| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 09.01.2001 |
| PATENT TITLE |
Stanniocalcin-2 |
| PATENT ABSTRACT | The present invention relates to polynucleotide and polypeptide molecules for stanniocalcin-2, a novel member of the stanniocalcin family. The polypeptides, and polynucleotides encoding them, modulate electrolyte homeostasis. The present invention also includes antibodies to the stanniocalcin-2 polypeptides |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | October 11, 1999 |
| PATENT REFERENCES CITED |
Chang et al., Mol.Cell. Endocrinol. 141(1-2): 95-99, Jun. 1998.* Ishibashi et al., AN JE0357, May 1999.* INC260632, LIFESEQ.TM. Clone Information Results, Incyte Pharmaceuticals, Inc., 1995. INC2484355, LIFESEQ.TM. Clone Information Results, Incyte Pharmaceuticals, Inc., 1997. INC2474784, LIFESEQ.TM. Clone Information Results, Incyte Pharmaceuticals, Inc., 1997. INC2474827, LIFESEQ.TM. Clone Information Results, Incyte Pharmaceuticals, Inc., 1997. INC2479915, LIFESEQ.TM. Clone Information Results, Incyte Pharmaceuticals, Inc., 1997. Genbank Acc. No. H98195, Hillier et al., WashU-Merck EST Project, 1995. Genbank Acc. No. AA195455, Hillier et al., WashU-Merck EST Project, 1995. Genbank Acc. No. AA223369, Hillier et al., WashU-Merck EST Project, 1995. HNT2RAT01, LIFESEQ.TM. Library Information Results, Incyte Pharmaceuticals, Inc., date unknown. SMCANOT01, LIFESEQ.TM. Library Information Results, Incyte Pharmaceuticals, Inc., date unknown |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. An isolated polynucleotide molecule encoding a stanniocalcin-2 (Stc2) polypeptide selected from the group consisting of: a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 73 to nucleotide 906 or as shown in SEQ ID NO: 13 from nucleotide 73 to nucleotide 888; b) molecules complementary to (a) c) polynucleotide molecules that encode a polypeptide that is the amino acid sequence of SEQ ID NO: 2 from amino acid residue 15 (Ser) to amino acid residue 184 (Leu); and d) degenerate nucleotide sequences of (a) and (b). 2. The isolated polynucleotide molecule of claim 1, wherein the polynucleotide is DNA. 3. The isolated polynucleotide molecule of claim 1, wherein the nucleotide sequence is the sequence as shown in SEQ ID NO: 1 from nucleotide 73 to nucleotide 906. 4. The isolated polynucleotide molecule of claim 1, wherein the nucleotide is the sequence as shown in SEQ ID NO: 13 from nucleotide 73 to nucleotide 888. 5. An expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment selected from the group consisting of: a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 73 to nucleotide 906 or as shown in SEQ ID NO: 13 from nucleotide 73 to nucleotide 888; b) polynucleotide molecules that encode a polypeptide that is the amino acid sequence of SEQ ID NO: 2 from amino acid residue 15 (Ser) to amino acid residue 184 (Leu); and c) degenerate nucleotide sequences of (a); and a transcription terminator. 6. A cultured cell into which has been introduced an expression vector according to claim 5, wherein said cell expresses the polypeptide encoded by the DNA segment. 7. A method of producing a stanniocalcin-2 comprising: culturing a cell into which has been introduced an expression vector according to claim 5, whereby said cell expresses a stanniocalcin-2 polypeptide encoded by the DNA segment; and recovering the stanniocalcin-2 polypeptide. |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION Maintaining electrolyte homeostasis is critical for preserving a cell's ability to interact with other cells and transduce signals that are mediated by changes in the environment. The levels of Ca.sup.++ and P.sub.i in extracellular fluid is tightly regulated, and excitability of nerve and muscle are dependent upon ion concentration. In vertebrates parathyroid hormone (PTH), active metabolites of vitamin D and calcitonin have been identified as regulators of plasma levels of Ca.sup.++ and P.sub.i. PTH stimulates the mobilization of Ca.sup.++ and P.sub.i from bone into plasma. In the kidneys, PTH decreases the urinary excretion of Ca.sup.++ and stimulates excretion of P.sub.i. P.sub.i reabsorption is reduced by decreasing the Na.sup.++ -dependent P.sub.i transport into the renal proximal tubule. Calcitonin is a hormone released by the thyroid that lowers Ca.sup.++ and P.sub.i levels in the blood to maintain the equilibrium when serum levels are elevated. Calcitonin is believed to counteract hypercalcemia by inhibiting osteoclast-mediated bone resorption. However, the role of calcitonin as regulator of transient influcuations in serum calcium levels or promotion of bone mineralization has not been demonstrated. In addition, there are no known regulators of transient changes P.sub.i levels in mammals, other than PTH, which only promotes P.sub.i excretion, thereby suggesting that our understanding and identification of factors involved in the calcium and phosphate homeostasis is incomplete. In fish, the environment can be either hypertonic (seawater) or hypotonic (fresh water) and is the primary source for Ca.sup.++ and P.sub.i, unlike terrestrial vertebrates whose uptake of Ca.sup.++ and P.sub.i are diet dependent. Some fish migrate to and from seawater and fresh water and have had to develop mechanisms to adapt to these radical changes in electrolyte concentrations. Stanniocalcin (also known as Stc, CS protein, teleocalcin or hypocalcin) is a homodimeric glycoprotein hormone secreted by endocrine glands found on the kidneys of bony fish called the corpuscles of Stannius (Stannius, H. Arch. Anat. Physiol. 6:97-101, 1839 and Wagner, G., in Biochemistry and Molecular Biology of Fishes, eds. Hochachka and Mommsen, Elsevier Science, Amersterdam, Vol 2, Chap. 21, pp. 419-434, 1993). The secretion of stanniocalcin in fishes is stimulated in response to rising plasma Ca.sup.++ levels (Wagner et al., Mol. Cell Endocrinol. 79:129-138, 1991) whereupon it acts to reduce Ca.sup.++ uptake by the gills (Wagner et al., Gen. Comp. Endocrinol. 63:481-491, 1986) and increase P.sub.i reabsorption by the kidneys (Lu et al., Am. J. Physiol. 36:R1356-R1362, 1994) with the net result being restoration of normal calcium levels. Stanniocalcin has been identified and isolated from several species of fish, such as Australian eel (Auguilla australis; Butkus et al., Mol. Cell. Endocrinol 54:123-134, 1987), rainbow trout (Oncorhynchus mykiss; Lafeber et al., Gen. Comp. Endocrinol. 69:19-30, 1988), coho salmon (Oncorhynchus kisutch; Wagner et al., Mol. Cell. Endocrinol. 90:7-15, 1992), and sockeye salmon (Oncorhychus nerka; Wagner et al., Gen. Comp. Endocrinol. 72:237-246, 1988. Studies using both in vivo and in vitro systems, show that in response to increased excellular Ca.sup.++ levels, Stc is released. Stc reduces plasma Ca.sup.++ concentration by inhibiting Ca.sup.++ absorption across the intestine (Sundell et al., J. Compl Physiol. B, Biochem Sys. Environ. Physiol. 162:489-495, 1992) and Ca.sup.++ transport across the gills (Wagner et al., ibid., 1988). In vivo, the rapid equilibration that occurs even when presented with massive increases in Ca.sup.++ suggests that there may be additional pathways involved that have not yet been elucidated. Human stanniocalcin has recently been discovered (Chang et al., Mol. Cell. Endocrinol. 112:241-247, 1995 and Olsen et al. WO 95/24411). The human protein is 247 amino acids, of which 214 amino acids constitute the mature polypeptide. It is believed that the first 33 amino acids represent a prepro region, consistent with Stc from fish, where the polypeptide is synthesized as a larger molecule and processed during secretion from the Corpuscles of Stannius. The human stanniocalcin discovered had a high degree of homology to the Australian eel; 119 identical amino acids in a 195 amino acid overlap (61% identity) and to coho salmon, 118 amino acids in a 204 amino acid overlap resulted in 57% identity. Eel and coho salmon Stc contain 15 and 12 cysteines respectively, while the human Stc has 11 cysteines. The spacing of the cysteines is highly conserved between all three proteins. Human stanniocalcin mRNA was found to be most abundant in ovary, prostate and thyroid as a 4 kb transcript (Chang et al., Mol. Cell. Endocrinol. 112:241-247, 1995). The 4 kb band was also seen in kidney, bone marrow, thymic stromal cells (Olsen et al., Proc. Natl. Acad. Sci. USA 93:1792-1796, 1996) and many other tissues, but no signal was found in brain, liver, spleen, peripheral blood leukocytes and adrenal medulla. In addition, a 2 kb transcript was identified with a probe designed to a 1 kb N-terminal portion of the molecule but was not seen with a probe containing C-terminal sequence (Chang et al., ibid., 1995). Isolation of human stanniocalcin has been reported from an early stage lung cDNA library (Olsen et al., WO 95/24441). Sera from normal humans was immunoreactive with salmon Stc antibodies. Analyses by immunocytochemistry suggested that cells in the human kidney tubules contained Stc-like proteins (Wagner et al., Proc. Natl. Acad. Sci. USA 92:1871-1875, 1995). Human stanniocalcin mRNA levels have been shown to increase in the presence of increasing amounts of excellular Ca.sup.++ in an immortalized liver fibroblast cell line SUSM-1, T98G human glioblastoma cell line and normal human foreskin fibroblasts (Chang et al., ibid., 1995). Because of the anti-hypercalcemic activity of stanniocalcin, members of the family, particularly human polypeptides will be very valuable in the understanding and treatment of diseases caused by electrolyte disorders. The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein. SUMMARY OF THE INVENTION Within one aspect, the present invention provides an isolated polynucleotide molecule encoding a stanniocalcin-2 (Stc2) polypeptide selected from the group consisting of (a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 73 to nucleotide 906 or as shown in SEQ ID NO: 13 from nucleotide 73 to nucleotide 888; (b) allelic variants of (a); (c) polynucleotide molecules that encode a polypeptide that is at least 60% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 15 (Ser) to amino acid residue 184 (Leu); and degenerate nucleotide sequences of (a), (b), or (c). Within a second aspect of the invention there is provided an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment selected from the group consisting of (a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO: 1 from nucleotide 73 to nucleotide 906 or as shown in SEQ ID NO: 13 from nucleotide 73 to nucleotide 888; (b) allelic variants of (a); (c) polynucleotide molecules that encode a polypeptide that is at least 60% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 15 (Ser) to amino acid residue 184 (Leu); and degenerate nucleotide sequences of (a), (b), or (c); and a transcription terminator. Within a third aspect of the present invention there is provided a cultured cell into which has been introduced the expression vector described above, wherein said cell expresses the polypeptide encoded by the DNA segment. A fourth aspect of the present invention provides an isolated and purified stanniocalcin-2 polypeptide. Within one embodiment, the isolated polypeptide is selected from the group consisting of (a) polypeptide molecules comprising an amino acid sequence as shown in SEQ ID NO: 2 from amino acid residue 1 to amino acid residue 278 or SEQ ID NO: 14 from amino acid residue 1 to amino acid residue 272; (b) allelic variants of (a); and polypeptides that are at least 60% identical to the amino acids of SEQ ID NO: 2 from amino acid residue 15 (Ser) to amino acid residue 184 (Leu). Within another aspect of the present invention there is provided a pharmaceutical composition comprising purified stanniocalcin-2 in combination with a pharmaceutically acceptable vehicle. Within another aspect of the present invention there is provided methods for producing stanniocalcin-2 comprising culturing a cell into which has been introduced the expression vector described previously, whereby said cell expresses a stanniocalcin-2 polypeptide encoded by the DNA segment and recovering the stanniocalcin-2 polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS The FIGURE illustrates a multiple alignment of salmon Stc (oncki); eel Stc (angau); human-1 Stc (stc1); human Stc2 (Zstc2); a partial sequence for hamster Stc2 and mouse Stc2. Within the FIGURE., certain amino acids in the hamster sequence were not determined but known to have a residue at that position. These undetermined amino acids are designated "X". DETAILED DESCRIPTION OF THE INVENTION The term "allelic variant" is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene. The term "expression vector" is used to denote a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. The term "isolated", when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. "Operably linked", when referring to DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in the promoter and proceeds through the coding segment to the terminator The term "promoter" is used herein for its art-recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5' non-coding regions of genes. The present invention is based in part upon the discovery of a novel DNA sequence that encodes a polypeptide having homology to hypocalcemic proteins of the stanniocalcin family. Analysis of the tissue distribution of the mRNA corresponding to this novel DNA showed that expression was highest in pancreas, followed by apparent but decreased expression levels in heart, placenta, skeletal muscle, thyroid and spleen. The polypeptide has been designated Stanniocalcin-2 (Stc2). The novel stanniocalcin-2 polypeptides of the present invention were initially identified by querying an EST database for homologous sequences to stanniocalcin. A single EST sequence was discovered and hypothesized to be related to the stanniocalcin family. The novel polypeptide encoded by the cDNA contained a cysteine motif of the formula: Cx{8}Cx{4}Cx{5}Cx{8}Cx{23}Cx{15}Cx{13}Cx{6}Cx{34}C wherein x{ } is the number of amino acid residues between cysteines (C). This cysteine motif occurs in all known members of the stanniocalcin family (for example, human, eel and salmon) and is unique to these proteins. Analysis of the DNA encoding a stanniocalcin-2 polypeptide (SEQ ID NO: 1) revealed an open reading frame encoding 302 amino acids (SEQ ID NO: 2) comprising a signal peptide of 24 amino acid residues (residue -24 to residue -1 of SEQ ID NO: 2) and a mature polypeptide of 278 amino acids (residue 1 to residue 278 of SEQ ID NO: 2). A potential N-linked glycosylation site is present at amino acid residue 49 of SEQ ID NO: 2 of the mature polypeptide. Multiple alignment of stanniocalcin-2 with other known stanniocalcins revealed a block of high percent identity corresponding to amino acid residue 15 to amino acid residue 184 of SEQ ID NO: 2 and as is shown in the FIGURE. A multiple alignment also revealed a greater degree of divergence at the C-terminus of stanniocalcin than seen at the N-terminus. The divergent region in human Stc2 corresponds to amino acid residues 184 to amino acid residue 278 of SEQ ID NO: 2 and is shown in the FIGURE. The mouse stanniocalcin-2 sequence was also identified from a mouse cDNA library generated from murina osteoblastic type cells. Analysis of the DNA encoding the mouse stanniocalcin-2 protein revealed an open reading from encoding 296 amino acids (SEQ ID NO: 14) comprising a signal peptide of 24 amino acids (amino acid residue -24 to residue -1 of SEQ ID NO: 14), and a mature polypeptide of 272 amino acids, residue 1 to residue 272 of SEQ ID NO): 14). Within the N-terminal block of high identity, the following percent identity figures are observed between members of the stanniocalcin family: oncki angau human STC2 oncki Stc 100 77.6 64.7 31.8 angau Stc 77.6 100 66.5 35.9 human Stc 64.7 66.5 100 37.6 human Stc2 31.8 35.9 37.6 100 Those skilled in the art will recognize that the sequences disclosed in SEQ ID NOS: 1 and 13 represent a single allele of the human and mouse Stc2 polypeptide, respectively. Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. The present invention further provides counterpart polypeptides and polynucleotides from other species ("species homologs"). Of particular interest are Stc2 polypeptides from other mammalian species, including porcine, ovine, bovine, canine, feline, equine and other primate proteins. Species homologs of the human proteins can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses the protein. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue of cell line. A Stc2-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202), using primers designed from the sequences disclosed herein. Within an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to the Stc2. Similar techniques can also be applied to the isolation of genomic clones. The present invention also provides isolated Stc2 polypeptides that are substantially homologous to the polypeptides of SEQ ID NOS: 2 and 14 and their species homologs. By "isolated" is meant a protein or polypeptide which is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. The term "substantially homologous" is used herein to denote polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequences shown in SEQ ID NOS: 2, 14 or their species homologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NOS: 2, 14 or their species homologs. Percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992, both incorporated herein by reference. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 1 (amino acids are indicated by the standard one-letter codes). TABLE 1 A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0 6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0 -2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4 The percent identity is then calculated as: ##EQU1## Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above. Substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 2) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification, such as a poly-histidine tract, an antigenic epitope or a binding domain. See, in general Ford et al., Protein Expression and Purification 2: 95-107, 1991, which is incorporated herein by reference. TABLE 2 Conservative amino acid substitutions Basic: arginine lysine histidine Acidic: glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244, 1081-1085, 1989). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g. calcium modulation) to identify amino acid residues that are critical to the activity of the molecule. Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988). Mutagenesis methods as disclosed above can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells. Mutagenized DNA molecules that encode active polypeptides (e.g., modulate calcium and phosphate levels) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure. Using the methods discussed above, one of ordinary skill in the art can prepare a variety of polypeptides that are substantially homologous to residues 1 to 278 of SEQ ID NO: 2, residues 1 to 272 of SEQ ID NO: 14 or allelic variants thereof and retain the electrolyte-modulating properties of the wild-type protein. The polypeptides of the present invention can be isolated by exploitation of their interaction with divalent ions. The polypeptides contain a histidine-rich region in the C-terminus of the molecule that confers an affinity for chelated metal ions. For example, immobilized metal ion adsorption (IMAC) chromatography can be used where a gel is first charged divalent metal ions to form a chelate (Sulkowski E., Trends in Biochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to the matrix with differing affinities, dependent upon the metal ion used, and eluted by competitive elution, lowering the pH, or use of strong chelating agents. Other methods of purification include, purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol., vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Acad. Press, San Diego, 1990, pp.529-539, incorporated herein by reference). The polypeptides of the present invention, including full-length proteins and fragments thereof, can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly culture cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., ibid., which are incorporated herein by reference. In general, a DNA sequence encoding a Stc2 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers. To direct a Stc2 polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector. The secretory signal sequence may be that of the Stc2 polypeptide, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo. The secretory signal sequence is joined to the Stc2 DNA sequence in the correct reading frame. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830). Cultured mammalian cells are also preferred hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., N.Y., 1987), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993), which are incorporated herein by reference. The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134, which are incorporated herein by reference. Preferred cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Md. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Pat. Nos. 4,579,821 and 4,601,978, which are incorporated herein by reference) and the adenovirus major late promoter. Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like. Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as "amplification." Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate. Other drug resistance genes (e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Other higher eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Pat. No. 5,162,222; Bang et al., U.S. Pat. No. 4,775,624; and WIPO publication WO 94/06463, which are incorporated herein by reference. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Fungal cells, including yeast cells, and particularly cells of the genus Saccharomyces, can also be used within the present invention, such as for producing Stc2 fragments or polypeptide fusions. Methods for transforming yeast cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075, which are incorporated herein by reference. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g. leucine). A preferred vector system for use in yeast is the POT1 vector system disclosed by Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092, which are incorporated herein by reference) and alcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454, which are incorporated herein by reference. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia guillermondii and Candida maltosa are known in the art. See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-3465, 1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Pat. No. 4,935,349, which is incorporated herein by reference. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228, which is incorporated herein by reference. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Pat. No. 4,486,533, which is incorporated herein by reference. Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co-transfected into the host cell. The activity of molecules of the present invention can be measured using a variety of assays that measure changes in electrolyte concentrations. Of particular interest are changes in calcium and phosphorus levels. Such assays are well known in the art. For a general reference, see Cellular Calcium: A Practical Approach, McCormack and Cobbold, eds., Oxford University Press, NY, 1991, which is incorporated herein by reference. Specific assays include, but are not limited to bioassays measuring calcium and phosphorus levels in urine and plasma of cannulated mice (Olsen et al., Proc. Natl. Acad. Sci. USA 93:1792-1796, 1996); P.sub.i resorption by kidney cells (Lu et al., 1994, ibid.); Ca.sup.++ uptake by osteoblasts (Whitson et al., J. Bone Miner. Res. 7:727-741, 1992), and Ca.sup.++ uptake by fish gills (Wagner et al., Proc. Natl. Acad. Sci. USA 92:1871-1875, 1995) and calvarial assays that measure bone resorption and increases in free Ca.sup.++ (Linkhart et al., Endocrinol. 125:1484-1491, 1989). Proteins of the present invention are useful for modulating calcium serum levels. Changes in calcium flux levels can be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to the appropriate animal model. For instance, Stc2 transfected (or co-transfected) expression host cells may be embedded in an alginate environment and injected (implanted) into recipient animals. Alginate-poly-L-lysine microencapsulation, permselective membrane encapsulation and diffusion chambers have been described as a means to entrap transfected mammalian cells or primary mammalian cells. These types of non-immunogenic "encapsulations" or microenvironments permit the transfer of nutrients into the microenvironment, and also permit the diffusion of proteins and other macromolecules secreted or released by the captured cells across the environmental barrier to the recipient animal. Most importantly, the capsules or microenvironments mask and shield the foreign, embedded cells from the recipient animal's immune response. Such microenvironments can extend the life of the injected cells from a few hours or days (naked cells) to several weeks (embedded cells). Alginate threads provide a simple and quick means for generating embedded cells. The materials needed to generate the alginate threads are readily available and relatively inexpensive. Once made, the alginate threads are relatively strong and durable, both in vitro and, based on data obtained using the threads, in vivo. The alginate threads are easily manipulable and the methodology is scalable for preparation of numerous threads. In an exemplary procedure, 3% alginate is prepared in sterile H.sub.2 O, and sterile filtered. Just prior to preparation of alginate threads, the alginate solution is again filtered. An approximately 50% cell suspension (containing about 5.times.105 to about 5.times.107 cells/ml) is mixed with the 3% alginate solution. One ml of the alginate/cell suspension is extruded into a 100 mM sterile filtered CaCl.sub.2 solution over a time period of .sup..about. 15 min, forming a "thread". The extruded thread is then transferred into a solution of 50 mM CaCl.sub.2, and then into a solution of 25 mM CaCl.sub.2. The thread is then rinsed with deionized water before coating the thread by incubating in a 0.01% solution of poly-L-lysine. Finally, the thread is rinsed with Lactated Ringer's Solution and drawn from solution into a syringe barrel (without needle attached). A large bore needle is then attached to the syringe, and the thread is intraperitoneally injected into a recipient in a minimal volume of the Lactated Ringer's Solution. Stc2 polypeptides can also be used to prepare antibodies that specifically bind to Stc2 proteins or polypeptides. Immunogens may be full-length or portions of molecules may be combined with a carrier, if "hapten-like". As used herein, the term "antibodies" includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab').sub.2 and Fab fragments, and the like, including genetically engineered antibodies. Antibodies are defined to be specifically binding if they bind to a Stc2 polypeptide with a K.sub.a of greater than or equal to 10.sup.7 /M. The affinity of an antibody can be readily determined by one of ordinary skill in the art (see, for example, Scatchard, ibid.). Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., 1989; and Hurrell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Inc., Boca Raton, Fla., 1982, which are incorporated herein by reference). As would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats. The immunogenicity of a Stc2 polypeptide may be increased through the use of an adjuvant such as Freund's complete or incomplete adjuvant. A variety of assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to Stc2 proteins or peptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations, enzyme-linked immunosorbent assays (ELISA), dot blot or Western blot assays, inhibition or competition assays, and sandwich assays. A preferred assay system employing a ligand-binding receptor fragment uses a commercially available biosensor instrument (BIAcore.TM., Pharmacia Biosensor, Piscataway, N.J.), wherein the receptor fragment is immobilized onto the surface of a receptor chip. Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145:229-240, 1991 and Cunningham and Wells, J. Mol. Biol. 234:554-563, 1993. A receptor fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell. A test sample is passed through the cell. If ligand is present in the sample, it will bind to the immobilized receptor polypeptide, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film. This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding. Antibodies to Stc2 may be used for isolating for affinity purification, for diagnostic assays for determining circulating levels of Stc2 polypeptides, and as antagonists to block Stc2 binding and signal transduction in vitro and in vivo. Molecules of the present invention can be used to identify and isolate receptors involved in electrolyte homeostasis. For example, proteins and peptides of the present invention can be immobilized on a column and membrane preparations run over the column (Immobilized Affinity Ligand Techniques, Hermanson et al., eds., Academic Press, San Diego, Calif., 1992, pp.195-202). Proteins and peptides can also be radiolabeled (Methods in Enzymol., vol. 182, "Guide to Protein Purification", M. Deutscher, ed., Acad. Press, San Diego, 1990, 721-737) or photoaffinity labeled (Brunner et al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedan et al., Biochem. Pharmacol. 33:1167-1180, 1984) and specific cell-surface proteins can be identified. The molecules of the present invention will be useful for removing Ni.sup.++, Ca.sup.++ and other divalent ions from solutions where the presence of such ions are a toxic contaminant. The polypeptides, nucleic acid and/or antibodies of the present invention may be used in treatment of disorders associated with changes in electrolyte homeostasis, particularly disorders caused by, or resulting in, changes in calcium, phosphate, magnesium, zinc and copper levels. The molecules of the present invention may used to modulate electrolyte imbalances or to treat or prevent development of pathological conditions in such diverse tissue as bone, heart, kidney, pancreas and the vascular system. In particular, certain bone diseases, hypertension, renal failure, hyperthyroidism, hyperparathyroidism, certain carcinomas, sarcoidosis, pancreatitis and drug-induced disorders that result in elevated levels of serum calcium, known as hypercalcemia, may be amenable to such diagnosis, treatment or prevention. Parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHRP) stimulate osteoclasts to resorb bone and are believed to provide the primary pathogenic mechanism for hypercalcemia. Certain cytokines have been shown to stimulate osteoclasts (including interleukin 1.alpha., interleukin 1.beta., tumor necrosis factor-.alpha., lymphotoxin, and transforming growth factor-.alpha.) and may play a role in hypercalcemia. Also, excessive gastrointestinal absorption of calcium can lead to hypercalcemia and has been correlated with pancreatitis. Increased serum phosphorus is also associated with hypercalcemia. Molecules of the present invention can be used to treat calcium-phosphorus imbalance. Elevated levels of serum calcium and phosphorus can result in bone and muscle pain, and molecules of the present invention would play a role in treating these disorders by modulating the serum calcium levels. In addition, calcium-phosphorus imbalances can lead to deposition of calcium in organs such as brain, eyes, myocardium and blood vessels. Depressed levels of serum calcium, or hypocalcemia, can lead to increased resorption of calcium from bone. Increased bone resorption can result in osteoporosis and Paget's disease. Molecules of the present invention can be used to identify and, in some cases treat, diseases where calcium regulation is abnormal. Hypomagnesium can occur because of reduced magnesium intake, reduced resorption and/or increased excretion. These conditions result from various disease states, that include hypercalcemia, hyperthyroidism, primary hyperparathyroidism, hungry bone syndrome, Bartter's syndrome, Gitelaman's syndrome, necrotizing enterocolitis and diabetes. Hypomagnesium (and the associated pertebation of mineral homeostasis that is caused by the imbalance) can contribute to the development of atherosclerosis, myocarial infarction, hypertension, cancer, renal stones, PMS, and has been correlated to insulin resistance. Hypomagnesium is often associated with renal failure. For pharmaceutical use, the proteins of the present invention are formulated for parenteral, particularly intravenous or subcutaneous, delivery according to conventional methods. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. In general, pharmaceutical formulations will include a Stc2 protein in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like. Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc. Methods of formulation are well known in the art and are disclosed, for example, in Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton Pa., 1990, which is incorporated herein by reference. Therapeutic doses will generally be in the range of 0.1 to 100 .mu.g/kg of patient weight per day, preferably 0.5-20 .mu.g/kg per day, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of the condition to be treated, patient traits, etc. Determination of dose is within the level of ordinary skill in the art. The proteins may be administered for acute treatment, over one week or less, often over a period of one to three days or may be used in chronic treatment, over several months or years. In general, a therapeutically effective amount of Stc2 is an amount sufficient to produce a clinically significant change in serum electrolyte levels. For example, normal ranges for serum calcium levels are in the range of 8.5-10.5 mg/dl or 2.1-2.5 mM. Treatment would generally begin when serum calcium levels drop below 7.5 mg/dL (1.9 mM) or above 12 mg/dL. Generally, inorganic phosphate serum levels below 1.0-2.0 mg/dl require treatment |
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