T. W. and Snutch, T. P. (1995) Determinants of PKC-dependent modulation of a family of neuronal calcium channels. Neuron 15:929-940; Bourinet, E., Soong, T. W., Stea, A. and Snutch, T. P. (1996) Determinants of the G-protein-dependent opioid modulation of neuronal calcium channels. Proc. Natl. Acad. Sci. (USA) 93: 1486-1491.)  
           [0009]    The electrophysiological and pharmacological properties of the calcium channels cloned to date can be summarized as shown in Table 1. While the cloned α 1  subunits identified to date correspond to several of the calcium channels found in cells, they do not account for all types of calcium conductances described in native cells. For example, they do not account for the various properties described for the heterogenous family described as T-type calcium channels. Furthermore, they do not account for novel calcium channels described in cerebellar granule cells or other types of cells. (Forti, L. and Pietrobon, D. (1993) Functional diversity of L-type calcium channels in rat cerebellar neurons. Neuron 10: 437-450; Tottene, A., Moretti, A., Pietrobon, A. 1996. Functional diversity of P-type and R-type calcium channels in rat cerebellar neurons. J. Neurosci. 16: 6353-6363).  
           [0010]    Because of the importance of calcium channels in cellular metabolism and human disease, it would be desirable to identify the remaining classes of α 1  subunits, and to develop expression systems for these subunits which would permit the study and characterization of these calcium channels, including the study of pharmacological modulators of calcium channel function. Thus, it is an object of the present invention to provide heretofor undisclosed calcium channels having novel α 1  subunits, including cell lines expressing these new calcium channels. It is a further object of the present invention to provide a method for testing these novel calcium channels using such cell lines.  
                                                                         TABLE 1                                   ω-conotoxin   1,4-       ω-agatoxin   ω-conotoxin   native           GVIA   dihydropyridines   cadmium   IVA   MVIIC   Ca 2+  channel type                                    α 1A     —   —   ✓   ✓   ✓   P/Q-type       α 1B     ✓   —   ✓   —   ✓   N-type       α 1C     —   ✓   ✓   —   —   L-type       α 1D     —   ✓   ✓   —   —   L-type       α 1E     —   —   ✓   —   —   novel       α 1S     —   ✓   ✓   —   —   L-type                  
 
         SUMMARY OF THE INVENTION  
         [0011]    The present invention provides partial sequences for a novel mammalian (human and rat sequences identified) calcium channel subunit which we have labeled as the α 1I  subunit, and an additional novel human calcium channel which we have labeled as the α 1H  subunit. This knowledge of the sequence of these two calcium channels permits the localization and recovery of the complete sequence from human cells, and the development of cell lines which express the novel calcium channels of the invention. These cells may be used for identifying compounds capable of acting as agonists or antagonists to the calcium channels. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0012]    [0012]FIG. 1 shows aligned amino acid sequences for the  C. elegans  C54D2.5 α 1  calcium channel subunit and initially identified portions of the calcium channel subunits of the invention.  
       DESCRIPTION OF THE INVENTION  
       [0013]    The present invention includes the following aspects for which protection is sought:  
         [0014]    (a) novel human calcium channel subunits and DNA fragments encoding such subunits. It will be appreciated that polymorphic variations may be made or may exist in the DNA of some individuals leading to minor deviations in the DNA or amino acids sequences from those shown which do not lead to any substantial alteration in the function of the calcium channel. Such variations, including variations which lead to substitutions of amino acids having similar properties are considered to be within the scope of the present invention.  
         [0015]    (b) polynucleotide sequences useful as probes in screening human cDNA libraries for genes encoding these novel calcium channel subunits. These probes can also be used in histological assay to determine the tissue distribution of the novel calcium channel subunits.  
         [0016]    (c) eukaryotic cell lines expressing the novel calcium channel subunits. These cell lines can be used to evaluate compounds as pharmacological modifiers of the function of the novel calcium channel subunits.  
         [0017]    (d) a method for evaluating compounds as pharmacological modifiers of the function of the novel calcium channel subunits using the cell lines expressing those subunits alone or in combination with other calcium channel subunits.  
         [0018]    Further, since defects in the novel calcium channel subunits may be associated with a human genetic disease including, but not limited to; epilepsy, migraine, ataxia, schizophrenia, hypertension, arrhythmia, angina, depression, small lung carcinoma, Lambert-Eaton syndrome, characterization of such associations and ultimately diagnosis of associated diseases can be carried out with probes which bind to the wild-type or defective forms of the novel calcium channels.  
         [0019]    In accordance with the present invention, we have identified human DNA sequences which code for novel calcium channel α 1  subunits. These subunits are believed to represent two new types of α 1  subunits of human voltage-dependent calcium channels which have been designated as type α 1I  and type α 1H .  
         [0020]    The novel α 1  subunits of the invention were identified by screening the  C. elegans  genomic DNA sequence data base for sequences homologous to previously identified mammalian calcium channel α 1  subunits. Specifically, the following twelve mammalian α 1  subunit sequences were used to screen the  C. elegans  genomic data bank:  
         [0021]    rat brain α 1A : GTCAAAACTC AGGCCTTCTA CTGG SEQ ID. No. 1  
         [0022]    rat brain α 1A : AACGTGTTCT TGGCTATCGC GGTG SEQ ID. No. 2  
         [0023]    rat brain α 1B : GTGAAAGCAC AGAGCTTCTA CTGG SEQ ID. No. 3  
         [0024]    rat brain α 1B : AACGTTTTCT TGGCCATTGC TGTG SEQ ID. No. 4  
         [0025]    rat brain α 1C : GTTAAATCCA ACGTCTTCTA CTGG SEQ ID. No. 5  
         [0026]    rat brain α 1C : AATGTGTTCT TGGCCATTGC GGTG SEQ ID. No. 6  
         [0027]    rat brain α 1D : GTGAAGTCTG TCACGTTTTA CTGG SEQ ID. No. 7  
         [0028]    rat brain α 1D : AAGCTCTTCT TGGCCATTGC TGTA SEQ ID. No. 8  
         [0029]    rat brain α 1E : GTCAAGTCGC AAGTGTTCTA CTGG SEQ ID. No. 9  
         [0030]    rat brain α 1E : AATGTATTCT TGGCTATCGC TGTG SEQ ID. No. 10  
         [0031]    rat brain consensus #1: ATCTAYGCYR TSATYGGSAT G SEQ ID. No. 11  
         [0032]    rat brain consensus #2: ATGGACAAYT TYGASTAYTC SEQ ID. No. 12  
         [0033]    This search identified four distinct  C. elegans  cosmids that contain open reading frames (coding regions) that exhibit homology to mammalian calcium channel α 1  subunits:  
         [0034]    cosmid and reading frame T02C5.5  
         [0035]    cosmid and reading frame C48A7.1  
         [0036]    cosmid and reading frame C54D2.5  
         [0037]    cosmid and reading frame C27F2.3  
         [0038]    Examination of the four  C. elegans  cosmid sequences by phylogeny analysis shows that two of these, T02C5.5 and C48A7.1, correspond closely with previously identified mammalian α 1  subunits. T02C5.5 appears to be an ancestral member related to the mammalian α 1A , α 1B  and α 1E  subunits. C48A7.1 appears to be an ancestral member related to the mammalian L-type channels encoded by α 1C , α 1D  and α 1S . In contrast, the  C. elegans  cosmids C54D2.5 and C27F2.3 identify novel types of calcium channel α 1  subunits distinct from the other mammalian subtypes.  
         [0039]    Mammalian counterparts of the  C. elegans  calcium channel α 1  subunit encoded by C54D2.5 were identified by screening of the GenBank expressed sequence tag (EST) data bank. This analysis identified a total of 13 mammalian sequences that exhibit some degree of DNA sequence and amino acid identity to C54D2.5, of which 8 are human sequences. (Table 2) Three of these sequences appear unlikely to encode novel calcium channel subunits because they either exhibit a significant degree of homology to previously identified mammalian α 1  subunits (clones H06096 and H14053) or exhibit homology in a region not considered to be diagnostic of calcium channel α 1  subunits specifically as opposed to other types of ion channel molecules in general (clone D20469). The five remaining sequences (H55225, H55617, H55223, H55544, and F07776), however, are believed to encode two previously unidentified calcium channel α 1  subunits because the degree of amino acid identity closely matches that of known calcium channel subunits in conserved regions but is sufficiently different to indicate that they do not encode previously identified mammalian calcium channel α 1  subunits α 1A , α 1B , α 1C , α 1D , α 1E , or α 1S . The expected amino acid sequence closely matches but is not identical to amino acid sequences in these known calcium channel subunits. The aligned amino acids sequences are shown in FIG. 1.  
                                                                 TABLE 2                       Query = C54D2.5 CE02562 CALCIUM CHANNEL ALPHA-1 SUBUNIT LG:6                                Database:   Non-redundant Database of GenBank EST Division           824,500 sequences; 302,742,428 total letter            Sequences producing High-scoring Segment Pairs:   Frame   Score   P(N)                    gb|AA183990|AA183990   ms53e02.r1 Life Tech mouse embry . . .   +1   108    1.8e-24       gb|H55225|H55225   CHR220164 Homo sapiens genomic c . . .   +1   136    2.5e-10       db|D68412|CELK131B1F   C.elegans cDNA clone yk131b1:5 . . .   +3   117    1.7e-06       gb|R75128|R75128   MDB1075 Mouse brain, Stratagene . . .   +3   113    7.2e-06       gb|H55617|H55617   CHR220556 Homo sapiens genomic c . . .   +2   102    2.8e-05       emb|FO7776|HSC2HD061   H. sapiens partial CDNA sequence . . .   +3   100    0.00057       gb|W76774|W76774   me84e08.rl Scares mouse embryo N . . .   +2   98   0.0012       gb|H06096|E06096   y177e01.rl Homo sapiens cDNA clo . . .   +3   98   0.0015       gb|H14053|H14053   ym65d10.r1 Homo sapiens cONA clo . . .   +2   91   0.0036       gb|H55223|H55223    CHR220162 Homo sapiens genoinic c . . .   +2   87   0.0039       dbj|D35703|CELK024D9F   C. elegans cDNA clone yk24d9: 5′. . .   +3   74   0.046       dbj|D20469|HUMGS01443   Human HL60 3′ directed MboI cDNA, . . .   −2   66   0.91       gb|H55544|H55544   CHR220483 Homo sapiens genomic c . . .   +1   65   0.98                  
 
         [0040]    Four of the five sequences (H55225, H55617, H55223, and H55544) are found on human chromosome 22, and are now believed to all be part of the same gene encoding the novel human calcium channel subunit all. The fifth sequence, F07776 is apparently distinct and associated with a further novel human calcium channel subunit designated O11H.  
         [0041]    The sequences of the five selected sequences and the references from which they are taken are given as follows:  
         [0042]    H55225 SOURCE human clone=C22 — 207 primer=T3 library=Chromosome 22 exon Trofatter, et al.,  Genome Res.  5 (3): 214-224 (1995) SEQ ID No. 13 1 GTGATCACTC TGGAAGGCTG GGTGGAGATC ATGTACTACG TGATGGATGC TCACTCCTTC 61 TACAACTTCA TCTACTTCAT CCTGCTTATC ATACCCCTCT TGCCTTGCAC CCCATATGGT 121 CTTCCCAGAG TGAGCTCATC CACCTCGTCA TGCCTGACTC GACGTTCA  
         [0043]    1155617 SOURCE human clone=C22 — 757 primer=T3 library=Chromosome 22 exon Trofatter, et al.,  Genome Res.  5 (3): 214-224 (1995) SEQ ID No. 14 1 GATGGTCGAG TACTCCCTGG ACCTTCAGAA CATCAALCCTG TCAGCCATCC GCACCGTGCG 61 CGTCCTGAGG CCCCTCAAAG CCATCAACCG CGTGCCCA  
         [0044]    H55223 SOURCE human clone=C22 — 204 primer=T3 library=Chromosome 22 exon Trofatter, et al,  Genome Res.  5 (3): 214-224 (1995) SEQ ID No. 15 1 CATGCTGGTG ATCCTGCTGA ACTGCGTGAC ACTTGGCATG TACCAGCCGT GCGACGACAT 61 GGACTGCCTG TCCGACCGCT GCAAGATCCT GCAG  
         [0045]    H55544 SOURCE human clone=C22 — 651 primer=T3 library=Chromosome 22 exon Trofatter, et al,  Genome Res.  5 (3): 214-224 (1995) SEQ ID No. 16 1 GTATCTCTGG TTACTTTAGT AGCCAACACT CTTGGCTACT CAGACCTTGG TCCCATTAA 61 TCCCTGCGAA CCTTGAGAGC ACTAAGACCT CTAAGAGCTT TGTCTAGATT TGAAGTAATG 121 AGG  
         [0046]    F07776 SOURCE human. Submitted (Jan. 19, 1995) Genethon, B. P. 60, 91002 Evry Cedex France and Genetique Moleculaire et Biologie du developpement, CNRS UPR420 B. P. 8, 94801 Villejuif Cedex France E-mail: genexpress@genethon.fr SEQ ID No. 17 1 TTCTCTCCAT TGTAGGAATG TTTCTGGCTG AACTGATAGA AAAGTATTTT GTGTGCCCTA 61 CCCTGTTNCG AGTGATCCGT CTTGCCAGGA TTGGCCGAAT CCTACGTCTG ATCAAAGGAG 121 CAAAGGGGAT CCGCACGCTG CTCTTTGCTT TGATGATGTC CCTTCCTGCG TTGTTTAACA 181 TCGGNCTCCT TCTTTTCCTG GTCATGTTCA TCTACGNCAT CTTTGGGATG TCCAATTTTG 241 CCTATGTTAA GAGGGAAGTT GGGATCGATG ACATGTTNAN CTTTGAGACC TTTGGCAACA 301 GCATGATCTG CCTGTTCCAA ATTACAACCT CTGCTGGCTG GGA  
         [0047]    A search of the Sanger Genome Sequencing Center (Cambridge, U.K.) and the Washington University Genome Sequencing Center (St. Louis. Mo.) sequences in progress revealed a Bacterial Artificial Chromosome (BAC) sequence (bK206c7) that contained matches to the  C. elegans  cosmid open reading frame, C54D2.5, and to the four human chromosome 22 ESTs, H55225, H55617, H55223,H55544. The  C. elegans  C54D2.5 cosmid sequence and the human EST sequences were then used to compare the translation of the bK206c7 BAC genomic sequence in all 6 reading frames. The analysis was performed using the graphical program Dotter (Eric Sohnhammer, NCBI). The analysis revealed a series of potential coding regions on one strand of the bK206c7 BAC sequence. These were subsequently translated in all 3 reading frames and the potential splice junctions identified. The translated sequence of this longer DNA fragment which is part of the human α 1I  subunit gene is given by Seq. ID No. 18.  
         [0048]    Using the sequence information from the five EST&#39;s, a full length gene can be recovered using any of several techniques. Polynucleotide probes having a sequence which corresponds to or hybridizes with the EST sequences or a distinctive portion thereof (for example oligonucleotide probes having a length of 18 to 100 nucleotides) can be used to probe a human cDNA library for identification of the full length DNA encoding the α 1I  and α 1H  subunits. The process of identifying cDNAs of interest using defined probes is well known in the art and is, for example, described in International Patent Publication No. WO95/04144, which is incorporated herein by reference. This process generally involves screening bacterial hosts (e.g.  E. coli ) harboring the library plasmids or infected with recombinant lambda phage with labeled probes, e.g. radiolabeled with  32 p, and selection of colonies or phage which bind the labeled probe. Each selected colony or phage is grown up, and the plasmids are recovered. Human cDNAs are recovered from the plasmids by restriction digestion, or can be amplified, for example by PCR. The recovered cDNA can be sequenced, and the position of the calcium channel subunit-encoding region further refined, although neither process is not necessary to the further use of the cDNA to produce cell lines expressing the novel calcium channel subunits.  
         [0049]    Longer portions of DNA-encoding the novel calcium channel subunits of the invention can also be recovered by PCR cloning techniques using primers corresponding to or based upon the EST sequences. Using this technique to identify relevant sequences within a human brain total RNA preparation confirmed that the novel α 1I  calcium channel subunit is present in human brain. Subcloning of the 567 nt PCR product and subsequent sequencing thereof showed that this product corresponds to the derived sequence form the bK206c7 BAC genomic sequence. The nucleotide sequence is given as SEQ ID No. 19. The same experiment was performed using a rat brain RNA preparation and resulted in recovery of a substantially identical PCR product. (SEQ ID. NO. 20). The protein encoded by the rat PCR product is 96% identical to the human PCR product.  
         [0050]    These sequences, which presumably encode a partial subunit can be used as a basis for constructing full length human or rat a,, clones. Briefly, the subcloned α 1I  PCR product is radiolabeled by random hexamer priming according to standard methods (See, Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Press) and used to screen commercial human brain cDNA libraries (Stratagene, La Jolla, Calif.). The screening of cDNA libraries follows standard methods and includes such protocols as infecting bacteria with recombinant lambda phage, immobilizing lambda DNA to nitrocellulose filters and screening under medium hybridization stringency conditions with radiolabeled probe. cDNA clones homologous to the probe are identified by autoradiography. Positive clones are purified by sequential rounds of screening.  
         [0051]    Following this protocol, most purified cDNA&#39;s are likely to be partial sequence clones due the nature of the cDNA library synthesis. Full length clones are constructed from cDNA&#39;s which overlap in DNA sequence. Restriction enzyme sites which overlap between cDNAs are used to ligate the individual cDNA&#39;s to generate a full-length cDNA. For subsequent heterologous expression, the full-length cDNA is subcloned directly into an appropriate vertebrate expression vector, such as pcDNA-3 (Invitrogen, San Diego, Calif.) in which expression of the cDNA is under the control of a promoter such as the CMV major intermediate early promoter/enhancer. Other suitable expression vectors include, for example, pMT2, pRCICMV, pcDNA3.1 and pCEP4.  
         [0052]    Once the full length cDNA is cloned into an expression vector, the vector is then transfected into a host cell for expression. Suitable host cells include Xenopus oocytes or mammalian cells such as human embryonic kidney cells as described in International Patent Publication No. WO 96/39512 which is incorporated herein by reference and Ltk cells as described in U.S. Pat. No. 5,386,025 which is incorporated herein by reference. Transfection into host cells may be accomplished by microinjection, lipofection, glycerol shock, electroporation calcium phosphate or particle-mediated gene transfer. The vector may also be transfected into host cells to provide coexpression of the novel α 1  subunits with a β and/or an α 2 δ subunit.  
         [0053]    The resulting cell lines expressing functional calcium channels including the novel α 1  subunits of the invention can be used test compounds for pharmacological activity with respect to these calcium channels. Thus, the cell lines are useful for screening compounds for pharmaceutical utility. Such screening can be carried out using several available methods for evaluation of the interaction, if any, between the test compound and the calcium channel. One such method involves the binding of radiolabeled agents that interact with the calcium channel and subsequent analysis of equilibrium binding measurements including but not limited to, on rates, off rates, K d  values and competitive binding by other molecules. Another such method involves the screening for the effects of compounds by electrophysiological assay whereby individual cells are impaled with a microelectrode and currents through the calcium channel are recorded before and after application of the compound of interest. Another method, high-throughput spectrophotometric assay, utilizes the loading the cell lines with a fluorescent dye sensitive to intracellular calcium concentration and subsequent examination of the effects of compounds on the ability of depolarization by potassium chloride or other means to alter intracellular calcium levels. Compounds to be tested as agonists or antagonists of the novel (α 1I  and α 1H  calcium channel subunits are combined with cells that are stably or transiently transformed with a DNA sequence encoding the α 1I  or α 1H  calcium channel subunits of the invention and monitored using one of these techniques.  
         [0054]    DNA fragments with sequences given by SEQ ID Nos. 13-19 may also be used for mapping the distribution of α 1I  and α 1H  calcium channel subunits within a tissue sample. This method follows normal histological procedures using a nucleic acid probe, and generally involves the steps of exposing the tissue to a reagent comprising a directly or indirectly detectable label coupled to a selected DNA fragment, and detecting reagent that has bound to the tissue. Suitable labels include fluorescent labels, enzyme labels, chromophores and radio-labels. 
     
    
     EXAMPLE 1  
       [0055]    In order to isolate novel human calcium channel α 1  subunits using standard molecular cloning protocols, synthetic DNA probes are prepared, radiolabeled with  32 P and utilized to screen human cDNA libraries commercially available in lambda phage vectors (Stratagene, La Jolla, Calif.) based on the human DNA sequences for H55225, H55617, H55223, H55544 and F07776. DNA fragments with the sequence of sequence ID NOs 18 and 19 may also be used for this purpose. Positive phage are purified through several rounds of screening involving immobilizing the phage DNA on nitrocellulose filters, hybridizing with the radiolabeled probe, washing off of excess probe and then selection of clones by autoradiography. Clones identified by this approach are expected to be partial length clones due to the nature of cDNA library synthesis and several rounds of screening for each calcium channel type may be necessary to obtain full-length clones.  
         [0056]    To characterize the clones, double stranded plasmid DNA is prepared from the identified clones and the sequences are determined using 35S dATP, Sequenase and standard gel electrophoresis methods. Regions of similarity and regions of overlap are determined by comparison of each cDNA sequence.  
         [0057]    Full-length clones are constructed by ligating overlapping cDNA fragments together at common restriction enzyme sites. The full-length clones are subsequently inserted into vectors suitable for expression in vertebrate cells (e.g. pMT2, pRC/CMV, pcDNA3.1, pCEP4, pREP7) by ligation into restriction sites in the vector polylinker region which is downstream of the promoter used to direct cDNA expression.  
         [0058]    DNA encoding the novel calcium channels can be stably or transiently introduced into eukaryotic cells (e.g. human embryonic kidney, mouse L cells, chinese hamster ovary, etc) by any number of available standard methods. Stable transfection is achieved by growing the cells under conditions that promote growth of cells expressing a marker gene which is contained in the expression vector (e.g. dihydrofolate reductase, thymidine kinase, or the like). The heterologous DNA encoding the human calcium channel may be integrated into the genome or may be maintained as an episomal element.  
         [0059]    Expression of the human calcium channel in transfected cells may monitored by any number of techniques, including Northern blot for RNA analysis, Southern blot for cDNA detection, electrophysiological assay for calcium channel function, the binding of radiolabeled agents thought to interact with the calcium channel, and fluorescent assay of dyes sensitive to intracellular calcium concentration.  
       EXAMPLE 2  
     Heterologous Expression of Human α 1I  Calcium Channels in Cells  
       [0060]    A. Transient Transfection in Mammalian Cells  
         [0061]    Host cells, such as human embryonic kidney cells, HEK 293 (ATCC# CRL 1573) are grown in standard DMEM medium supplemented with 2 mM glutamine and 10% fetal bovine serum. HEK 293 cells are transfected by a standard calcium-phosphate-DNA co-precipitation method using the full-length human α 1I  calcium channel cDNA in a vertebrate expression vector (for example see Current protocols in Molecular Biology). The human α 1I  calcium channel cDNA may be transfected alone or in combination with other cloned subunits for mammalian calcium channels, such as α2δ and β subunits, and also with clones for marker proteins such the jellyfish green fluorescent protein.  
         [0062]    Electrophysiological Recording: After an incubation period of from 24 to 72 hrs the culture medium is removed and replaced with external recording solution (see below). Whole cell patch clamp experiments are performed using an Axopatch 200B amplifier (Axon Instruments, Burlingame, Calif.) linked to an IBM compatible personal computer equipped with pCLAMP software. Microelectrodes are filled with 3 M CsCl and have typical resistances from 0.5 to 2.5 MΩ. The external recording solution is 20 mM BaCl 2 , 1 mM MgCl 2 , 10 mM HEPES, 40 mM TEACl, 10 mM Glucose, 65 mM CsCl, (pH 7.2). The internal pipette solution is 105 mM CsCl, 25 mM TEACl, 1 mM CaCl 2 , 11 mM EGTA, 10 mM HEPES (pH 7.2). Currents are typically elicited from a holding potential of −100 mV to various test potentials. Data are filtered at 1 kHz and recorded directly on the harddrive of a personal computer. Leak subtraction is carried out on-line using a standard P/5 protocol. Currents are analyzed using pCLAMP versions 5.5 and 6.0. Macroscopic current-voltage relations are fitted with the equation I={1/(1+exp(−(V m −V h )/S)}×G−(V m −E rev ), where V m  is the test potential, V h  is the voltage at which half of the channels are activated, and S reflects the steepness of the activation curve and is an indication of the effective gating charge movement. Inactivation curves are normalized to 1 and fitted with I=(1/l+exp ((V m −V h )/S) with V m  being the holding potential. Single channel recordings are performed in the cell-attached mode with the following pipette solution (in mM): 100 BaCl 2 , 10 HEPES, pH 7.4 and bath solution: 100 KCl, 10 EGTA, 2 MgCl 2 , 10 HEPES, pH 7.4.  
         [0063]    B. Transient Transfection in Xenopus Oocytes  
         [0064]    Stage V and VI Xenopus oocytes are prepared as described by Dascal et al (1986), Expression and modulation of voltage-gated calcium channels after RNA injection into Xenopus oocytes. Science 231:1147-1150. After enzymatic dissociation with collagenase, oocytes nuclei are microinjected with the human α 1I  calcium channel cDNA expression vector construct (approximately 10 ng DNA per nucleus) using a Drummond nanoject apparatus. The human α 1I  calcium channel may be injected alone, or in combination with other mammalian calcium channel subunit cDNAs, such as the α2-δ and β1b subunits. After incubation from 48 to 96 hrs macroscopic currents are recorded using a standard two microelectrode voltage-clamp (Axoclamp 2A, Axon Instruments, Burlingame, Calif.) in a bathing medium containing (in mM): 40 Ba(OH) 2 , 25 TEA-OH, 25 NaOH, 2 CsOH, 5 HEPES (pH titrated to 7.3 with methan-sulfonic acid). Pipettes of typical resistance ranging from 0.5 to 1.5 mΩ are filled with 2.8M CsCl, 0.2M CsOH, 10 mM HEPES, 10 mM BAPTA free acid. Endogenous Ca (and Ba)—activated Cl currents are suppressed by systematically injecting 10-30 nl of a solution containing 100 mM BAPTA-free acid, 10 mM HEPES (pH titrated to 7.2 with CsOH) using a third pipette connected to a pneumatic injector. Leak currents and capacitive transients are subtracted using a standard P/5 procedure.  
       EXAMPLE 3  
     Construction of Stable Cell Lines Expressing Human α 1I  Calcium Channels  
       [0065]    Mammalian cell lines stably expressing human α 1I  calcium channels are constructed by transfecting the α 1I  calcium channel cDNA into mammalian cells such as HEK 293 and selecting for antibiotic resistance encoded for by an expression vector. Briefly, the full-length human α 1I  calcium channel cDNA subcloned into a vertebrate expression vector with a selectable marker, such as the pcDNA3 (InvitroGen, San Diego, Calif.), is transfected into HEK 293 cells by calcium phosphate coprecipitation or lipofection or electroporation or other method according to well known procedures (Methods in Enzymology, Volume 185, Gene Expression Technology (1990) Edited by Goeddel, D. V.). The human α 1I  calcium channel may be transfected alone, or in combination with other mammalian calcium channel subunit cDNAs, such as the α2-δ and β1b subunits, either in a similar expression vector or other type of vector using different selectable markers. After incubation for 2 days in nonselective conditions, the medium is supplemented with Geneticin (G418) at a concentration of between 600 to 800 ug/ml. After 3 to 4 weeks in this medium, cells which are resistant to G418 are visible and can be cloned as isolated colonies using standard cloning rings. After growing up each isolated colony to confluency to establish cell lines, the expression of human α 1I  calcium channels can be determined at with standard gene expression methods such as Northern blotting, RNase protection and reverse-transcriptase PCR.  
         [0066]    The functional detection of human α 1I  calcium channels in stably transfected cells can be examined electrophysiologically, such as by whole patch clamp or single channel analysis (see above). Other means of detecting functional calcium channels include the use of radiolabeled  45 Ca uptake, fluorescence spectroscopy using calcium sensitive dyes such as FURA-2, and the binding or displacement of radiolabeled ligands that interact with the calcium channel.  
     
       
       
         1 
         
           
             33  
           
           
             1  
             24  
             DNA  
             Rat  
           
            1 

gtcaaaactc aggccttcta ctgg                                            24 

 
           
             2  
             24  
             DNA  
             Rat  
           
            2 

aacgtgttct tggctatcgc ggtg                                            24 

 
           
             3  
             24  
             DNA  
             Rat  
           
            3 

gtgaaagcac agagcttcta ctgg                                            24 

 
           
             4  
             24  
             DNA  
             Rat  
           
            4 

aacgttttct tggccattgc tgtg                                            24 

 
           
             5  
             24  
             DNA  
             Rat  
           
            5 

gttaaatcca acgtcttcta ctgg                                            24 

 
           
             6  
             24  
             DNA  
             Rat  
           
            6 

aatgtgttct tggccattgc ggtg                                            24 

 
           
             7  
             24  
             DNA  
             Rat  
           
            7 

gtgaagtctg tcacgtttta ctgg                                            24 

 
           
             8  
             24  
             DNA  
             Rat  
           
            8 

aagctcttct tggccattgc tgta                                            24 

 
           
             9  
             24  
             DNA  
             Rat  
           
            9 

gtcaagtcgc aagtgttcta ctgg                                            24 

 
           
             10  
             24  
             DNA  
             Rat  
           
            10 

aatgtattct tggctatcgc tgtg                                            24 

 
           
             11  
             21  
             DNA  
             Rat  
           
            11 

atctaygcyr tsatyggsat g                                               21 

 
           
             12  
             20  
             DNA  
             Rat  
           
            12 

atggacaayt tygastaytc                                                 20 

 
           
             13  
             168  
             DNA  
             Human  
             
               Expressed sequence tag H55225  
             
           
            13 

gtgatcactc tggaaggctg ggtggagatc atgtactacg tgatggatgc tcactccttc     60 

tacaacttca tctacttcat cctgcttatc atacccctct tgccttgcac cccatatggt    120 

cttcccagag tgagctcatc cacctcgtca tgcctgactc gacgttca                 168 

 
           
             14  
             98  
             DNA  
             Human  
             
               Expressed sequence tag H55617  
             
           
            14 

gatggtcgag tactccctgg accttcagaa catcaacctg tcagccatcc gcaccgtgcg     60 

cgtcctgagg cccctcaaag ccatcaaccg cgtgccca                             98 

 
           
             15  
             94  
             DNA  
             Human  
             
               Expressed sequence tag H55223  
             
           
            15 

catgctggtg atcctgctga actgcgtgac acttggcatg taccagccgt gcgacgacat     60 

ggactgcctg tccgaccgct gcaagatcct gcag                                 94 

 
           
             16  
             123  
             DNA  
             Human  
             
               Expressed sequence tag H55544  
             
           
            16 

gtatctctgg ttactttagt agccaacact cttggctact cagaccttgg tcccattaaa     60 

tccctgcgaa ccttgagagc actaagacct ctaagagctt tgtctagatt tgaaggaatg    120 

agg                                                                  123 

 
           
             17  
             343  
             DNA  
             Human  
             
               Expressed sequence tag F07776  
             
           
            17 

ttctctccat tgtaggaatg tttctggctg aactgataga aaagtatttt gtgtgcccta     60 

ccctgttncg agtgatccgt cttgccagga ttggccgaat cctacgtctg atcaaaggag    120 

caaaggggat ccgcacgctg ctctttgctt tgatgatgtc ccttcctgcg ttgtttaaca    180 

tcggnctcct tcttttcctg gtcatgttca tctacgncat ctttgggatg tccaattttg    240 

cctatgttaa gagggaagtt gggatcgatg acatgttnan ctttgagacc tttggcaaca    300 

gcatgatctg cctgttccaa attacaacct ctgctggctg gga                      343 

 
           
             18  
             5562  
             DNA  
             Human  
             
               CDS  
               (1)...(5562)  
               Human alpha-1 partial sequence from BAC bK206c7  
             
           
            18 

atg ttt ttc gtc tca gcc aat ccc tgg gtg agt ttc acc agt ttt gat       48 
Met Phe Phe Val Ser Ala Asn Pro Trp Val Ser Phe Thr Ser Phe Asp 
 1               5                   10                  15 

tta aac gtg gcc aat atg gac aac ttc ttc gcc ccc gtt ttc acc atg       96 
Leu Asn Val Ala Asn Met Asp Asn Phe Phe Ala Pro Val Phe Thr Met 
             20                  25                  30 

ggc aaa tat tat acg caa ggc gac aag gtg ctg atg ccg ctg gcg att      144 
Gly Lys Tyr Tyr Thr Gln Gly Asp Lys Val Leu Met Pro Leu Ala Ile 
         35                  40                  45 

cag gct ctg aaa cag ctg atg ttc aaa ttg gtg gcc act gtt gct cga      192 
Gln Ala Leu Lys Gln Leu Met Phe Lys Leu Val Ala Thr Val Ala Arg 
     50                  55                  60 

aca cat gct aca ccg tca cac atc acg ggt ggt cct gga aca ggg atg      240 
Thr His Ala Thr Pro Ser His Ile Thr Gly Gly Pro Gly Thr Gly Met 
 65                  70                  75                  80 

cac acg ggc acc ttc cag gaa gga gct gag cct ggt tca tct cag cac      288 
His Thr Gly Thr Phe Gln Glu Gly Ala Glu Pro Gly Ser Ser Gln His 
                 85                  90                  95 

cct gag gca cag gcc acg tat aca gca ggg tgc acc cca gcc ccc acg      336 
Pro Glu Ala Gln Ala Thr Tyr Thr Ala Gly Cys Thr Pro Ala Pro Thr 
            100                 105                 110 

ggc gat ccc acc tgc tgc ttt gtc ctt gac ttg gtg tgc acg tgg ttt      384 
Gly Asp Pro Thr Cys Cys Phe Val Leu Asp Leu Val Cys Thr Trp Phe 
        115                 120                 125 

gaa tgt gtc agc atg ctg gtg atc ctg ctg aac tgc gtg aca ctt ggc      432 
Glu Cys Val Ser Met Leu Val Ile Leu Leu Asn Cys Val Thr Leu Gly 
    130                 135                 140 

atg tac cag ccg tgc gac gac atg gac tgc ctg tcc gac cgc tgc aag      480 
Met Tyr Gln Pro Cys Asp Asp Met Asp Cys Leu Ser Asp Arg Cys Lys 
145                 150                 155                 160 

atc ctg cag gtc ttt gat gac ttc atc ttt atc ttc ttt gcc atg gag      528 
Ile Leu Gln Val Phe Asp Asp Phe Ile Phe Ile Phe Phe Ala Met Glu 
                165                 170                 175 

atg gtg ctc aag atg gtg gcc ctg ggg att ttt ggc aag aag tgc tac      576 
Met Val Leu Lys Met Val Ala Leu Gly Ile Phe Gly Lys Lys Cys Tyr 
            180                 185                 190 

ctc ggg gac aca tgg aac cgc ctg gat ttc ttc atc gtc atg gca ggc      624 
Leu Gly Asp Thr Trp Asn Arg Leu Asp Phe Phe Ile Val Met Ala Gly 
        195                 200                 205 

aac atc aac ctg tca gcc atc cgc acc gtg cgc gtc ctg agg ccc ctc      672 
Asn Ile Asn Leu Ser Ala Ile Arg Thr Val Arg Val Leu Arg Pro Leu 
    210                 215                 220 

aaa gcc atc aac cgc gtg ccc agt atg cgg atc ctg gtg aac ctg ctc      720 
Lys Ala Ile Asn Arg Val Pro Ser Met Arg Ile Leu Val Asn Leu Leu 
225                 230                 235                 240 

ctg gac aca ctg ccc atg ctg ggg aat gtc ctg ctg ctc tgc ttc ttt      768 
Leu Asp Thr Leu Pro Met Leu Gly Asn Val Leu Leu Leu Cys Phe Phe 
                245                 250                 255 

gtc ttc ttc atc ttt ggc atc ata ggt gtg cag ctc tgg gcg ggc ctg      816 
Val Phe Phe Ile Phe Gly Ile Ile Gly Val Gln Leu Trp Ala Gly Leu 
            260                 265                 270 

ctg cgt aac cgc tgc ttc ctg gag gag aac ttc acc ata caa ggg gat      864 
Leu Arg Asn Arg Cys Phe Leu Glu Glu Asn Phe Thr Ile Gln Gly Asp 
        275                 280                 285 

gtg gcc ttg ccc cca tac tac cag ccg gag gag gat gat gag atg ccc      912 
Val Ala Leu Pro Pro Tyr Tyr Gln Pro Glu Glu Asp Asp Glu Met Pro 
    290                 295                 300 

ttc atc tgc tcc ctg tcg ggc gac aat ggg ata atg ggc tgc cat gag      960 
Phe Ile Cys Ser Leu Ser Gly Asp Asn Gly Ile Met Gly Cys His Glu 
305                 310                 315                 320 

atc ccc ccg ctc aag gag cag ggc cgt gag tgc tgc ctg tcc aag gac     1008 
Ile Pro Pro Leu Lys Glu Gln Gly Arg Glu Cys Cys Leu Ser Lys Asp 
                325                 330                 335 

gac gtc tac gac ttt ggg gcg ggg cgc cag gac ctc aat gcc agc ggc     1056 
Asp Val Tyr Asp Phe Gly Ala Gly Arg Gln Asp Leu Asn Ala Ser Gly 
            340                 345                 350 

ctc tgt gtc aac tgg aac cgt tac tac aat gtg tgc cgc acg ggc agc     1104 
Leu Cys Val Asn Trp Asn Arg Tyr Tyr Asn Val Cys Arg Thr Gly Ser 
        355                 360                 365 

gcc aac ccc cac aag ggt gcc atc aac ttt gac aac atc ggt tat gct     1152 
Ala Asn Pro His Lys Gly Ala Ile Asn Phe Asp Asn Ile Gly Tyr Ala 
    370                 375                 380 

tgg att gtc atc ttc cag gtg atc act ctg gaa ggc tgg gtg gag atc     1200 
Trp Ile Val Ile Phe Gln Val Ile Thr Leu Glu Gly Trp Val Glu Ile 
385                 390                 395                 400 

atg tac tac gtg atg gat gct cac tcc ttc tac aac ttc atc tac ttc     1248 
Met Tyr Tyr Val Met Asp Ala His Ser Phe Tyr Asn Phe Ile Tyr Phe 
                405                 410                 415 

atc ctg ctt atc ata agt gag ctc atc cac ctc gtc atg cct gac tgc     1296 
Ile Leu Leu Ile Ile Ser Glu Leu Ile His Leu Val Met Pro Asp Cys 
            420                 425                 430 

agc ttc agc aca gca cag tcc cca aaa tgt caa ggt gat tca ctc cca     1344 
Ser Phe Ser Thr Ala Gln Ser Pro Lys Cys Gln Gly Asp Ser Leu Pro 
        435                 440                 445 

gga gtc gct gct gaa tcc ctg ctg ctg cga gac tct agc tcc tca gtc     1392 
Gly Val Ala Ala Glu Ser Leu Leu Leu Arg Asp Ser Ser Ser Ser Val 
    450                 455                 460 

atc act gat gag gct gca gcc atg gag aac ctc ctg gcg ggc acc tcc     1440 
Ile Thr Asp Glu Ala Ala Ala Met Glu Asn Leu Leu Ala Gly Thr Ser 
465                 470                 475                 480 

aag ggg gat gaa agc tat ctg ctc agg ctg gcc ggc agc caa gtt cac     1488 
Lys Gly Asp Glu Ser Tyr Leu Leu Arg Leu Ala Gly Ser Gln Val His 
                485                 490                 495 

tcc cag gct cag caa atg ctg ggg agg ggg ctg ggc cct gaa agc ctg     1536 
Ser Gln Ala Gln Gln Met Leu Gly Arg Gly Leu Gly Pro Glu Ser Leu 
            500                 505                 510 

gaa act gga gag gag ccc cac tcg tgg agc cct cgg gcc aca agg aga     1584 
Glu Thr Gly Glu Glu Pro His Ser Trp Ser Pro Arg Ala Thr Arg Arg 
        515                 520                 525 

tgg gat ccc caa tgc caa cca ggg cag cct ctc ccc ctt cat ttc atg     1632 
Trp Asp Pro Gln Cys Gln Pro Gly Gln Pro Leu Pro Leu His Phe Met 
    530                 535                 540 

caa gca cag gtg ggc tcc ttc ttc atg atc aac ctg tgc ctc gtt gtc     1680 
Gln Ala Gln Val Gly Ser Phe Phe Met Ile Asn Leu Cys Leu Val Val 
545                 550                 555                 560 

ata gcg acc cag ttc tcg gag acc aag caa cgg gag cac cgg ctg atg     1728 
Ile Ala Thr Gln Phe Ser Glu Thr Lys Gln Arg Glu His Arg Leu Met 
                565                 570                 575 

ctg gag cag cgg cag cgc tac ctg tcc tcc agc acg gtg gcc agc tac     1776 
Leu Glu Gln Arg Gln Arg Tyr Leu Ser Ser Ser Thr Val Ala Ser Tyr 
            580                 585                 590 

gcc gag cct ggc gac tgc tac gag gag atc ttc cag tat gtc tgc cac     1824 
Ala Glu Pro Gly Asp Cys Tyr Glu Glu Ile Phe Gln Tyr Val Cys His 
        595                 600                 605 

atc ctg cgc aag gcc aag cgc cgc gcc ctg ggc ctc tac cag gcc ctg     1872 
Ile Leu Arg Lys Ala Lys Arg Arg Ala Leu Gly Leu Tyr Gln Ala Leu 
    610                 615                 620 

cag agc cgg cgc cag gcc ctg ggc ccg gag gcc ccg gcc ccc gcc aaa     1920 
Gln Ser Arg Arg Gln Ala Leu Gly Pro Glu Ala Pro Ala Pro Ala Lys 
625                 630                 635                 640 

cct ggg ccc cac gcc aag gag ccc cgg cac tac cct ctc aca gtc tgg     1968 
Pro Gly Pro His Ala Lys Glu Pro Arg His Tyr Pro Leu Thr Val Trp 
                645                 650                 655 

gaa tcg att ctt ggg agg caa gca gaa gaa tgc acg ctc aga gct gcc     2016 
Glu Ser Ile Leu Gly Arg Gln Ala Glu Glu Cys Thr Leu Arg Ala Ala 
            660                 665                 670 

gcc cac ccg tcc tcg ggt gcc agc cat cca ggc gtg ggc tcg gag gag     2064 
Ala His Pro Ser Ser Gly Ala Ser His Pro Gly Val Gly Ser Glu Glu 
        675                 680                 685 

gcc cca gag ctg tgc ccg caa cat agc ccc ctg gat gcg acg ccc cac     2112 
Ala Pro Glu Leu Cys Pro Gln His Ser Pro Leu Asp Ala Thr Pro His 
    690                 695                 700 

acc ctg gtg cag ccc atc ccc gcc acg ctg gct tcc gat ccc gcc agc     2160 
Thr Leu Val Gln Pro Ile Pro Ala Thr Leu Ala Ser Asp Pro Ala Ser 
705                 710                 715                 720 

tgc cct tgc tgc cag cat gag gac ggc cgg cgg ccc tcg ggc ctg ggc     2208 
Cys Pro Cys Cys Gln His Glu Asp Gly Arg Arg Pro Ser Gly Leu Gly 
                725                 730                 735 

agc acc gac tcg ggc cag gag ggc tcg ggc tcc ggg agc tcc gct ggt     2256 
Ser Thr Asp Ser Gly Gln Glu Gly Ser Gly Ser Gly Ser Ser Ala Gly 
            740                 745                 750 

ggc gag gac gag gcg gat ggg gac ggg gcc cgg agc agc gag gac gga     2304 
Gly Glu Asp Glu Ala Asp Gly Asp Gly Ala Arg Ser Ser Glu Asp Gly 
        755                 760                 765 

gcc tcc tca gaa ctg ggg aag gag gag gag gag gag gag cag gcg gat     2352 
Ala Ser Ser Glu Leu Gly Lys Glu Glu Glu Glu Glu Glu Gln Ala Asp 
    770                 775                 780 

ggg gcg gtc tgg ctg tgc ggg gat gtg tgg cgg gag acg cga gcc aag     2400 
Gly Ala Val Trp Leu Cys Gly Asp Val Trp Arg Glu Thr Arg Ala Lys 
785                 790                 795                 800 

ctg cgc ggc atc gtg gac agc aag tac ttc aac cgg ggc atc atg atg     2448 
Leu Arg Gly Ile Val Asp Ser Lys Tyr Phe Asn Arg Gly Ile Met Met 
                805                 810                 815 

gcc atc ctg gtc aac acc gtc agc atg ggc atc gag cac cac gag cag     2496 
Ala Ile Leu Val Asn Thr Val Ser Met Gly Ile Glu His His Glu Gln 
            820                 825                 830 

gcc agt gca gcg cag ccg ggc cgg gcc tgc ggg aga gga caa aat cca     2544 
Ala Ser Ala Ala Gln Pro Gly Arg Ala Cys Gly Arg Gly Gln Asn Pro 
        835                 840                 845 

gac ctt tgc atg acc ctc aag gcc cct tgt ctc tgt cac aac gtc cct     2592 
Asp Leu Cys Met Thr Leu Lys Ala Pro Cys Leu Cys His Asn Val Pro 
    850                 855                 860 

tca cca ggc cag ggt gtc ctg tcc cat cca gtg act cca ccc cat aca     2640 
Ser Pro Gly Gln Gly Val Leu Ser His Pro Val Thr Pro Pro His Thr 
865                 870                 875                 880 

gcc cca tgg cgc atg gag aca gga aag cag gga cac gga tgt gaa gaa     2688 
Ala Pro Trp Arg Met Glu Thr Gly Lys Gln Gly His Gly Cys Glu Glu 
                885                 890                 895 

gga cca gga caa cga agc agt gac atg ttt gcc ctg gag atg atc ctg     2736 
Gly Pro Gly Gln Arg Ser Ser Asp Met Phe Ala Leu Glu Met Ile Leu 
            900                 905                 910 

aag ctg gct gca ttt ggg ctc ttc gac tac ctg cgt aac ccc tac aac     2784 
Lys Leu Ala Ala Phe Gly Leu Phe Asp Tyr Leu Arg Asn Pro Tyr Asn 
        915                 920                 925 

atc ttc gac agc atc att gtc atc atc agc atc tgg gag atc gtg ggg     2832 
Ile Phe Asp Ser Ile Ile Val Ile Ile Ser Ile Trp Glu Ile Val Gly 
    930                 935                 940 

cag gcg gac ggt ggg ctg tcg gtg ctg cgg acc ttc cgg ctg ctg cgc     2880 
Gln Ala Asp Gly Gly Leu Ser Val Leu Arg Thr Phe Arg Leu Leu Arg 
945                 950                 955                 960 

gtg ctg aaa ctg gtg cgc ttc atg cct gcc ctg cgg cgc cag ctc gtg     2928 
Val Leu Lys Leu Val Arg Phe Met Pro Ala Leu Arg Arg Gln Leu Val 
                965                 970                 975 

gtg ctc atg aag acc atg gac aac gtg gcc acc ttc tgc atg ctg ctc     2976 
Val Leu Met Lys Thr Met Asp Asn Val Ala Thr Phe Cys Met Leu Leu 
            980                 985                 990 

atg ctc ttc atc ttc atc ttc agc atc ctt ggg atg cat att ttt ggc     3024 
Met Leu Phe Ile Phe Ile Phe Ser Ile Leu Gly Met His Ile Phe Gly 
         995                1000                1005 

tgc aag ttc agc ctc cgc acg gac act gga gac acg gtg ccc gac agg     3072 
Cys Lys Phe Ser Leu Arg Thr Asp Thr Gly Asp Thr Val Pro Asp Arg 
    1010                1015                1020 

aag aac ttc gac tcc ctg ctg tgg gcc atc gtc act gtg ttc cag atc     3120 
Lys Asn Phe Asp Ser Leu Leu Trp Ala Ile Val Thr Val Phe Gln Ile 
1025                1030                1035                1040 

ctc acc cag gag gac tgg aac gtc gtt ctc tac aat ggc atg gcc tcc     3168 
Leu Thr Gln Glu Asp Trp Asn Val Val Leu Tyr Asn Gly Met Ala Ser 
                1045                1050                1055 

act tct ccc tgg gcc tcc ctc tac ttt gtc gcc ctc atg acc ttc ggc     3216 
Thr Ser Pro Trp Ala Ser Leu Tyr Phe Val Ala Leu Met Thr Phe Gly 
            1060                1065                1070 

aac tat gtg ctc ttc aac ctg ctg gtg gcc atc ctg gtg gag ggc ttc     3264 
Asn Tyr Val Leu Phe Asn Leu Leu Val Ala Ile Leu Val Glu Gly Phe 
        1075                1080                1085 

cag gcg gag gtg act gtg gtc ttg gca gag gaa gca ccc cca cag ggc     3312 
Gln Ala Glu Val Thr Val Val Leu Ala Glu Glu Ala Pro Pro Gln Gly 
    1090                1095                1100 

ctg cga aag act ggg cga ggg aga ggt ggc ctg gat ggg gga ggg ctg     3360 
Leu Arg Lys Thr Gly Arg Gly Arg Gly Gly Leu Asp Gly Gly Gly Leu 
1105                1110                1115                1120 

caa ttc aaa ctt cta gca ggc aac cta tcc cta aag gag ggg gtt gct     3408 
Gln Phe Lys Leu Leu Ala Gly Asn Leu Ser Leu Lys Glu Gly Val Ala 
                1125                1130                1135 

gat gag gtg ggt gac gcc aat cgc tcc tac tcg gac gag gac cag agc     3456 
Asp Glu Val Gly Asp Ala Asn Arg Ser Tyr Ser Asp Glu Asp Gln Ser 
            1140                1145                1150 

tca tcc aac ata gaa gag ttt gat aag ctc cag gaa ggc ctg gac agc     3504 
Ser Ser Asn Ile Glu Glu Phe Asp Lys Leu Gln Glu Gly Leu Asp Ser 
        1155                1160                1165 

agc gga gat ccc aag ctc tgc cca atc ccc atg acc ccc aat ggg cac     3552 
Ser Gly Asp Pro Lys Leu Cys Pro Ile Pro Met Thr Pro Asn Gly His 
    1170                1175                1180 

ctg gac ccc agt ctc cca ctg ggt ggg cac cta ggt cct gct ggg gct     3600 
Leu Asp Pro Ser Leu Pro Leu Gly Gly His Leu Gly Pro Ala Gly Ala 
1185                1190                1195                1200 

gcg gga cct gcc ccc cga ctc tca ctg cag ccg gac ccc atg ctg gtg     3648 
Ala Gly Pro Ala Pro Arg Leu Ser Leu Gln Pro Asp Pro Met Leu Val 
                1205                1210                1215 

gcc ctg ggc tcc cga aag agc agc gtc atg tct cta ggg agg atg agc     3696 
Ala Leu Gly Ser Arg Lys Ser Ser Val Met Ser Leu Gly Arg Met Ser 
            1220                1225                1230 

tat gac cag cgc tcc ctg gtg ggt ggt ctt aga gcc aca gcg ggg gtg     3744 
Tyr Asp Gln Arg Ser Leu Val Gly Gly Leu Arg Ala Thr Ala Gly Val 
        1235                1240                1245 

cag gct gcc ttt ggg cac ctg gtg ccc cag ccg tgg gtg tgc ctg tgg     3792 
Gln Ala Ala Phe Gly His Leu Val Pro Gln Pro Trp Val Cys Leu Trp 
    1250                1255                1260 

ggc gct gac ccg aac ggg aac tcc ttc cag tcc agc tcc cgg agc tcc     3840 
Gly Ala Asp Pro Asn Gly Asn Ser Phe Gln Ser Ser Ser Arg Ser Ser 
1265                1270                1275                1280 

tac tac ggg cca tgg ggc cgc agc gcg gcc tgg gcc agc cgt cgc tcc     3888 
Tyr Tyr Gly Pro Trp Gly Arg Ser Ala Ala Trp Ala Ser Arg Arg Ser 
                1285                1290                1295 

agc tgg aac agc ctc aag cac aag ccg ccg tcg gcg gag cat gag tcc     3936 
Ser Trp Asn Ser Leu Lys His Lys Pro Pro Ser Ala Glu His Glu Ser 
            1300                1305                1310 

ctg ctc tct gcg gag cgc ggc ggc ggc gcc cgg gtc tgc gag gtt gcc     3984 
Leu Leu Ser Ala Glu Arg Gly Gly Gly Ala Arg Val Cys Glu Val Ala 
        1315                1320                1325 

gcg gac gag ggg ccg ccg cgg gcc gca ccc ctg cac acc cca cac gcc     4032 
Ala Asp Glu Gly Pro Pro Arg Ala Ala Pro Leu His Thr Pro His Ala 
    1330                1335                1340 

cac cac gtt cat cac ggg ccc cat ctg gcg cac cgc cac cgc cac cac     4080 
His His Val His His Gly Pro His Leu Ala His Arg His Arg His His 
1345                1350                1355                1360 

cgc cgg acg ctg tcc ctc gac aac agg gac tcg gtg gac ctg gcc gag     4128 
Arg Arg Thr Leu Ser Leu Asp Asn Arg Asp Ser Val Asp Leu Ala Glu 
                1365                1370                1375 

ctg gtg ccc gcg gtg ggc gcc cac ccc cgg gcc gcc tgg agg gcg gca     4176 
Leu Val Pro Ala Val Gly Ala His Pro Arg Ala Ala Trp Arg Ala Ala 
            1380                1385                1390 

ggc ccg gcc ccc ggg cat gag gac tgc aat ggc agg atg ccc agc atc     4224 
Gly Pro Ala Pro Gly His Glu Asp Cys Asn Gly Arg Met Pro Ser Ile 
        1395                1400                1405 

gcc aaa gac gtc ttc acc aag atg ggc gac cgc ggg gat cgc ggg gag     4272 
Ala Lys Asp Val Phe Thr Lys Met Gly Asp Arg Gly Asp Arg Gly Glu 
    1410                1415                1420 

gat gag gag gaa atc gac tac gtg agt ggg ggc ggg gcc gaa ggg gac     4320 
Asp Glu Glu Glu Ile Asp Tyr Val Ser Gly Gly Gly Ala Glu Gly Asp 
1425                1430                1435                1440 

ctg acc ctg tgc ttc cgc gtc cgc aag atg atc gac gtc tat aag ccc     4368 
Leu Thr Leu Cys Phe Arg Val Arg Lys Met Ile Asp Val Tyr Lys Pro 
                1445                1450                1455 

gac tgg tgc gag gtc cgc gaa gac tgg tct gtc tac ctc ttc tct ccc     4416 
Asp Trp Cys Glu Val Arg Glu Asp Trp Ser Val Tyr Leu Phe Ser Pro 
            1460                1465                1470 

gag aac agg ctc agg gat ctg ggc tgg gta agc ctc gag tgc cag gga     4464 
Glu Asn Arg Leu Arg Asp Leu Gly Trp Val Ser Leu Glu Cys Gln Gly 
        1475                1480                1485 

aag gtg ggt gac ctc gtg gtg tgg gtg tat ggt cag agg agg cag cgc     4512 
Lys Val Gly Asp Leu Val Val Trp Val Tyr Gly Gln Arg Arg Gln Arg 
    1490                1495                1500 

cag acc att att gcc cac aaa ctc ttc gac tac gtc gtc ctg gcc ttc     4560 
Gln Thr Ile Ile Ala His Lys Leu Phe Asp Tyr Val Val Leu Ala Phe 
1505                1510                1515                1520 

atc ttt ctc aac tgc atc acc atc gcc ctg gag cgg cct cag atc gag     4608 
Ile Phe Leu Asn Cys Ile Thr Ile Ala Leu Glu Arg Pro Gln Ile Glu 
                1525                1530                1535 

gcc ggc agc acc gaa cgc atc ttt ctc acc gtg tcc aac tac atc ttc     4656 
Ala Gly Ser Thr Glu Arg Ile Phe Leu Thr Val Ser Asn Tyr Ile Phe 
            1540                1545                1550 

acg gcc atc ttc gtg ggc gag atg aca ttg aag gta gtc tcg ctg ggc     4704 
Thr Ala Ile Phe Val Gly Glu Met Thr Leu Lys Val Val Ser Leu Gly 
        1555                1560                1565 

ctg tac ttc ggc gag cag gcg tac cta cgc agc agc tgg aac gtg ctg     4752 
Leu Tyr Phe Gly Glu Gln Ala Tyr Leu Arg Ser Ser Trp Asn Val Leu 
    1570                1575                1580 

gat ggc ttt ctt gtc ttc gtg tcc atc atc gac atc gtg gtg tcc ctg     4800 
Asp Gly Phe Leu Val Phe Val Ser Ile Ile Asp Ile Val Val Ser Leu 
1585                1590                1595                1600 

gcc tca gcc ggg gga gcc aag atc ttg ggg gtc ctc cga gtc ttg cgg     4848 
Ala Ser Ala Gly Gly Ala Lys Ile Leu Gly Val Leu Arg Val Leu Arg 
                1605                1610                1615 

ctc ctg cgc acc cta cgc ccc ctg cgt gtc atc agc cgg gcg ccg ggc     4896 
Leu Leu Arg Thr Leu Arg Pro Leu Arg Val Ile Ser Arg Ala Pro Gly 
            1620                1625                1630 

ctg aag ctg gtg gtg gag aca ctc atc tcc tcc ctc aag ccc atc ggc     4944 
Leu Lys Leu Val Val Glu Thr Leu Ile Ser Ser Leu Lys Pro Ile Gly 
        1635                1640                1645 

aac atc gtg ctc atc tgc tgt gcc ttc ttc atc atc ttt ggc atc ctg     4992 
Asn Ile Val Leu Ile Cys Cys Ala Phe Phe Ile Ile Phe Gly Ile Leu 
    1650                1655                1660 

gga gtg cag ctc ttc aag ggc aag ttc tac cac tgt ctg ggc gtg gac     5040 
Gly Val Gln Leu Phe Lys Gly Lys Phe Tyr His Cys Leu Gly Val Asp 
1665                1670                1675                1680 

acc cgc aac atc acc aac cgc tcg gac tgc atg gcc gcc aac tac cgc     5088 
Thr Arg Asn Ile Thr Asn Arg Ser Asp Cys Met Ala Ala Asn Tyr Arg 
                1685                1690                1695 

tgg gtc cat cac aaa tac aac ttc gac aac ctg ggc cag gct ctg atg     5136 
Trp Val His His Lys Tyr Asn Phe Asp Asn Leu Gly Gln Ala Leu Met 
            1700                1705                1710 

tcc ctc ttt gtc ctg gca tcc aag gat ggt tgg gtg aac atc atg tac     5184 
Ser Leu Phe Val Leu Ala Ser Lys Asp Gly Trp Val Asn Ile Met Tyr 
        1715                1720                1725 

aat gga ctg gat gct gtt gct gtg gac cag cag cct gtg acc aac cac     5232 
Asn Gly Leu Asp Ala Val Ala Val Asp Gln Gln Pro Val Thr Asn His 
    1730                1735                1740 

aac ccc tgg atg ctg ctg tac ttc atc tcc ttc ctg ctc atc gtc agc     5280 
Asn Pro Trp Met Leu Leu Tyr Phe Ile Ser Phe Leu Leu Ile Val Ser 
1745                1750                1755                1760 

ttc ttt gtg ctc aac atg ttt gtg ggt gtc gtg gtg gag aac ttc cac     5328 
Phe Phe Val Leu Asn Met Phe Val Gly Val Val Val Glu Asn Phe His 
                1765                1770                1775 

aag tgc cgg cag cac cag gag gct gaa gag gca cgg cgg cgt gag gag     5376 
Lys Cys Arg Gln His Gln Glu Ala Glu Glu Ala Arg Arg Arg Glu Glu 
            1780                1785                1790 

aag cgg ctg cgg cgc ctg gag aag aag cgc cgg aag gcc cag cgg ctg     5424 
Lys Arg Leu Arg Arg Leu Glu Lys Lys Arg Arg Lys Ala Gln Arg Leu 
        1795                1800                1805 

ccc tac tat gcc acc tat tgt cac acc cgg ctg ctc atc cac tcc atg     5472 
Pro Tyr Tyr Ala Thr Tyr Cys His Thr Arg Leu Leu Ile His Ser Met 
    1810                1815                1820 

tgc acc agc cac tac ctg gac atc ttc atc acc ttc atc atc tgc ctc     5520 
Cys Thr Ser His Tyr Leu Asp Ile Phe Ile Thr Phe Ile Ile Cys Leu 
1825                1830                1835                1840 

aac gtg gtc acc atg tcc ctg gag cac tac aat cag ccc acg             5562 
Asn Val Val Thr Met Ser Leu Glu His Tyr Asn Gln Pro Thr 
                1845                1850 

 
           
             19  
             1853  
             PRT  
             Human  
           
            19 

Phe Phe Val Ser Ala Asn Pro Trp Val Ser Phe Thr Ser Phe Asp Leu 
 1               5                  10                  15 

Asn Val Ala Asn Met Asp Asn Phe Phe Ala Pro Val Phe Thr Met Gly 
            20                  25                  30 

Lys Tyr Tyr Thr Gln Gly Asp Lys Val Leu Met Pro Leu Ala Ile Gln 
        35                  40                  45 

Ala Leu Lys Gln Leu Met Phe Lys Leu Val Ala Thr Val Ala Arg Thr 
    50                  55                  60 

His Ala Thr Pro Ser His Ile Thr Gly Gly Pro Gly Thr Gly Met His 
65                  70                  75                  80 

Thr Gly Thr Phe Gln Glu Gly Ala Glu Pro Gly Ser Ser Gln His Pro 
                85                  90                  95 

Glu Ala Gln Ala Thr Tyr Thr Ala Gly Cys Thr Pro Ala Pro Thr Gly 
            100                 105                 110 

Asp Pro Thr Cys Cys Phe Val Leu Asp Leu Val Cys Thr Trp Phe Glu 
        115                 120                 125 

Cys Val Ser Met Leu Val Ile Leu Leu Asn Cys Val Thr Leu Gly Met 
    130                 135                 140 

Tyr Gln Pro Cys Asp Asp Met Asp Cys Leu Ser Asp Arg Cys Lys Ile 
145                 150                 155                 160 

Leu Gln Val Phe Asp Asp Phe Ile Phe Ile Phe Phe Ala Met Glu Met 
                165                 170                 175 

Val Leu Lys Met Val Ala Leu Gly Ile Phe Gly Lys Lys Cys Tyr Leu 
            180                 185                 190 

Gly Asp Thr Trp Asn Arg Leu Asp Phe Phe Ile Val Met Ala Gly Asn 
        195                 200                 205 

Ile Asn Leu Ser Ala Ile Arg Thr Val Arg Val Leu Arg Pro Leu Lys 
    210                 215                 220 

Ala Ile Asn Arg Val Pro Ser Met Arg Ile Leu Val Asn Leu Leu Leu 
225                 230                 235                 240 

Asp Thr Leu Pro Met Leu Gly Asn Val Leu Leu Leu Cys Phe Phe Val 
                245                 250                 255 

Phe Phe Ile Phe Gly Ile Ile Gly Val Gln Leu Trp Ala Gly Leu Leu 
            260                 265                 270 

Arg Asn Arg Cys Phe Leu Glu Glu Asn Phe Thr Ile Gln Gly Asp Val 
        275                 280                 285 

Ala Leu Pro Pro Tyr Tyr Gln Pro Glu Glu Asp Asp Glu Met Pro Phe 
    290                 295                 300 

Ile Cys Ser Leu Ser Gly Asp Asn Gly Ile Met Gly Cys His Glu Ile 
305                 310                 315                 320 

Pro Pro Leu Lys Glu Gln Gly Arg Glu Cys Cys Leu Ser Lys Asp Asp 
                325                 330                 335 

Val Tyr Asp Phe Gly Ala Gly Arg Gln Asp Leu Asn Ala Ser Gly Leu 
            340                 345                 350 

Cys Val Asn Trp Asn Arg Tyr Tyr Asn Val Cys Arg Thr Gly Ser Ala 
        355                 360                 365 

Asn Pro His Lys Gly Ala Ile Asn Phe Asp Asn Ile Gly Tyr Ala Trp 
    370                 375                 380 

Ile Val Ile Phe Gln Val Ile Thr Leu Glu Gly Trp Val Glu Ile Met 
385                 390                 395                 400 

Tyr Tyr Val Met Asp Ala His Ser Phe Tyr Asn Phe Ile Tyr Phe Ile 
                405                 410                 415 

Leu Leu Ile Ile Ser Glu Leu Ile His Leu Val Met Pro Asp Cys Ser 
            420                 425                 430 

Phe Ser Thr Ala Gln Ser Pro Lys Cys Gln Gly Asp Ser Leu Pro Gly 
        435                 440                 445 

Val Ala Ala Glu Ser Leu Leu Leu Arg Asp Ser Ser Ser Ser Val Ile 
    450                 455                 460 

Thr Asp Glu Ala Ala Ala Met Glu Asn Leu Leu Ala Gly Thr Ser Lys 
465                 470                 475                 480 

Gly Asp Glu Ser Tyr Leu Leu Arg Leu Ala Gly Ser Gln Val His Ser 
                485                 490                 495 

Gln Ala Gln Gln Met Leu Gly Arg Gly Leu Gly Pro Glu Ser Leu Glu 
            500                 505                 510 

Thr Gly Glu Glu Pro His Ser Trp Ser Pro Arg Ala Thr Arg Arg Trp 
        515                 520                 525 

Asp Pro Gln Cys Gln Pro Gly Gln Pro Leu Pro Leu His Phe Met Gln 
    530                 535                 540 

Ala Gln Val Gly Ser Phe Phe Met Ile Asn Leu Cys Leu Val Val Ile 
545                 550                 555                 560 

Ala Thr Gln Phe Ser Glu Thr Lys Gln Arg Glu His Arg Leu Met Leu 
                565                 570                 575 

Glu Gln Arg Gln Arg Tyr Leu Ser Ser Ser Thr Val Ala Ser Tyr Ala 
            580                 585                 590 

Glu Pro Gly Asp Cys Tyr Glu Glu Ile Phe Gln Tyr Val Cys His Ile 
        595                 600                 605 

Leu Arg Lys Ala Lys Arg Arg Ala Leu Gly Leu Tyr Gln Ala Leu Gln 
    610                 615                 620 

Ser Arg Arg Gln Ala Leu Gly Pro Glu Ala Pro Ala Pro Ala Lys Pro 
625                 630                 635                 640 

Gly Pro His Ala Lys Glu Pro Arg His Tyr Pro Leu Thr Val Trp Glu 
                645                 650                 655 

Ser Ile Leu Gly Arg Gln Ala Glu Glu Cys Thr Leu Arg Ala Ala Ala 
            660                 665                 670 

His Pro Ser Ser Gly Ala Ser His Pro Gly Val Gly Ser Glu Glu Ala 
        675                 680                 685 

Pro Glu Leu Cys Pro Gln His Ser Pro Leu Asp Ala Thr Pro His Thr 
    690                 695                 700 

Leu Val Gln Pro Ile Pro Ala Thr Leu Ala Ser Asp Pro Ala Ser Cys 
705                 710                 715                 720 

Pro Cys Cys Gln His Glu Asp Gly Arg Arg Pro Ser Gly Leu Gly Ser 
                725                 730                 735 

Thr Asp Ser Gly Gln Glu Gly Ser Gly Ser Gly Ser Ser Ala Gly Gly 
            740                 745                 750 

Glu Asp Glu Ala Asp Gly Asp Gly Ala Arg Ser Ser Glu Asp Gly Ala 
        755                 760                 765 

Ser Ser Glu Leu Gly Lys Glu Glu Glu Glu Glu Glu Gln Ala Asp Gly 
    770                 775                 780 

Ala Val Trp Leu Cys Gly Asp Val Trp Arg Glu Thr Arg Ala Lys Leu 
785                 790                 795                 800 

Arg Gly Ile Val Asp Ser Lys Tyr Phe Asn Arg Gly Ile Met Met Ala 
                805                 810                 815 

Ile Leu Val Asn Thr Val Ser Met Gly Ile Glu His His Glu Gln Ala 
            820                 825                 830 

Ser Ala Ala Gln Pro Gly Arg Ala Cys Gly Arg Gly Gln Asn Pro Asp 
        835                 840                 845 

Leu Cys Met Thr Leu Lys Ala Pro Cys Leu Cys His Asn Val Pro Ser 
    850                 855                 860 

Pro Gly Gln Gly Val Leu Ser His Pro Val Thr Pro Pro His Thr Ala 
865                 870                 875                 880 

Pro Trp Arg Met Glu Thr Gly Lys Gln Gly His Gly Cys Glu Glu Gly 
                885                 890                 895 

Pro Gly Gln Arg Ser Ser Asp Met Phe Ala Leu Glu Met Ile Leu Lys 
            900                 905                 910 

Leu Ala Ala Phe Gly Leu Phe Asp Tyr Leu Arg Asn Pro Tyr Asn Ile 
        915                 920                 925 

Phe Asp Ser Ile Ile Val Ile Ile Ser Ile Trp Glu Ile Val Gly Gln 
    930                 935                 940 

Ala Asp Gly Gly Leu Ser Val Leu Arg Thr Phe Arg Leu Leu Arg Val 
945                 950                 955                 960 

Leu Lys Leu Val Arg Phe Met Pro Ala Leu Arg Arg Gln Leu Val Val 
                965                 970                 975 

Leu Met Lys Thr Met Asp Asn Val Ala Thr Phe Cys Met Leu Leu Met 
            980                 985                 990 

Leu Phe Ile Phe Ile Phe Ser Ile Leu Gly Met His Ile Phe Gly Cys 
        995                 1000                1005 

Lys Phe Ser Leu Arg Thr Asp Thr Gly Asp Thr Val Pro Asp Arg Lys 
    1010                1015                1020 

Asn Phe Asp Ser Leu Leu Trp Ala Ile Val Thr Val Phe Gln Ile Leu 
1025                1030                1035                1040 

Thr Gln Glu Asp Trp Asn Val Val Leu Tyr Asn Gly Met Ala Ser Thr 
                1045                1050                1055 

Ser Pro Trp Ala Ser Leu Tyr Phe Val Ala Leu Met Thr Phe Gly Asn 
            1060                1065                1070 

Tyr Val Leu Phe Asn Leu Leu Val Ala Ile Leu Val Glu Gly Phe Gln 
        1075                1080                1085 

Ala Glu Val Thr Val Val Leu Ala Glu Glu Ala Pro Pro Gln Gly Leu 
    1090                1095                1100 

Arg Lys Thr Gly Arg Gly Arg Gly Gly Leu Asp Gly Gly Gly Leu Gln 
1105                1110                1115                1120 

Phe Lys Leu Leu Ala Gly Asn Leu Ser Leu Lys Glu Gly Val Ala Asp 
                1125                1130                1135 

Glu Val Gly Asp Ala Asn Arg Ser Tyr Ser Asp Glu Asp Gln Ser Ser 
            1140                1145                1150 

Ser Asn Ile Glu Glu Phe Asp Lys Leu Gln Glu Gly Leu Asp Ser Ser 
        1155                1160                1165 

Gly Asp Pro Lys Leu Cys Pro Ile Pro Met Thr Pro Asn Gly His Leu 
    1170                1175                1180 

Asp Pro Ser Leu Pro Leu Gly Gly His Leu Gly Pro Ala Gly Ala Ala 
1185                1190                1195                1200 

Gly Pro Ala Pro Arg Leu Ser Leu Gln Pro Asp Pro Met Leu Val Ala 
                1205                1210                1215 

Leu Gly Ser Arg Lys Ser Ser Val Met Ser Leu Gly Arg Met Ser Tyr 
            1220                1225                1230 

Asp Gln Arg Ser Leu Val Gly Gly Leu Arg Ala Thr Ala Gly Val Gln 
        1235                1240                1245 

Ala Ala Phe Gly His Leu Val Pro Gln Pro Trp Val Cys Leu Trp Gly 
    1250                1255                1260 

Ala Asp Pro Asn Gly Asn Ser Phe Gln Ser Ser Ser Arg Ser Ser Tyr 
1265                1270                1275                1280 

Tyr Gly Pro Trp Gly Arg Ser Ala Ala Trp Ala Ser Arg Arg Ser Ser 
                1285                1290                1295 

Trp Asn Ser Leu Lys His Lys Pro Pro Ser Ala Glu His Glu Ser Leu 
            1300                1305                1310 

Leu Ser Ala Glu Arg Gly Gly Gly Ala Arg Val Cys Glu Val Ala Ala 
        1315                1320                1325 

Asp Glu Gly Pro Pro Arg Ala Ala Pro Leu His Thr Pro His Ala His 
    1330                1335                1340 

His Val His His Gly Pro His Leu Ala His Arg His Arg His His Arg 
1345                1350                1355                1360 

Arg Thr Leu Ser Leu Asp Asn Arg Asp Ser Val Asp Leu Ala Glu Leu 
                1365                1370                1375 

Val Pro Ala Val Gly Ala His Pro Arg Ala Ala Trp Arg Ala Ala Gly 
            1380                1385                1390 

Pro Ala Pro Gly His Glu Asp Cys Asn Gly Arg Met Pro Ser Ile Ala 
        1395                1400                1405 

Lys Asp Val Phe Thr Lys Met Gly Asp Arg Gly Asp Arg Gly Glu Asp 
    1410                1415                1420 

Glu Glu Glu Ile Asp Tyr Val Ser Gly Gly Gly Ala Glu Gly Asp Leu 
1425                1430                1435                1440 

Thr Leu Cys Phe Arg Val Arg Lys Met Ile Asp Val Tyr Lys Pro Asp 
                1445                1450                1455 

Trp Cys Glu Val Arg Glu Asp Trp Ser Val Tyr Leu Phe Ser Pro Glu 
            1460                1465                1470 

Asn Arg Leu Arg Asp Leu Gly Trp Val Ser Leu Glu Cys Gln Gly Lys 
        1475                1480                1485 

Val Gly Asp Leu Val Val Trp Val Tyr Gly Gln Arg Arg Gln Arg Gln 
    1490                1495                1500 

Thr Ile Ile Ala His Lys Leu Phe Asp Tyr Val Val Leu Ala Phe Ile 
1505                1510                1515                1520 

Phe Leu Asn Cys Ile Thr Ile Ala Leu Glu Arg Pro Gln Ile Glu Ala 
                1525                1530                1535 

Gly Ser Thr Glu Arg Ile Phe Leu Thr Val Ser Asn Tyr Ile Phe Thr 
            1540                1545                1550 

Ala Ile Phe Val Gly Glu Met Thr Leu Lys Val Val Ser Leu Gly Leu 
        1555                1560                1565 

Tyr Phe Gly Glu Gln Ala Tyr Leu Arg Ser Ser Trp Asn Val Leu Asp 
    1570                1575                1580 

Gly Phe Leu Val Phe Val Ser Ile Ile Asp Ile Val Val Ser Leu Ala 
1585                1590                1595                1600 

Ser Ala Gly Gly Ala Lys Ile Leu Gly Val Leu Arg Val Leu Arg Leu 
                1605                1610                1615 

Leu Arg Thr Leu Arg Pro Leu Arg Val Ile Ser Arg Ala Pro Gly Leu 
            1620                1625                1630 

Lys Leu Val Val Glu Thr Leu Ile Ser Ser Leu Lys Pro Ile Gly Asn 
        1635                1640                1645 

Ile Val Leu Ile Cys Cys Ala Phe Phe Ile Ile Phe Gly Ile Leu Gly 
    1650                1655                1660 

Val Gln Leu Phe Lys Gly Lys Phe Tyr His Cys Leu Gly Val Asp Thr 
1665                1670                1675                1680 

Arg Asn Ile Thr Asn Arg Ser Asp Cys Met Ala Ala Asn Tyr Arg Trp 
                1685                1690                1695 

Val His His Lys Tyr Asn Phe Asp Asn Leu Gly Gln Ala Leu Met Ser 
            1700                1705                1710 

Leu Phe Val Leu Ala Ser Lys Asp Gly Trp Val Asn Ile Met Tyr Asn 
        1715                1720                1725 

Gly Leu Asp Ala Val Ala Val Asp Gln Gln Pro Val Thr Asn His Asn 
    1730                1735                1740 

Pro Trp Met Leu Leu Tyr Phe Ile Ser Phe Leu Leu Ile Val Ser Phe 
1745                1750                1755                1760 

Phe Val Leu Asn Met Phe Val Gly Val Val Val Glu Asn Phe His Lys 
                1765                1770                1775 

Cys Arg Gln His Gln Glu Ala Glu Glu Ala Arg Arg Arg Glu Glu Lys 
            1780                1785                1790 

Arg Leu Arg Arg Leu Glu Lys Lys Arg Arg Lys Ala Gln Arg Leu Pro 
        1795                1800                1805 

Tyr Tyr Ala Thr Tyr Cys His Thr Arg Leu Leu Ile His Ser Met Cys 
    1810                1815                1820 

Thr Ser His Tyr Leu Asp Ile Phe Ile Thr Phe Ile Ile Cys Leu Asn 
1825                1830                1835                1840 

Val Val Thr Met Ser Leu Glu His Tyr Asn Gln Pro Thr 
                1845                1850 

 
           
             20  
             567  
             DNA  
             Human  
             
               CDS  
               (1)...(567)  
               Human alpha-1 partial sequence  
             
           
            20 

atg cgg atc ctg gtg aac ctg ctc ctg gac aca ctg ccc atg ctg ggg       48 
Met Arg Ile Leu Val Asn Leu Leu Leu Asp Thr Leu Pro Met Leu Gly 
 1               5                   10                  15 

aat gtc ctg ctg ctc tgc ttc ttt gtc ttc ttc acc ttt ggc atc ata       96 
Asn Val Leu Leu Leu Cys Phe Phe Val Phe Phe Thr Phe Gly Ile Ile 
             20                  25                  30 

ggt gtg cag ctc tgg gcg ggc ctg ctg cgt aac cgc tgc ttc ctg gag      144 
Gly Val Gln Leu Trp Ala Gly Leu Leu Arg Asn Arg Cys Phe Leu Glu 
         35                  40                  45 

gag aac ttc acc ata caa ggg gat gtg gcc ttg ccc cca tac tac cag      192 
Glu Asn Phe Thr Ile Gln Gly Asp Val Ala Leu Pro Pro Tyr Tyr Gln 
     50                  55                  60 

ccg gag gag gat gat gag atg ccc ttc atc tgc tcc ctg tcg ggc gac      240 
Pro Glu Glu Asp Asp Glu Met Pro Phe Ile Cys Ser Leu Ser Gly Asp 
 65                  70                  75                  80 

aat ggg ata atg ggc tgc cat gag atc ccc ccg ctc aag gag cag ggc      288 
Asn Gly Ile Met Gly Cys His Glu Ile Pro Pro Leu Lys Glu Gln Gly 
                 85                  90                  95 

cgt gag tgc tgc ctg tcc aag gac gac gtc tac gac ttt ggg gcg ggg      336 
Arg Glu Cys Cys Leu Ser Lys Asp Asp Val Tyr Asp Phe Gly Ala Gly 
            100                 105                 110 

cgc cag gac ctc aat gcc agc ggc ctc tgt gtc aac tgg aac cgt tac      384 
Arg Gln Asp Leu Asn Ala Ser Gly Leu Cys Val Asn Trp Asn Arg Tyr 
        115                 120                 125 

tac aat gtg tgc cgc acg ggc agc gcc aac ccc cac aag ggt gcc atc      432 
Tyr Asn Val Cys Arg Thr Gly Ser Ala Asn Pro His Lys Gly Ala Ile 
    130                 135                 140 

agc ttt gac aac atc ggt tat gct tgg att gtc atc ttc cag gtg atc      480 
Ser Phe Asp Asn Ile Gly Tyr Ala Trp Ile Val Ile Phe Gln Val Ile 
145                 150                 155                 160 

act ctg gaa ggc tgg gtg gcg atc atg tac tac gtg atg gat gct ctc      528 
Thr Leu Glu Gly Trp Val Ala Ile Met Tyr Tyr Val Met Asp Ala Leu 
                165                 170                 175 

tcc ttc tac aac ttc gtc tac ttc atc ctg ctt atc ata                  567 
Ser Phe Tyr Asn Phe Val Tyr Phe Ile Leu Leu Ile Ile 
            180                 185 

 
           
             21  
             188  
             PRT  
             Human  
           
            21 

Arg Ile Leu Val Asn Leu Leu Leu Asp Thr Leu Pro Met Leu Gly Asn 
 1               5                  10                  15 

Val Leu Leu Leu Cys Phe Phe Val Phe Phe Thr Phe Gly Ile Ile Gly 
            20                  25                  30 

Val Gln Leu Trp Ala Gly Leu Leu Arg Asn Arg Cys Phe Leu Glu Glu 
        35                  40                  45 

Asn Phe Thr Ile Gln Gly Asp Val Ala Leu Pro Pro Tyr Tyr Gln Pro 
    50                  55                  60 

Glu Glu Asp Asp Glu Met Pro Phe Ile Cys Ser Leu Ser Gly Asp Asn 
65                  70                  75                  80 

Gly Ile Met Gly Cys His Glu Ile Pro Pro Leu Lys Glu Gln Gly Arg 
                85                  90                  95 

Glu Cys Cys Leu Ser Lys Asp Asp Val Tyr Asp Phe Gly Ala Gly Arg 
            100                 105                 110 

Gln Asp Leu Asn Ala Ser Gly Leu Cys Val Asn Trp Asn Arg Tyr Tyr 
        115                 120                 125 

Asn Val Cys Arg Thr Gly Ser Ala Asn Pro His Lys Gly Ala Ile Ser 
    130                 135                 140 

Phe Asp Asn Ile Gly Tyr Ala Trp Ile Val Ile Phe Gln Val Ile Thr 
145                 150                 155                 160 

Leu Glu Gly Trp Val Ala Ile Met Tyr Tyr Val Met Asp Ala Leu Ser 
                165                 170                 175 

Phe Tyr Asn Phe Val Tyr Phe Ile Leu Leu Ile Ile 
            180                 185 

 
           
             22  
             567  
             DNA  
             Rat  
             
               CDS  
               (1)...(567)  
               Rat alpha-1 partial sequence  
             
           
            22 

atg cgg atc ctg gtg aac ctg ctg ctc gac acg ctg ccc atg ctg ggg       48 
Met Arg Ile Leu Val Asn Leu Leu Leu Asp Thr Leu Pro Met Leu Gly 
 1               5                   10                  15 

aac gtg ctc ctg ctc tgt ttc ttc gtc ttc ttc atc ttc ggc atc att       96 
Asn Val Leu Leu Leu Cys Phe Phe Val Phe Phe Ile Phe Gly Ile Ile 
             20                  25                  30 

ggc gtg cag ctc tgg gca ggc ctg cta cgg aac cgc tgc ttc ctg gaa      144 
Gly Val Gln Leu Trp Ala Gly Leu Leu Arg Asn Arg Cys Phe Leu Glu 
         35                  40                  45 

gaa aac ttc acc ata caa ggg gat gtg gcc ctg ccc cct tat tac caa      192 
Glu Asn Phe Thr Ile Gln Gly Asp Val Ala Leu Pro Pro Tyr Tyr Gln 
     50                  55                  60 

cca gag gag gat gac gag atg ccc ttt atc tgc tcc ctg act ggg gac      240 
Pro Glu Glu Asp Asp Glu Met Pro Phe Ile Cys Ser Leu Thr Gly Asp 
 65                  70                  75                  80 

aat ggc atc atg ggc tgc cac gag atc ccc cca ctg aag gag cag ggc      288 
Asn Gly Ile Met Gly Cys His Glu Ile Pro Pro Leu Lys Glu Gln Gly 
                 85                  90                  95 

cgg gaa tgc tgc ctg tcc aaa gat gat gtg tat gac ttc ggg gcg ggg      336 
Arg Glu Cys Cys Leu Ser Lys Asp Asp Val Tyr Asp Phe Gly Ala Gly 
            100                 105                 110 

cgc cag gac ctc aac gcc agc ggt ctg tgc gtc aac tgg aac cgc tac      384 
Arg Gln Asp Leu Asn Ala Ser Gly Leu Cys Val Asn Trp Asn Arg Tyr 
        115                 120                 125 

tac aac gtc tgc cgc acg ggc aac gcc aac cct cac aag ggc gcc atc      432 
Tyr Asn Val Cys Arg Thr Gly Asn Ala Asn Pro His Lys Gly Ala Ile 
    130                 135                 140 

aac ttt gac aac att ggc tat gcc tgg att gtg att ttc cag gtg atc      480 
Asn Phe Asp Asn Ile Gly Tyr Ala Trp Ile Val Ile Phe Gln Val Ile 
145                 150                 155                 160 

act ctg gaa ggc tgg gtg gag atc atg tac tat gtg atg gac gca cat      528 
Thr Leu Glu Gly Trp Val Glu Ile Met Tyr Tyr Val Met Asp Ala His 
                165                 170                 175 

tct ttc tac aac ttc atc tac ttc atc ctg ctt atc ata                  567 
Ser Phe Tyr Asn Phe Ile Tyr Phe Ile Leu Leu Ile Ile 
            180                 185 

 
           
             23  
             188  
             PRT  
             Rat  
           
            23 

Arg Ile Leu Val Asn Leu Leu Leu Asp Thr Leu Pro Met Leu Gly Asn 
 1               5                  10                  15 

Val Leu Leu Leu Cys Phe Phe Val Phe Phe Ile Phe Gly Ile Ile Gly 
            20                  25                  30 

Val Gln Leu Trp Ala Gly Leu Leu Arg Asn Arg Cys Phe Leu Glu Glu 
        35                  40                  45 

Asn Phe Thr Ile Gln Gly Asp Val Ala Leu Pro Pro Tyr Tyr Gln Pro 
    50                  55                  60 

Glu Glu Asp Asp Glu Met Pro Phe Ile Cys Ser Leu Thr Gly Asp Asn 
65                  70                  75                  80 

Gly Ile Met Gly Cys His Glu Ile Pro Pro Leu Lys Glu Gln Gly Arg 
                85                  90                  95 

Glu Cys Cys Leu Ser Lys Asp Asp Val Tyr Asp Phe Gly Ala Gly Arg 
            100                 105                 110 

Gln Asp Leu Asn Ala Ser Gly Leu Cys Val Asn Trp Asn Arg Tyr Tyr 
        115                 120                 125 

Asn Val Cys Arg Thr Gly Asn Ala Asn Pro His Lys Gly Ala Ile Asn 
    130                 135                 140 

Phe Asp Asn Ile Gly Tyr Ala Trp Ile Val Ile Phe Gln Val Ile Thr 
145                 150                 155                 160 

Leu Glu Gly Trp Val Glu Ile Met Tyr Tyr Val Met Asp Ala His Ser 
                165                 170                 175 

Phe Tyr Asn Phe Ile Tyr Phe Ile Leu Leu Ile Ile 
            180                 185 

 
           
             24  
             31  
             PRT  
             Human  
           
            24 

Val Ile Ser Leu Glu Gly Trp Thr Asp Ile Met Tyr Tyr Val Gln Asp 
 1               5                  10                  15 

Ala His Ser Phe Trp Asn Trp Ile Tyr Phe Val Leu Leu Ile Val 
            20                  25                  30 

 
           
             25  
             31  
             PRT  
             C. elegans  
           
            25 

Val Ile Thr Leu Glu Gly Trp Val Glu Ile Met Tyr Tyr Val Met Asp 
 1               5                  10                  15 

Ala His Ser Phe Tyr Asn Phe Ile Tyr Phe Ile Leu Leu Ile Ile 
            20                  25                  30 

 
           
             26  
             23  
             PRT  
             Human  
           
            26 

Asn Ile Asn Leu Thr Ala Ile Arg Thr Val Arg Val Leu Arg Pro Leu 
 1               5                  10                  15 

Arg Ala Val Asn Arg Ile Pro 
            20 

 
           
             27  
             23  
             PRT  
             C. elegans  
           
            27 

Asn Ile Asn Leu Ser Ala Ile Arg Thr Val Arg Val Leu Arg Pro Leu 
 1               5                  10                  15 

Lys Ala Ile Asn Arg Val Pro 
            20 

 
           
             28  
             19  
             PRT  
             Human  
           
            28 

Met Ala Val Ile Met Ile Asn Cys Val Thr Leu Gly Met Tyr Arg Pro 
 1               5                  10                  15 

Cys Glu Asp 

 
           
             29  
             19  
             PRT  
             C. elegans  
           
            29 

Met Leu Val Ile Leu Leu Asn Cys Val Thr Leu Gly Met Tyr Gln Pro 
 1               5                  10                  15 

Cys Asp Asp 

 
           
             30  
             23  
             PRT  
             Human  
           
            30 

Leu Thr Ala Ile Arg Thr Val Arg Val Leu Arg Pro Leu Arg Ala Val 
 1               5                  10                  15 

Asn Arg Ile Pro Ser Met Arg 
            20 

 
           
             31  
             23  
             PRT  
             C. elegans  
           
            31 

Ile Lys Ser Leu Arg Thr Leu Arg Ala Leu Arg Pro Leu Arg Ala Leu 
 1               5                  10                  15 

Ser Arg Phe Glu Gly Met Arg 
            20 

 
           
             32  
             41  
             PRT  
             Human  
           
            32 

Pro Thr Ile Ile Arg Val Met Arg Val Leu Arg Ile Ala Arg Val Leu 
 1               5                  10                  15 

Lys Leu Leu Lys Met Ala Lys Gly Ile Arg Ser Leu Leu Asp Thr Val 
            20                  25                  30 

Gly Glu Ala Leu Pro Gln Val Gly Asn 
        35                  40 

 
           
             33  
             40  
             PRT  
             C. elegans  
             
               VARIANT  
               (1)...(40)  
               Xaa = Any Amino Acid  
             
           
            33 

Pro Thr Leu Xaa Arg Val Ile Arg Leu Ala Arg Ile Gly Arg Ile Leu 
 1               5                  10                  15 

Arg Leu Ile Lys Ala Lys Gly Ile Arg Thr Leu Leu Phe Ala Leu Met 
            20                  25                  30 

Met Ser Leu Pro Ala Leu Phe Asn 
        35                  40

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