CN1330664A - Platelet-derived growth factor C, DNA coding therefor and uses thereof - Google Patents

Abstract

A method for modulating vasculogenesis or angiogenesis using the core domain protein of PDGF-C, a new member of the PDGF/VEGF family of growth factors, or a homodimer or a heterodimer comprising the core domain. Also disclosed are pharmaceutical compositions comprising the core protein, nucleotide sequences encoding the protein, and uses thereof in medical and diagnostic applications.

Description

Platelet-derived growth factor C, its coding DNA and its application

The present invention relates to a growth factor of connective tissue cells, fibroblasts, myofibroblasts and neurocollagen cells and/or a growth factor of endothelial cells, and in particular relates to a new platelet-derived growth factor/vascular endothelial growth factor-like growth factor encoding this The polynucleotide sequences of the factors, and pharmaceutical and diagnostic compositions and methods utilizing or derived from the factors.

Background of the invention

In the developing embryo, the formation of the nascent vascular network is achieved by in situ differentiation of mesoderm cells, a process known as angiogenesis. All subsequent processes involving the generation of new blood vessels in the embryo or in the adult are determined by the budding or division of capillaries in the established vasculature, a process called angiogenesis (Pepper et al. , Enzyme & Protein, 1996 49 138-162; Breier et al., Dev.Dyn.1995 204 228-239; Risau, Nature, 1997 386 671-674). The process of angiogenesis is not only related to embryonic development and the growth, repair and regeneration of normal tissues, but also involves the reproductive cycle of female organisms, the formation and maintenance of pregnancy, and the recovery process of trauma and fractures. Angiogenesis not only occurs in normal individuals, but is also associated with a large number of pathological processes, especially the growth and metastasis of tumors, and other pathological processes related to the proliferation of blood vessels, especially the proliferation of the microvasculature, such as diabetic retinopathy , psoriasis and arthritis. Then, inhibiting angiogenesis can help prevent the occurrence of the disease or alleviate the symptoms of the disease.

On the other hand, in some occasions where vascularization or vascularization needs to be promoted, such as after tissue or organ transplantation, or commonly seen in coronary heart disease and vasculitis obliterans, when it is necessary to establish branches next to traumatic tissue or stenotic arteries, Promoting the angiogenic process is again desirable.

The process of angiogenesis is highly complex, including maintaining endothelial cells in the cell cycle, degrading the extracellular matrix, migrating into and inserting into surrounding tissues, and finally forming ducts. The complex molecular mechanism of angiogenesis is far beyond what people know about it now.

Due to the important role of angiogenesis in many physiological and pathological processes, factors related to the control of angiogenesis have been studied in detail. Many growth factors have been shown to regulate the angiogenic process. These growth factors include fibroblast growth factors (FGFs), platelet-derived growth factors (PDGF), transforming growth factor alpha (TGFα) and hepatocyte growth factor (HGF). See Folkman et al., J. Biol. Chem., 1992267 10931-10934 (review).

Some people think that a special kind of endothelial cell-specific growth factor family, namely, vascular endothelial growth factors (VEGFs), VEGFs and their corresponding receptors, stimulate the growth and differentiation of endothelial cells, and thus differentiate into specific functions. Cellular processes are directly related. These factors all belong to the PDGF family and mainly function through endothelial receptor tyrosine kinases (RTKs).

Nine different proteins in the PDGF family have been identified, named PDGF-A, PDGF-B, and VEGF respectively. The remaining six proteins are very similar to VEGF, namely: VEGF-B, see the international patent PCT/US96/ 02957 (WO 96/26736) and U.S. Patents 5,840,693 and 5,607,918 (Ludwig Cancer Institute and Helsinki University); VEGF-C, see Joukov et al., EMBO J., 1996 15 290-298 and Lee et al., Proc. Natl .Acad.Sci.USA, 1996 93 1988-1992; VEGF-D, see International Patent PCT/US97/14696 (WO 98/07832) and Achen et al., Proc.Natl.Acad.Sci.USA, 1998 95 548 -553; Placental Growth Factor (P1GF), see Maglione et al., Proc.Natl.Acad.Sci.USA, 1991 88 9267-9271; VEGF2, see International Patent PCT/US94/05291 (WO 95/24473) ( Human Genome Sciences, Inc.) and VEGF3, see international patent PCT/US95/07283 (WO 96/39421) (Human Genome Sciences, Inc.). Compared with VEGF, members of the VEGF family have 30%-45% identical amino acid sequences. Members of the VEGF family all contain a VEGF homology domain, a cysteine knot composed of six cysteine residues. The functions of the VEGF family include altering the degree of endothelial cell mitosis, causing changes in vascular permeability, angiogenesis, and lymphatic vessel properties.

Vascular endothelial growth factor (VEGF) is a homodimeric glycoprotein that can be isolated from a variety of sources. VEGF can highly specific endothelial cells to produce mitotic activity. VEGF plays a very important regulatory role in the formation of new blood vessels during embryonic angiogenesis or in adult angiogenesis (Carmeliet et al., Nature, 1996 380 435-439; Ferrara et al., Nature 1996 380 439 -442; Freeara, Davis-Smyth, Endocrine Rev., 1997 18 4-25). If one allele of VEGF is inactivated, the embryo will die due to the failure of the vasculature to develop, showing the importance of the function performed by VEGF (Carmeliet et al., Nature, 1996 380 435-439; Ferrara et al., Nature 1996 380 439-442). In addition, VEGF also has a strong chemical induction effect on monocytes, can induce the production of plasminogen activator and plasminogen activator inhibitor in endothelial cells, and can also promote the permeability of microvessels . Because it can promote microvascular permeability, it is often also called vascular permeability factor (VPF). The isolation method and properties of VEGF can be found in Ferrara et al., J.Cellular Biochem., 1991 47 211.218 and Connolly et al., J.Cellular Biochem..1991 47 219-223. Truncation of the VEGF gene at the mRNA level produces five isoforms of VEGF.

Compared with VEGF, VEGF-B has similar angiogenic and other properties, but the tissues expressing VEGF-B are different from those expressing VEGF. VEGF-B is abundantly expressed in the heart and very little in the lung, whereas VEGF is just the opposite. This indicates that although VEGF-B and VEGF are both expressed in many tissues, they may perform different functions.

VEGF-B was isolated by yeast co-hybrid interaction capture screening technique. Cellular proteins that can interact with type I cellular resinous acid-binding protein (CRABP-I) were screened. Concrete separation process and its properties can be seen in patent PCT/US96/02957 and Olofsson et al., Proc.Natl.Acad.Sci.USA, 1996 932576-2581.

VEGF-C is obtained from the culture medium of PC-3 prostate cancer cell line (CRL1435), according to whether it can tyrosine-phosphorylate the specific receptor tyrosine kinase VEGFR-3 (Flt4) on endothelial cells. The phenomenon is obtained by screening. Among them, VEGFR-3 is expressed by the transfected cell line. Then, VEGF-C was purified by using an affinity chromatography column connected with recombinant VEGFR-3, and then cloned from the PC-3 cDNA library. The specific separation process and its related properties can be found in Joukov et al., EMBO J., 1996 15 290-298.

VEGF-D can be isolated from a human breast cDNA library, which can be purchased directly from Clontech. The EST "Soares Breast3NbHBst" (Achen et al., Proc. Natl. Acad. Sci. USA, 1998 95 548-553) in the human cDNA library was used as a hybridization probe for screening. The specific separation process and its related properties can be found in the international patent PCT/US97/14696 (WO98/07832).

The VEGF-D gene is widely expressed in adults, but certainly not everywhere. VEGF-D is abundantly expressed in heart, lung and skeletal muscle. VEGF-D intermediates are also expressed in the spleen, ovary, small intestine, and colon, with reduced expression in the kidney, pancreas, thymus, prostate, and testis. However, no VEGF-D mRNA was detected in brain, placenta, liver or peripheral leukocytes.

PlGF can be isolated from a cDNA library of term placenta. Concrete separation process and its relevant properties can be seen Maglione et al., Proc.Natl.Acad.Sci.USA, 1991 88 9267-9271. Its specific biological function is still unclear.

VEGF 2 can be isolated from estrogen-independent human breast cancer cell lines. VEGF 2 shares 22% homology with PDGF and 30% homology with VEGF. There is currently no method for isolating the gene encoding VEGF2 and no data on the biological activity of VEGF2.

VEGF 3 can be isolated from a cDNA library derived from colon tissue. VEGF3 is 36% identical and 66% similar to VEGF. There is currently no method for isolating the gene encoding VEGF3 and no data on the biological activity of VEGF3.

The degree of similarity between two proteins is determined by comparing the amino acid sequence and the conservative amino acid changes between the two proteins, and comparing the similarity of the two proteins is only comparing the amino acid sequences, not comparing the conservative amino acid changes between the two proteins.

Members of the PDGF/VEGF family can bind to tyrosine kinase receptors. Five endothelial cell-specific tyrosine kinase receptors have now been discovered, named VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), VEGFR-3 (Flt-4), Tie and Tek/Tie-2. All of them possess the tyrosine kinase activity necessary for signaling. VEGFR-1, VEGFR-2, VEGFR-3, Tie and Tek/Tie-2 play critical and specific roles in angiogenesis and angiogenesis, which has been demonstrated by site-directed mutagenesis of these receptors in mouse embryos Inactivation tests were demonstrated.

The known tyrosine kinase receptors that can bind to VEGFs are VEGFR-1, VEGFR-2 and VEGFR-3. VEGFR-1 and VEGFR-2 can be combined with VEGF with high specificity, and VEGFR-1 can also be combined with VEGF-B and PlGF. VEGF-C is the ligand of VEGFR-3, and can also activate VEGFR-2 (Joukov et al., TheEMBO Journal, 1996 15 290-298). VEGF-D can bind both VEGFR-2 and VEGFR-3. Ligands for Tek/Tie-2 can be found in International Patent No. PCT/US95/12935 (WO 96/11269) (Regeneron Pharmaceuticals, Inc.). Ligands for Tie have not yet been discovered.

Recently, a specific receptor for a new VEGF isoform has been purified and cloned, with a size of 130-135Kda (Soker et al., Cell, 1998 92 735-745). This VEGF receptor can bind VEGF ₁₆₅ very specifically through the sequence encoded by exon 7, but it has only weak binding ability to heparin (Soker et al., Cell, 1998 92 735-745). Surprisingly, this receptor is identical to human NP-1, a receptor involved in early neurogenesis. PlGF-2 can also interact with NP-1 (Migdal et al., J Biol. Chem., 1998 273 22272-22278).

VEGFR-1, VEGFR-2 and VEGFR-3 are all expressed by endothelial cells. VEGFR-1 and VEGFR-2 are expressed by vascular endothelium (Oelrichs et al., Oncogene, 1992 8 11-18; Kaipainen et al., J.Exp.Med., 1993 178 2077-2088; Dumont et al., Dev. Dyn., 1995 203 80-92; Fong et al., Dev. Dyn., 1996 207 1-10). VEGFR-3 is expressed by the lymphatic endothelium of adult tissues (Kaipanen et al., Proc. Natl. Acad. Sic. USA, 1995 9 3566-3570). VEGFR-3 is also expressed by blood vessels surrounding tumors.

Deletion of the VEGFR gene leads to abnormal development of the vascular system, resulting in embryonic death in the second trimester. Analysis of embryos with a complete inactivation of the VEGFR-1 gene revealed that this receptor is required for the functional organization of the endothelium (Fong et al., Nature 1995 376 66-70). However, deletion of only part of the gene encoding the intracellular tyrosine kinase domain of VEGFR-1 produced viable mice with normal vasculature (Hiratsuka et al., Proc. Natl. Acad. Sci. USA 1998 95 9349- 9354). The reasons for these differences remain to be elucidated, but it can be speculated that receptor signaling through tyrosine kinases is not essential for the normal function of VEGFR-1. Studies on homozygous mice with an inactivated VEGFR-2 gene have shown that this receptor is essential for cell proliferation, hematopoiesis, and vasculature (Shalaby et al., Nature, 1995 376 62-66; Shalaby et al., Cell, 1997 89 981-990). Inactivation of VEGFR-3 can cause errors in the cardiovascular system, as it leads to abnormal reorganization of large blood vessels (Dumont et al., Science, 1998 282 946-949).

Although VEGFR-1 is primarily expressed in endothelial cells during development, it is also produced in hematopoietic precursor cells early in embryogenesis (Fong et al., Nature 1995 37666-70). In adults, monocytes and macrophages also express this receptor (Barleon et al., Blood, 1996 87 3336-3343). In embryos, VEGFR-1 is expressed by most, but not all, blood vessels (Breier et al., Dev. Dyn., 1995 204 228-239; Fong et al., Dev. Dyn., 1996 207 1-10) .

The receptor VEGFR-3 is widely expressed in endothelial cells in early embryonic development, but with the progress of embryogenesis, its expression in venous endothelium and lymphatic endothelium is limited (Kaipainen et al., Cancer Res., 1994 54 6571-6577; Kaipainen et al., Proc. Natl. Acad. Sci. USA 1995 92 3566-3570). VEGFR-3 is expressed in lymphatic endothelial cells of adult tissues. This receptor is critical for vascular development during embryogenesis. Targeted inactivation of the VEGFR-3 gene in mice, due to abnormal self-organization of large blood vessels with defective cavities, will lead to the formation of defective blood vessels, resulting in fluid retention in the pericardial cavity, 9.5 years after sexual intercourse Dysfunction of the cardiovascular system. Based on these findings, it can be speculated that VEGFR-3 is required for the development of the primary vascular network to larger vessels. However, the function of VEGFR-3 in the development of lymphatic vessels could not be studied now because the mouse embryos mentioned above died before the formation of the lymphatic system. Scientists speculate that VEGFR-3 is expressed in lymphogenesis and lymphatic endothelial cells in adults, thus playing a role in lymphatic vessel development and lymphogenesis. This is based on the speculation that ectopic expression of VEGF-C was found in the skin of transgenic mice, resulting in a ligand of VEGFR-3, which is produced due to the proliferation of lymphatic endothelial cells in the dermis and the proliferation of blood vessels. increase. This finding further shows that perhaps the most basic function of VEGF-C is its role in the lymphatic endothelium, and the auxiliary function is to play a role in angiogenesis together with VEGF, and to regulate vascular osmolarity (Joukov et al ., EMBO J., 1996 15 290-298).

Due to the anti-angiogenesis mechanism of tumor tissue, some inhibitors of VEGF and VEGF receptor system can inhibit the growth of tumor. See Kim et al., Nature, 1993362 841-844; Saleh et al., Cancer Res., 1996 56 393-401.

From the above-mentioned content, the VEGF family of growth factors are all members of the PDGF family. PDGF plays an important role in the growth and motility of connective tissue cells, fibroblasts, myofibroblasts and glial cells (Heldin et al., "Structure of platelet-derived growth factor: Implications for functional properties", Growth Factor, 1993 8 245-252). In adults, PDGF can promote the healing of wound tissue (Robson et al., Lancet, 1992 339 23-25). Structurally, PDGF is a homodimer (PDGF-AA or PDGF-BB) or a heterodimer (PDGF-AB) composed of homologous polypeptide chains A or B linked by disulfide bonds.

PDGF and its isoforms realize their functions by binding to two structurally associated receptor tyrosine kinases (RTKs) on target cells. Receptor α can bind to A chain and B chain of PDGF, while receptor β can only bind to B chain. These two receptors can be expressed by many cell lines cultured in vitro, but are mainly expressed by mesenchymal cells in vivo. PDGFs can regulate cell proliferation, cell survival and chemotaxis to cells in vitro (see review Heldin et al., Biochim Biophys Acta., 1998 1378 F79-113). In vivo, since PDGFs are expressed in epithelial cells (PDGF-A) or endothelial cells (PDGF-B), close to the stroma where PDGFR is expressed, PDGF can exert its effect in a faster manner. In tumor cells or cell lines cultured in vitro, the co-expression of PDGFs and PDGF receptors will create an automatic signal transmission loop, which is very important for cell transformation (Betsholtz et al., Cell, 1984 39 447-57; Keating et al ., J.R. Coll Surg Edinb., 1990 35 172-4). Overexpression of PDGFs has been found in some pathological conditions, including malignant tumors, arteriosclerosis and fibroproliferative diseases (Heldin et al., The Molecular and Cellular Biology of Wound Repair, New York: Plenum Press, 1996, 249-273).

The importance of PDGFs as regulators of cell proliferation and cell survival was recognized in a recent directed study of the associated genes in mice, which showed that in addition to some overlap between ligands of different PDGFRs, PDGFs and Their receptors play a unique role in physiological processes. Homozygous for PDGF ligand or its receptor with two null mutations are not viable. Approximately 50% of PDGF-A-deficient homozygous mice had an early lethal phenotype, whereas the remaining surviving mice had a complex postnatal phenotype characterized by abnormal formation of alveolar septa due to lack of alveolar myofibroblasts , thus suffering from emphysema (Bostrom et al., Cell, 1996 85 863-873). In addition, mice deficient in PDGF-A also have an epidermal defect phenotype characterized by thinner dermal tissue, malformed hair follicles, and sparse hair (Karlsson et al., Development, 1999 126 2611-2). PDGF-A is also required during the development of oligodendrocytes and myelination in the central nervous system (Fruttiger et al., Development, 1999 126 457-67). The PDGFR-α-deficient phenotype is more prone to early embryonic death, incomplete head closure, abnormal neural crown development, cardiovascular system defects, and skeletal defects (Soriano et al., Development, 1997 124 2691-70). Mice deficient in PDGF-B and PDGFR-β also have a phenotype similar to that described above, often characterized by abnormalities in the kidney, blood, and cardiovascular systems (Leveen et al., Genes Dev., 1994 8 1875-1887; Soriano et al. al., Genes Dev., 1994 8 1888-96; Lindahi et al., Science, 1997277 242-5; Lindahi, Development, 1998 125 3313-2). Among them, abnormalities of the kidney and cardiovascular system are at least partly related to the lack of proper reorganization of parietal cells (cardiovascular smooth muscle cells, adventitial cells or mesangial cells) into blood vessels (Leveen et al., GenesDev., 1994 8 1875- 1887; Lindahl et al., Science, 1997 277 242-5; Lindahl al., Development, 1998 125 3313-2).

SUMMARY OF THE INVENTION

The present invention discloses a novel growth factor capable of stimulating and/or enhancing the proliferation or differentiation and/or growth and/or motility of cells expressing PDGF-C receptors, including, but not limited to, endothelial cells, connective tissue cells, myofibroblasts, and glial cells. In addition, novel growth factors expressed by polynucleotide sequences and formulations of pharmaceutical compositions for treatment and diagnosis all belong to the scope of the present invention.

First, the present invention discloses an isolated and purified polynucleotide molecule comprising a nucleotide sequence at least as shown in Figure 1 (SEQ ID NO: 2) 37 to 1071, 6 to 956 of the nucleotide sequence (SEQ ID NO: 3) shown in Figure 3 or 196 to 1233 of the nucleotide sequence shown in Figure 5 (SEQ ID NO: 6) at least 85 % the same, 90% the same better, preferably 95% the same. 37 to 1071 of the nucleotide sequence (SEQ ID NO: 2) shown in Figure 1, 6 to 956 of the nucleotide sequence (SEQ ID NO: 3) shown in Figure 3 or the nucleus shown in Figure 5 The nucleotide sequence (SEQ ID NO: 6) from 196 to 1233 encodes a nascent polypeptide named PDGF-C (formerly named "VEGF-F"), which is structurally related to PDGF-A, PDGF-B, VEGF, VEGF-B, VEGF-C and VEGF-D are homologous. In the specific example proposed, the nucleic acid molecule is 6 of the nucleotide sequence (SEQ ID NO: 3) shown in Fig. 956 or the 196 to 1233 cDNA molecule of the nucleotide sequence (SEQ ID NO: 6) shown in Figure 5. Simultaneously, the present invention also includes such nucleic acid molecule, it can be with the nucleotide sequence (SEQ ID NO: 3) 6 to 956 positions or 196 to 1233 positions of the nucleotide sequence shown in Figure 5 (SEQ ID NO: 6) or their fragments are hybridized under stringent conditions.

Second, the polypeptide molecule of the present invention has the function of stimulating and/or enhancing cell proliferation or differentiation and/or growth and/or motility that can express PDGF-C receptors, these cells include, but are not limited to, endothelial cells, Connective tissue cells, myofibroblasts and glial cells. Its amino acid sequence is as shown in Figure 2 (SEQ ID NO: 3), Figure 4 (SEQ ID NO: 5), Figure 6 (SEQ ID NO: 7) or their fragments and has the ability to stimulate and/or enhance the expression of PDGF-C receptors Functional analogs of proliferation or differentiation and/or growth and/or motility of cells including, but not limited to, endothelial cells, connective tissue cells (such as fibroblasts), myofibroblasts, and glia cell. The amino acid sequence of the polypeptide should be the same as that in Figure 2 (SEQ ID NO: 3), Figure 4 (SEQ ID NO: 5), Figure 6 (SEQ ID NO: 7), their fragments or analogs with PDGF-C biological activity 85% of the amino acid sequences are identical, more preferably 90% identical, most preferably 95% identical. In an embodiment of the present invention, the polypeptide molecule is a PDGF-C truncated form comprising the PDGF/VEGF homology domain (PVHD) portion of PDGF-C. This domain is from amino acid residue 230 to 345. However, this region can be extended N-terminally up to residue 164. Here, PVHD is defined as a truncated form of PDGF-C. The truncated form of PDGF-C is an active form of PDGF-C.

In the present invention, the comparison of the same degree of sequence is carried out by the comparison tool "MEGALIGN" in the Lasergene software package (DNASTAR, Ltd. Abacus House, Manor Road, West Ealing, London W130AS United Kingdom). The comparison process has been artificially refined. The proportion of the same degree is calculated based on the series in the comparison.

These polypeptides or polypeptides expressed by polynucleotides should have the function of stimulating and/or enhancing the proliferation, differentiation, motility, survival or permeability of cardiovascular cells that can express PDGF-C receptors, these cells include, but not only Limited to, cardiovascular endothelial cells, lymphatic endothelial cells, connective tissue cells (eg fibroblasts), myofibroblasts and glial cells. In addition, it should also promote wound healing. PDGF-C also has angiogenic effects, which is one of the activities of PDGF-C. These properties are collectively referred to as "biological activity of PDGF-C" hereinafter, and can be identified by common methods in the art.

Here, "PDGF-C" refers to the polypeptide molecules of Fig. 2 (SEQ ID NO: 3), Fig. 4 (SEQ ID NO: 5), Fig. 6 (SEQ ID NO: 7) and their fragments or have the above-mentioned PDGF- C Biologically active analogs. It can also refer to polynucleotides encoding PDGF-C or its fragments or analogs with PDGF-C biological activity. The polynucleotide can be native or present in a carrier or liposome.

In another aspect, the present invention discloses a polypeptide having the following amino acid sequence: PXCLLVXRCGGXCXCC (SEQ ID NO: 1). These sequences exist only in PDGF-C, which can be distinguished from other members of the growth factor PDGF/VEGF family, the difference lies in the third and fourth cysteine (see Figure 9, SEQ ID NO: 8- 17) There are three amino acid residues (NCA) inserted between them.

Conservative substitution, insertion or deletion mutations are performed on the polypeptide, but the biological activity of PDGF-C is still maintained. Such mutated polypeptide molecules obviously also belong to the scope of the present invention. Those skilled in the art should know the methods for obtaining the above polypeptides, such as site-directed mutagenesis, specific enzyme digestion and ligation. In addition, it is also possible to obtain a protein complex that still has PDGF-C activity by replacing the amino acid residues in the complex composed of such polypeptides with unnatural amino acids or amino acid analogs. These complexes can be prepared and characterized using methods common in the art. Obviously, these polypeptide complexes also belong to the scope of the present invention.

In addition, possible variants of PDGF-C polypeptide molecules due to selective truncation (VEGF and VEGF-B are produced due to selective truncation) and allelic variants naturally occurring in the nucleic acid sequence encoding PDGF-C Also belong to the category of the present invention. Allelic variant is a standard term in the field, which refers to a nucleic acid sequence that has substitution, deletion or insertion mutations at one or several nucleotide positions, but these changes do not affect the function of the expressed polypeptide .

Such PDGF-C variants can be obtained by modifying non-essential regions of PDGF-C polypeptides. These non-essential regions should be outside the highly conserved regions indicated in Figure 9 (SEQ ID NO: 8-17). In particular, the PDGF family of growth factors, including VEGF, are dimers, and VEGF, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-A, and PDGF-B all have fully conserved eight Cysteine regions, such as PDGF/VEGF-like regions (Olofsson et al., Proc. Natl. Acad. Sci. USA, 1996 93 2576-2581; Joukov et al., EMBO J., 1996 15290-298) . These cysteines may be involved in the formation of intermolecular and intramolecular disulfide bonds. In addition, there is a highly conserved, but not completely conserved, cysteine residue in the C-terminal region. Intramolecular disulfide bonds form loops 1, 2 and 3 within each subunit, and these sites are associated with receptor binding for the PDGF/VEGF family of growth factors (Andersson et al., GrowthFactors. 1995 12 159-164).

Those skilled in the art will appreciate that these cysteine residues should be conserved in those variants that are active, as should the active site amino acids in loops 1, 2 and 3. Other regions in the molecule are not so important in biological function and can be modified to a certain extent. These modified polypeptides should also be able to identify PDGF-C activity by conventional activity detection methods, for example, using the fibroblast proliferation assay method in Example 6.

Some modified PDGF-C polypeptides have the ability to bind to PDGF-C receptors on cells, including, but not limited to, endothelial cells, connective tissue cells, myofibroblasts and/or glial cells, However, it cannot stimulate cell proliferation, differentiation, migration, motility enhancement and survival, or induce vascular proliferation, connective tissue development, and wound healing. These modified polypeptides can be used as competitive or non-competitive inhibitors of PDGF-C polypeptides and growth factors of the PDGF/VEGF family, and can be used in situations where it is necessary to inhibit or reduce the effects of PDGF-C polypeptides and growth factors of the PDGF/VEGF family . These PDGF-C polypeptide variants that have binding ability, but cannot induce mitosis, induce differentiation, induce migration, enhance mobility, increase survival rate, promote vascular proliferation and wound healing also belong to the scope of the present invention. It is referred to herein as a "variant that binds to the receptor but has no other activity". To activate its sole receptor, PDGF-C forms a dimer. However, if the dimer is composed of the above-mentioned variant monomer that can bind to the receptor but has no other activity and another wild-type PDGF-C or a growth factor monomer of the wild-type PDGF/VEGF family, such a dimer The aggregate should be able to bind the receptor, but not trigger downstream signaling.

At the same time, there are other modified PDGF-C polypeptides that can prevent the ability of wild-type PDGF-C polypeptides and PDGF/VEGF family growth factors to bind to the corresponding receptors on cells, including, but not limited to, endothelial cells. cells, connective tissue cells (such as fibroblasts), myofibroblasts, and/or glial cells. As such, the dimer will not be able to stimulate increased proliferation, differentiation, migration, and survival of endothelial cells, nor the proliferation, differentiation, and motility of connective tissue cells, myofibroblasts, and/or glial cells . These modified polypeptides can be used as competitive or non-competitive inhibitors of PDGF-C polypeptides and growth factors of the PDGF/VEGF family, and can be used in situations where it is necessary to inhibit or reduce the effects of PDGF-C polypeptides and growth factors of the PDGF/VEGF family . These occasions include tissue remodeling processes that occur when primary or migratory tumor cells invade normal cell populations. These have the ability to bind to PDGF-C receptors and growth factor PDGF/VEGF family receptors, but do not induce mitosis, do not induce differentiation, do not induce migration, do not enhance mobility, do not improve survival, do not promote vascular proliferation and wound healing PDGF-C growth factor variants also fall within the scope of the present invention. It is referred to herein as a "variant or intermediate that can form a PDGF-C growth factor dimer but has no other activity".

An example of a variant or intermediate that forms PDGF-C growth factor dimers but has no other activity is a mutant PDGF-C that prevents cleavage of the CUB domain by proteases. Variants or intermediates that can form PDGF-C growth factor dimers but have no other activity can be made from active monomers, such as VEGF, VEGF-B, VEGF-C, VEGF-D, PDGF-C, PDGF- A, PDGF-B or PlGF, formed by linking with a mutated CUB domain that can tolerate proteolysis. Dimers composed of the aforementioned variants or intermediates capable of forming PDGF-C growth factor dimers but having no other activity and monomers linked to mutated CUB domains should not be able to bind to their corresponding receptors.

A variant of the above-mentioned dimer is made from an active monomer such as VEGF, VEGF-B, VEGF-C, VEGF-D, PDGF-C, PDGF-A, PDGF-B or PlGF and a mutated A protease cleavage site is inserted between the CUB domains. The inserted protease cleavage site can cut the connection between the CUB domain and the active monomer, releasing the active body that can bind to the corresponding receptor. In this way, dimers with controlled release of active monomers can be prepared.

Thirdly, the present invention discloses a nucleic acid molecule encoding the above polypeptide or polypeptide fragment. Nucleic acid molecules can be DNA, genomic DNA, cDNA or RNA, and can be single-stranded or double-stranded. The source of nucleic acid can be extracted from cells or tissues, or can be recombinantly obtained, or directly synthesized. Due to the degeneracy of the genetic code, the polypeptide molecules and their fragments shown in Figure 2 (SEQ ID NO: 3), Figure 4 (SEQ ID NO: 5), and Figure 6 (SEQ ID NO: 7) can be expressed , analogues with corresponding biological activities, variants that can bind to receptors but have no other activities, or variants or intermediates that can form PDGF-C growth factor dimers but have no other activities or intermediates There should be many types of nucleic acid molecules.

Fourth, the present invention discloses vectors carrying cDNA molecules or nucleic acid molecules as described in the third aspect, and host cells that can be transformed or transfected by the vectors or nucleic acid molecules. They can be of eukaryotic or prokaryotic origin. Host cells should be suitable for expressing the polypeptide molecules of the present invention, including insect cells such as Sf9 cells, which can be obtained from the American Type Culture Collection (ATCC SRL-171), and can be transfected with baculovirus vectors; or human embryonic kidney The cell line 293-EBNA, can be transformed with an appropriate expression plasmid. The preferred vector of the present invention is an expression vector formed by linking the above-mentioned nucleic acid sequence with one or several suitable promoters or other regulatory sequences, and then transforming or transfecting host cells with the vector capable of expressing the polypeptide molecule of the present invention . Other recommended vectors are those suitable for transfection of less animal cells, or for gene therapy, such as adenovirus, vaccinia virus, retrovirus vectors or liposomes.

The present invention also discloses a method for preparing a vector capable of expressing a polypeptide from a nucleic acid molecule, which includes the following steps: linking the nucleic acid molecule with one or several suitable promoters or regulatory sequences in the vector.

The present invention also discloses a method for preparing the polypeptide molecule of the present invention, comprising the steps of: expressing the nucleic acid molecule or vector in a host cell, and then isolating the expressed nucleic acid molecule from the culture medium of the host cell or the cultured cell. Polypeptide molecule.

Fifth, the present invention obtains an antibody molecule that can specifically react with the polypeptide molecule or its fragment described in the present invention. This antibody recognizes variants, immunologically active fragments, analogs and recombinants of PDGF-C. Such antibodies can be used as inhibitors or agonists of PDGF-C, and can detect and quantify PDGF-C. Antibodies can be polyclonal or monoclonal. Such monoclonal or polyclonal antibodies can be used to collect the polypeptide molecules of the present invention and their variants, fragments and analogs. An epitope tag, such as FLAG octapeptide (Sigma, St. Louis, MO), can be attached to the polypeptide sequence to facilitate affinity chromatography enrichment. In some cases, for example, when it is necessary to use monoclonal antibodies to inhibit PDGF-C activity in clinical practice, human monoclonal antibodies should be used. Cytotoxic or cytostatic agents may be further added to these antibodies. The methods for preparing the above substances, including recombinant DNA, are common techniques in the art.

At the same time, antibodies that can specifically recognize PDGF-C and are labeled themselves also belong to the scope of the present invention.

In order to facilitate detection, the polypeptide molecule or antibody of the present invention may have a detectable label. In this way, labeled polypeptide molecules can detect their corresponding receptors in situ. Polypeptide molecular antibodies can be covalently or non-covalently linked to supermagnetic substances, paramagnetic substances, electron-dense substances or radioactive substances, so as to facilitate detection. If used in a diagnostic assay, radioactive or non-radioactive labels may be used. Radioactive labels include ^radioactive isotopes such as125I ^or32P . Non-radioactive labels include enzyme-labeled reagents such as horseradish peroxidase and fluorescent reagents such as FITC. Labeling can be direct or indirect, covalent or non-covalent.

The clinical applications of the present invention include accelerating the angiogenesis process of transplanted tissues or organs, stimulating the healing of wounded tissues, stimulating the development of connective tissues, establishing bypass connections between damaged tissues and arterial blockages (such as coronary heart disease), inhibiting tumor tissue Or the angiogenic process in diabetic retinopathy and the tissue transformation that occurs during inhibition of the invasion of primary or migratory tumor cells into normal cell populations. In addition, in tumor biopsy, detecting the amount of PDGF-C is very helpful for judging the risk of tumor metastasis.

In addition, PDGF-C is associated with many physiological phenomena of the lung. The detection of PDGF-C can be used in the diagnosis of many lung diseases. PDGF-C can also be used in the treatment of many lung diseases to enhance the circulation of blood vessels in the lungs and improve the exchange of air and blood in the lungs. Similarly, for patients with heart defects, PDGF-C can also be used to enhance the blood vessel flow and oxygen permeability of the heart. Similarly, for patients with chronic airway obstruction, PDGF-C can also enhance blood and gas exchange.

The present invention discloses a method for stimulating angiogenesis, lymphangiogenesis, neural network formation, connective tissue development and wound healing in mammals, comprising the steps of: applying an effective dose of PDGF-C, its fragments or Biologically active analogs of PDGF-C. PDGF-C, its fragments or analogs can be applied to the test animals.

PDGF-C agonists (such as antibodies or inhibitors that can competitively or non-competitively bind PDGF-C to form dimers and bind to receptors) can be used to treat conditions such as cardiac congestion disorders, by , such as increasing the permeability of blood vessels in the lungs to cause fluid accumulation that counteracts the problem of vascular permeability in the heart. PDGF-C also addresses fibrotic conditions that can be found in the lungs, kidneys and liver. Applying PDGF-C in the intestinal tract can treat dyspepsia, but at the same time the blood circulation and vascular permeability of the liver and kidney are enhanced.

The present invention also discloses a method capable of inhibiting angiogenesis, lymphangiogenesis, neural network formation, connective tissue development and wound healing in mammals, comprising the steps of: applying an effective dose of PDGF-C agonist to mammals. The agonist can be any substance that can inhibit the activity of PDGF-C, and the mechanism can be to inhibit the binding of PDGF-C to the corresponding receptor on the cell, or to inhibit the activation of the receptor, for example, by using "can bind to the receptor but not variants with other activities". Agonists include, but are not limited to, anti-PDGF-C antibodies, inhibitors that can competitively or non-competitively inhibit PDGF-C and its receptor binding, such as the above-mentioned "PDGF-C growth factor II Polymers but no other active variants or intermediates", complexes that can bind to PDGF-C and modify PDGF-C to inactivate them, and antisense nucleic acid molecules of the above polynucleotide sequences.

The present invention also provides a method for determining the reagent combined with the active fragment of PDGF-C, the steps are: mixing the active fragment of PDGF-C with the detection reagent, and monitoring the degree of combination with an appropriate method. Reagents may contain complexes and other proteins.

The present invention also proposes a screening system for searching active fragments that can bind to PDGF-C. First prepare the active fragment of PDGF-C, mix it with the reagent to be detected, and then quantitatively measure the binding degree of the reagent to be detected and the active fragment of PDGF-C by using an appropriate method. This screening system can also be used to identify substances that inhibit the proteolytic cleavage of full-length PDGF-C, thus preventing the release of the active fragment of PDGF-C that is active. Of course, full-length PDGF-C must be prepared first.

Use this screening system to find compounds that may alter the biological activity of PDGF-C. This screening method can be transformed into a large-scale, automated device similar to the PANDEX (Baxter-Dade Diagnostics) system to accommodate efficient high-throughput screening of possible drugs.

In this screening system, active fragments of PDGF-C or full-length PDGF-C should be prepared in advance, preferably using recombinant DNA technology. Detection reagents, such as complexes and proteins, are introduced into reaction vessels containing active fragments of PDGF-C or full-length PDGF-C. The binding of the detection reagent to the active fragment of PDGF-C or the full-length PDGF-C can be determined using appropriate methods, these methods include, but not limited to, labeling the detection reagent with radioactive isotopes or chemical labels. Other methods for detecting the binding degree of reagents to active fragments of PDGF-C or full-length PDGF-C can also refer to U.S. Patent No. 5,585,277, which determines the ratio of detection reagents and PDGF-C by monitoring the ratio of folded and unfolded proteins. The degree of binding of active fragments of C or full-length PDGF-C. Examples of such detection of protein folding include, but are not limited to, monitoring the resistance of active fragments of PDGF-C or full-length PDGF-C to specific proteases, or detection of proteins and antibodies that can specifically bind to protein folds. degree of bonding.

Workers in the field should understand that the size of the IC ₅₀ value is related to the selected detection reagent. For example, assay reagents with _IC50 values less than 10 nM are well suited for use in drug therapy situations. However, reagents with higher specificity and lower binding strength are better used in this situation. Those skilled in the art will recognize that information on the binding capacity, inhibitory activity and specific selectivity of reagents is very useful for researching corresponding drugs.

When PDGF-C or a PDGF-C agonist is used in therapy, the dosage and mode of use will be determined by the individual administered and the specific situation being treated, at the discretion of the physician or veterinarian. The administration methods include oral administration, subcutaneous injection, intramuscular injection, intraperitoneal injection, intravenous injection, implantation, topical application, and application that does not pass through the digestive system. The method of topical application of PDGF-C is similar to the method of application of VEGF. For example, when promoting wound healing, or other occasions where it is necessary to enhance angiogenesis, PDGF-C can be directly applied to the required tissue or organ, and the effective dose is between 0.1 and 1000 μg/Kg according to the ratio of the dose/body weight between.

PDGF-C or PDGF-C agonists can be used in combination with appropriate drug carriers. The final composition is an appropriate amount of PDGF-C or PDGF-C agonist with medicinal effect, biocompatible non-toxic salts and biocompatible solid or liquid carrier or adjuvant. Examples of such carriers or adjuvants include, but are not limited to, alkali salts, buffered alkali salts, Ringer's solution, mineral oil, talc, starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, phosphoric acid Dicalcium, sodium chloride, dextrose, water, glycerin, ethanol, thickener, stabilizer, suspending agent, or a combination of the above. They can be prepared as solutions, suspensions, tablets, capsules, ointments, elixirs, syrups, wafers, salves or in other forms. The form of the drug should be determined according to the mode of administration. Here, the components containing PDGF-C can also be replaced with one or more of the following substances: PDGF-A, PDGF-B, VEGF, VEGF-B, VEGF-C, VEGF-D, PlGF and/or heparin. The weight ratio of the active substance PDGF-C contained in the medicine is between 0.1% and 90%, generally between 10% and 30%.

For intramuscular preparations, it should be in sterile form, and the active fragment of PDGF-C exists in the form of soluble salts such as hydrochloride, dissolved in pharmaceutical diluents, such as pyrogen-free distilled water, normal saline or 5% glucose solution. Insoluble compounds can be prepared as suspensions in aqueous solutions, or suspended in biocompatible oleic bases, such as esters containing long-chain fatty acids, such as ethyl oleate.

The inventive idea of the present invention can also be used to manufacture diagnostic/disease prediction devices, for example in the form of test kits. In a specific example of the kit of the present invention, the kit includes an anti-PDGF-C antibody, and a method for detecting, more precisely, measuring and estimating the binding status between PDGF-C and its antibody. In another specific example, the above-mentioned anti-PDGF-C antibody is called a primary antibody, and besides the site capable of binding to PDGF-C, it also has a certain animal antigen. In addition, the kit also provides a Secondary antibody antibody molecule, anti-animal antigen on the primary antibody. There is a detectable label on the secondary antibody, and PDGF-C or unlabeled primary antibody is bound to the surface of the substrate, so that the degree of interaction between PDGF-C and the primary antibody can be determined according to the amount of the corresponding label on the substrate. The label was introduced by binding the secondary antibody to the PDGF-C-conjugated primary antibody. In a specific example, the diagnosis/disease prediction device of the present invention is provided in the form of an enzyme-linked immunosorbent assay (ELISA) kit.

In another specific example of the present invention, this diagnostic/disease prediction device may also include a polymerase chain reaction method to amplify the individual to be detected, and compare its sequence structure with the sequence structure published in the present invention Comparison to detect whether there is abnormality, and determine whether the abnormality of PDGF-C expression is related to the specific stage of certain diseases.

In addition, in the specific example of the present invention, the restriction length polymorphism (RFLP) analysis method can also be used, and the genomic DNA of the sample to be tested is cut with a restriction endonuclease, and electrophoresis is performed to generate a specific DNA band. These bands are then compared with the sequence bands published in this patent to detect whether there is any abnormality, and to determine whether the abnormal expression of PDGF-C is related to the specific stage of certain diseases.

The present invention includes a method capable of detecting whether the PDGF-C gene of the sample to be tested that may be related to the disease stage is abnormal. The steps of this method are: obtain DNA or RNA samples from the samples, add specific primers, and selectively amplify the genes related to PDGF-C by PCR, and amplify the nucleic acid sequences amplified from the samples and The sequences in Figure 1 (SEQ ID NO: 2) or Figure 3 (SEQ ID NO: 5) were compared. Those kits comprising primers capable of specifically binding to the PDGF-C gene of the sample and a polymerase capable of amplifying the PDGF-C gene from the DNA sample also belong to the scope of the present invention.

The invention provides a method for detecting PDGF-C in biological samples. The specific steps are: mix and react the reagent capable of binding to PDGF-C with the sample, and then detect the degree of binding. The part that can bind to PDGF-C is preferably an anti-PDGF-C antibody, preferably a monoclonal anti-PDGF-C antibody. In one embodiment, the degree of binding is detected by a detectable label, suitable labels being discussed above.

The present invention relates to protein dimers composed of PDGF-C, especially protein dimers connected by disulfide bonds. The dimer can be a homodimer composed of two PDGF-C polypeptides, or a PDGF-C polypeptide and a VEGF, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-A Or a heterodimer composed of PDGF-B.

The present invention also provides a method for isolating PDGF-C, including the steps of: treating cells expressing PDGF-C with heparin, so that the cells release PDGF-C, and then purifying the released PDGF-C.

The present invention also provides a carrier containing antisense nucleic acid sequence. The antisense nucleic acid sequence is at least partially complementary to the DNA sequence encoding PDGF-C, PDGF-C fragments or analogues with PDGF-C biological activity. Alternatively, the antisense nucleic acid sequence can also interact with the promoter region of the PDGF-C gene, or interact with other non-coding regions of the gene, thereby achieving inhibition, at least reducing the expression level of PDGF-C.

According to the above, such vectors containing antisense nucleic acid sequences can be used to suppress, at least reduce the expression level of PDGF-C. The use of vectors that inhibit PDGF-C expression is quite effective in those cases where PDGF-C expression is associated with disease, for example, tumors produce PDGF-C to promote angiogenesis, or tissue remodeling, which occurs When primary or migrating tumor cells invade the normal cell population. Transformation of the tumor cells with the vector carrying the antisense nucleic acid sequence will inhibit or hinder tumor growth or tissue remodeling.

Another aspect of the invention relates to the discovery that the full length PDGF-C protein may be a potential growth factor. This growth factor requires proteolytic release of the active PDGF/VEGF homology domain for activation. The known proteolytic site is located at residues 231-234 of the full-length protein, residues -RKSR-. This is a binary motif. This site is structurally conserved in murine PDGF-C. Known -RKSR-proteolytic sites are also found in PDGF-A, PDGF-B, PDGF-C and PDGF-D. In these four proteins, a known proteolytic site was also found in the preceding sequence of the smallest domain of the PDGF/VEGF homology domain. All these facts suggest that -RKSR- is the site of proteolysis.

Preferred proteases include, but are not limited to, plasmin, factor X and enterokinase. The N-terminal CUB domain may function as an inhibitory domain, which may be used to keep PDGF-C in a latent form in some extracellular compartments, and this domain can pass specific protein levels when PDGF-C is required. Remove.

The invention provides a method of producing an activated truncated form of PDGF-C or modulating the specificity of receptor binding of PDGF-C. These methods include expressing an expression vector comprising a polynucleotide encoding a polypeptide having PDGF-C biological activity, and supporting hydrolysis by at least one enzyme for processing the expressed polypeptide to produce a truncated form of active PDGF-C .

The invention also provides a method for selectively activating the polypeptide with growth factor activity. The method comprises an expression vector expressing a polynucleotide encoding a polypeptide having growth factor activity and a CUB domain, wherein a proteolytic site is contained between the CUB domain and the polypeptide fragment having activity, the method supports Hydrolysis by at least one enzyme for processing the expressed polypeptide produces an activated polypeptide having growth factor activity.

In addition, the present invention also includes methods for isolating nucleic acid molecules encoding polypeptides with PDGF-C biological activity, and isolating nucleic acid molecules encoding polypeptides that contain a proteolytic site with an amino acid sequence of RKSR or a polypeptide with a structurally conserved amino acid sequence. .

This aspect also includes an isolated dimer consisting of a monomer of active PDGF-C and activated VEGF, VEGF-B, VEGF-C, VEGF-D, PDGF with a CUB domain linked -C, PDGF-A, PDGF-B and PlGF monomers, or conversely, from activated VEGF, VEGF-B, VEGF-C, VEGF-D, PDGF-C, PDGF-A, PDGF-B and The monomer of PlGF and the active monomer of PDGF-C with CUB domain are formed. This dimer may or may not include a proteolytic site located at the junction of the activated monomer and the CUB domain.

Polynucleotides as described above, fragments of these polynucleotides, and polynucleosides having sufficient similarity to non-coding polynucleotide strands that can hybridize under stringent conditions to such polynucleotides or polynucleotide fragments The acid variants can be used to identify, purify, and isolate polynucleotides encoding other non-human mammalian PDGF-C. These polynucleotide fragments and variants are therefore useful in aspects of the invention. Examples of stringent hybridization conditions are as follows: hybridization temperature 42°C, 5X SSC, 20 mM Na ₃ PO ₄ , pH 6.8, 50% formamide; washing conditions 42°C, 0.2X SSC. Those skilled in the art should understand that specific conditions need to be changed according to the length and GC base pair content of the nucleic acid sequence to be hybridized. For specific examples see "Molecular Cloning: A Laboratory Manual", Second Edition, pp. 9.47-9.51, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory (1989).

In addition, purified and isolated polynucleotides that encode other, non-human, mammalian PDGF-C are also within the scope of the present invention, as are purified and isolated polypeptides encoded thereby and those that can be combined with non-human PDGF-C. Variant antibodies that mount a specific immune response. Accordingly, the invention includes the purification and isolation of mammalian PDGF-C polypeptides, as well as the purification and isolation of polynucleotides encoding the polypeptides.

Obviously, the nucleic acid and polypeptide in this invention can be obtained through synthetic or recombinant methods, and can also be extracted and purified from natural substances.

Here, "including" means "including, but not limited to", when specific examples are given.

Brief description of the drawings

Fig. 1 (SEQ ID NO: 2) shows the complete nucleotide sequence of the cDNA of encoding human PDGF-C (hPDGF-C) (2108bp);

Figure 2 (SEQ ID NO: 3) shows the deduced one-dimensional sequence of the full-length hPDGF-C containing 345 amino acid residues (the translated part of the cDNA corresponds to nucleotides 37 to 1071 in Figure 1);

Figure 3 (SEQ ID NO: 4) shows the cDNA sequence of the fragment encoding human PDGF-C (bPDGF-C) (1536bp);

Figure 4 (SEQ ID NO:5) shows the amino acid sequence of the fragment of hPDGF-C (ie the translation of the nucleotide sequence from the 3rd to the 956th in Figure 3);

Figure 5 (SEQ ID NO: 6) shows the nucleotide sequence of the cDNA of mouse PDGF-C (mPDGF-C);

Figure 6 (SEQ ID NO:7) shows the amino acid sequence of the fragment of mouse PDGF-C (translated from the cDNA of the 196th to 1233rd nucleotide in Figure 5);

Figure 7 shows a comparison of the amino acid sequence of human PDGF-C and the amino acid sequence of mouse PDGF-C in Figure 2;

Figure 8 shows the schematic structure of mouse PDGF-C, which consists of signal peptide (indicated by the slash box), N-terminal Clr/Cls/embryonic sea urchin protein Uegf/bone morphogenetic protein 1 (CUB) domain, and C-terminal PDGF/ VEGF-homology domain (indicated by open boxes);

Figure 9 shows a sequence comparison of the PDGF/VEGF homology domains of human and mouse PDGF-C, and compares with other members of the VEGF/PDGF family of growth factors (SEQ ID NO: 8-17);

Figure 10 shows the phylogenetic tree of several growth factors belonging to the VEGF/PDGF family;

Figure 11 provides the amino acid sequence comparison (SEQ ID NO: 18 and 19) of the CUB domain that exists in human and mouse PDGF-Cs and others exist in human osteogenetic protein-1 (hBMP-1, 3 CUB domains Amino acid sequences of the CUB domains of CUB1-3) (SEQ ID NOs: 20-22) and human NP-1 (2 CUB domains) (SEQ ID NOs: 23 and 24);

Figure 12 shows the analysis of the Northern hybridization of the transcript expressed by PDGF-C in several human tissues;

Figure 13 shows the change of the expression level of the mRNA of PDGF-C induced by hypoxia;

Figure 14 shows the expression of PDGF-C in human tumor cell lines;

Figure 15 shows the results of detecting full-length human PDGF-C by immunoblotting in transfected COS-1 cells;

Figure 16 shows the isolation process and partial features of full-length PDGF-C;

Figure 17 shows the isolation and partial characterization of a truncated body of human PDGF-C containing only the PDGF/VEGF homology domain;

Figure 18 is the standard curve obtained by the PDGF-BB homodimer binding to the PAE-1 cells expressing PDGF α-receptor;

Figure 19 provides the graph that the PDGF-BB of representative inhibition label is bound to on the PAE-1 cell expressing PDGF α-acceptor, and this inhibition can be by increasing the PDGF-CC albumen of a large amount of purification full-length and truncated;

Figure 20 shows the effect of full-length and truncated PDGF-CC homodimers on the phosphorylation of PDGF-α-receptors;

Figure 21 shows the mitogenic activity of full-length and truncated PDGF-CC homodimers on fibroblasts;

Figure 22 shows the identification results of truncated PDGF-CC binding to PDGF receptors;

Figure 23 shows the immunohybridization of the 26-28kDa protein fragment of undigested full-length PDGF-C protein and plasmin;

Figure 24 shows the identification results of the competitive binding of full-length PDGF-C and truncated PDGF-C to the PDGER-α receptor;

Figure 25 shows the result of analyzing the human PDGF-C CUB domain by SDS-PAGE under reducing and non-reducing conditions;

Figures 26A-26V show the expression status of PDGF-C in developing mouse embryos;

Figures 27A-27F show the expression status of PDGF-C, PDGF-A and PDGFR-α in developing kidney;

Figure 28A-28F shows that from wild type (Figure 28A and 28c), PDGFR-α-/- (Figure 28B and 28F), PDGF-A-/- (Figure 28D) and PDGF-A/PDGF-B double-/- (FIG. 28E) Histology of E 16.5 kidney from kidney.

Detailed description of the preferred embodiment

Fig. 1 (SEQ ID NO: 2) shows the nucleotide sequence of the cDNA of complete coding human PDGF-C (hPDGF-C) (2108bp), and this hPDGF-C is the new member of VEGF/PDGF family. Comparing clone #4 encoding hPDGF-C (see Figure 3 and Figure 4-SEQ ID NO: 4 and 5) with the mouse protein (corresponding to 27 amino acids) shows that clone #4 is not a full-length sequence, lacking about 80 base pair coding sequence. Additional cDNA clones isolated from a human fetal lung cDNA library included inserts of the above-mentioned deleted sequences. Clone #10 has a longer insertion than clone #4. The insertion of clone #10 begins at the 5' region and contains the deleted sequence. Clone 10# includes the full sequence of human PDGF-C. List some 5'-untranslated sequences in Fig. 1 (SEQID NO:2), the sequence of the cDNA of some translated coding human PDGF-C and some 3'-untranslated nucleic acid sequences. The stop codon is located 21 bp upstream of the start ATG (underlined under the start ATG in FIG. 1 ).

By searching NCBI's EST database and dbEST, a human EST sequence (W21436) was found, which encodes the human homologous part of mouse PDGF-C. From this, the work of isolating the undiscovered human PDGF/VEGF can be started. According to the obtained human EST sequence, two oligonucleotide chains were designed as follows:

5'-GAA GTT GAG GAA CCC AGT G-3'5' Primer (SEQ ID NO: 25)

5'-CTT GCC AAG AAG TTG CCA AG-3'3' Primer (SEQ ID NO: 26)

Using the above-mentioned oligonucleotide primers, a polynucleotide with a length of 348 bp in the human fetal lung 5'-STRETCH PLUS λgt10 cDNA library can be amplified by PCR technology. The PCR product was then cloned into the PCR 2.1-vector of the TA cloning kit (Invitrogen). Subsequently, PCR product clones with a length of 348 bp were used to construct hPDGF-C probes according to standard techniques.

10 ⁶ λ-clones from the human fetal lung 5'-STRETCH PLUS gt10 cDNA library (Clontech) were screened with the hPDGF-C probe according to standard methods. Of the several positive clones, clone #4 was focused on analyzing the inserted nucleotide sequence using internal and vector oligonucleotides according to standard methods. The insert of clone #4 contained a partial nucleotide sequence of cDNA encoding full-length human PDGF-C (hPDGF-C). Figure 3 (SEQ ID NO: 4) shows the nucleotide sequence (1536 bp) of the clone #4 insert. The translated portion of this cDNA includes nucleotides 6 to 956. Figure 4 (SEQ ID NO: 5) shows the amino acid sequence that is presumed to be the translated portion of the inserted sequence. The amino acid sequence constitutes a polypeptide lacking the first 28 amino acid residues of the full-length hPDGF-C polypeptide, but the polypeptide includes a proteolytic fragment that can fully activate the PDGF alpha receptor. It should be noted that the first glycine (Gly) of SEQ ID NO 5 is not found in full-length hPDGF-C.

After searching the dbEST database of NCBI, a mouse EST sequence (AI020581) was found, which coded part of a new mouse PDGF, a part of PDGF-C. The vast majority of mouse cDNA was obtained by PCR amplification using DNA from the mouse embryonic λgt10 cDNA library as a template. In order to amplify the 3' end of the cDNA, it is necessary to use a sense strand primer obtained according to the mouse EST sequence (the sequence of the primer is 5'-CTT CAG TAC CTT GGA AGA G, primer 1 (SEQ ID NO: 27)), In order to amplify the 5' end of the cDNA, it is necessary to use a nonsense strand primer (the sequence of the primer is 5'-CGC TTGACC AGG AGA CAAC, primer 2 (SEQ ID NO: 28)) obtained according to the mouse EST sequence. λgt10 carrier primers are sense strand primer 5'-ACG TGA ATT CGA CAA GTT CAG CCTGGT TAA (primer 3 (SEQ ID NO: 29)) and nonsense strand primer 5'-ACG TGGATC CTG AGT ATT TCT TCC AGG GTA (primer 4 (SEQ ID NO: 30)). Vector primers and internal primers derived from mouse ESTs were used in standard PCR reactions. The sizes of the fragments amplified by PCR were about 750 bp (3' fragment) and 800 bp (5' fragment), respectively. These fragments were cloned into PCR2.1 vector and subjected to nucleic acid sequence analysis using vector primers and internal primers. These fragments do not contain the full-length sequence of mPDF-C, and the mouse liver ZAP cDNA library was screened using standard methods. A 32P-labeled 261 bp PCR fragment was constructed as a probe using primers 1 and 2 and using DNA from the mouse embryo lambda gt10 library as template (see above). The positive phage plaques were purified, and then the inserted nucleotide sequence was obtained by subcloning. Vector-specific primers and internal primers were used. By analyzing the nucleotide sequence information of the PCR clones produced and the isolated clones, the full-length amino acid sequence of mPDGF-C can be deduced (see Figure 6) (SEQ ID NO: 7).

Figure 7 shows the comparison of the amino acid sequences of mouse and human PDGF-C (SEQ ID NO: 6 and 2, respectively). This comparison shows that human and mouse PDGF-Cs have approximately 87% identity, and 45 amino acids have been substituted in the full-length 345 amino acid residues of the protein. Almost all amino acid substitutions observed were conservative in nature. The predicted site of signal peptidase hydrolysis in mPDGF-C is located between residues G19 and T20. This results in a murine secretable polypeptide of 326 residues.

Figure 8 provides a schematic domain structure of murine PDGF-C with a signal sequence (represented by a slashed box), an N-terminal CUB domain, and a C-terminal PDGF/VEGF homology domain (represented by an open box). structure. Underlined amino acid sequences show no significant similarity to the CUB domain or to the VEGF-homology domain.

A high degree of sequence identity indicates that human and mouse PDGF-C have almost identical domain structures. The comparison of amino acid sequences showed that both mouse and human PDGF-C had a new domain structure. In addition to the PDGF/VEGF-homology domain located in the C-terminal region of both proteins (residues 164 to 345), there is a domain at the N-terminal region of both PDGF-Cs that can be assigned to the CUB domain (Bork and Beckmann, J. Mol. Biol., 1993231, 539-545). This approximately 110 amino acid domain was originally identified from the complement factor Clr/Cls, but has recently been found in several other extracellular proteins, including signaling molecules such as bone-forming protein 1 (BMP-1) (Wozney et al. al., Science, 1988 242,1528-1534), receptor molecules, such as NP-1 (Soker et al., Cell, 1998 92 735-745). Although the function of the CUB domain is unclear, it is possible that it is involved in protein-protein interactions or in interactions with carbohydrates such as the proteoglycan heparan sulfate.

Figure 9 shows the comparison of the amino acid sequences of the C-terminal PDGF/VEGF-homology domains of human and mouse PDGF-Cs and the comparison with PDGF/VEGF family members such as VEGF ₁₆₅ , PLGF-2, VEGF-B ₁₆₇ , Pox Orf VEGF Comparison of the amino acid sequences of the C-terminal PDGF/VEGF-homology domains of , VEGF-C, VEGF-D, PDGF-A and PDGF-B (SEQ ID NO: 8-17). Some amino acid sequences of the N-terminal and C-terminal regions of VEGF-C and VEGF-D are omitted in this figure. Gaps are introduced to optimize comparisons. The comparison was obtained using the method of J. Hein (Methods Enzymol., 1990 183 626-45) and the PAM250 residue weight table. Boxed residues show amino acids that match PDGF-Cs located inside the two distant units.

This comparison shows that PDGF-C has the expected structure consisting of cysteine residues, with one exception. Between cysteines 3 and 4, usually separated by 2 residues, there is an insertion of three amino acids (NCA). The characterization of this sequence in PDGF-C was unexpected.

According to the amino acid sequence comparison in Fig. 9, the phylogenetic tree shown in Fig. 10 can be constructed. The data indicate that the PDGF-C homology domain is closely related to the PDGF/VEGF-homology domain of VEGF-C and VEGF-D.

As shown in Figure 11, the amino acid sequences from several CUB-containing proteins were compared. The results show that the simple CUB domains of human and murine PDGF-C share significant identity, as compared to most closely related CUB domains. Derived from human BMP-1, with the sequence of 3 CUB domains (SEQ ID NO: 20-22) and human NP with 2 CUB domains (CUB1-2) (SEQ ID NO: 23 and 24, respectively) -1 is also shown in the sequence diagram. Gaps are introduced to optimize comparisons. The comparison was made using the method of J. Hein (Methods Enzymol., 1990 183 626-45) and the PAM250 residue weight table.

Fig. 12 shows the analysis results of Northen hybridization of expression products of PDGF-C transcript in several human tissues. This analysis indicated that PDGF-C is encoded by a major transcript of approximately 3.8-3.9 kb and a minor transcript of 2.8 kb. Numbers on the right refer to the size of the mRNA (expressed in kb). Tissue expression of PDGF-C was detected by the commercialized MTN technology (Clontech). The hybridization assay can be performed according to the supplier's instructions, incubated at 68°C for one hour using ExpressHyb solution (highly stringent conditions), with the 353bp hPDGF-C EST probe derived from the fetal lung cDNA library screened as described above detection. The hybridization was then washed in 50°C 2X SSC containing 0.05% SDS for 30 minutes and then in 50°C 0.1X SSC containing 0.1% SDS for 40 minutes. The hybridization was then transferred to a membrane and exposed at -70°C. Hybridization results showed that PDGF-C transcripts were most abundant in heart, liver, kidney, pancreas and ovary, while in other tissues, such as placenta, skeletal muscle and prostate, its content level was very low. In the spleen, colon, and peripheral blood leukocytes, the PDGF-C content was lower than the detectable level.

Figure 13 shows the regulation of the expression of PDGF-C mRNA caused by hypoxia. The Marker (expressed in kb) is located on the left side of the panel below. The estimated sizes of PDGF-C mRNAs are shown on the left of the upper panel (2.7 and 3.5 kbs, respectively). To investigate whether PDGF-C is induced by hypoxia, cultured human skin fibroblasts were exposed to hypoxia for 0, 4, 8, and 24 hours, respectively. Poly(A)+ mRNA was isolated from cells using oligo-dT cellulose affinity chromatography purification. The isolated mRNA was electrophoresed in 12% agarose, and 4 μg mRNA was loaded on each lane, and then Northern hybridization was performed, that is, hybridization with the probe of PDGF-C. The size of these two bands was determined on the one hand by hybridizing the same filter to a mixture of hVEGF, hVEGF-B and hVEGF-C probes (Enholmetal. Oncogene, 1997 14 2475-2483), on the other hand according to The interpolation of the size of the mRNA is determined. The results shown in Figure 13 demonstrate that PDGF-C is not regulated by hypoxia in human skin fibroblasts.

Figure 14 shows expression of PDGF-C mRNA in human tumor cell lines. To investigate whether PDGF-C is expressed in human tumor cell lines, Poly(A)+ mRNA was isolated from several known tumor cell lines, followed by 12% agarose electrophoresis, and then hybridized with PDGF-C probe The Northem hybridization method was used for analysis. The results shown in Figure 14 show that PDGF-C mRNA is expressed in several types of human tumor cell lines, such as JEG3 (human choriocarcinoma, ATCC #HTB-36), G401 (Wilms' tumor, ATCC #CRL-1441) , DAMI (megakaryoblastic leukemia), A549 (human lung cancer, ATCC #CCL-185) and HEL (human erythroleukemia, ATCC #TID-180). It should be noted that further growth of these PDGF-C expressing tumors can be inhibited by inhibiting PDGF-C. Likewise, PDGF-C expression can also be detected as a method to identify specific tumor types.

Example 1: Generation of specific anti-peptide antibodies against human PDGF-C

Two synthetic peptides were used to raise antibodies against human PDGF-C. The first synthetic peptide corresponds to residues 29-48 of the N-terminus of full-length PDGF-C, including N-terminal and C-terminal extra cysteine residues: CKFQFSSNKEQNGVQDPQHERC (SEQ ID NO: 31). The second synthetic peptide corresponds to residues 230-250 of the internal region of full-length PDGF-C, which includes an additional cysteine residue at the C-terminus: GRKSRVVDLNLLTEEVRLYSC (SEQ ID NO: 32). The two synthetic peptides were linked to each other by using N-succinimidyl-1-3-(2-pyridyldithio)propionate (SPDP) (Pharmacia Inc.) according to the supplier's instructions. Tethered pores were entangled on hemocyanin (KLH, Calbiochem), and 200-300 mg of the ligation product was emulsified in phosphate-buffered saline (PBS), respectively, in Freund's complete adjuvant and injected subcutaneously at multiple sites in rabbits. Rabbits were injected subcutaneously with the same amount of the ligation product emulsified in Freund's complete adjuvant every two weeks. Rabbit blood was drawn and serum was prepared by known standard methods.

Example 2: Expression of full-length human PDGF-C in mammalian cells

The full-length cDNA encoding human PDGF-C was cloned into a mammalian expression vector with the SV40 promoter, pSG5 (Stratagene, La Jolla, CA). COS-1 cells transfected with this vector were harvested using DEAE-dextran, and the pSG5 vector without cDNA inserted was used as a negative control. Serum-free medium was added to the transfected COS-1 cells 24 hours after transfection, and the fluid containing secreted proteins was collected 24 hours after the addition of the medium. These liquids tend to coagulate after 30 minutes in ice-bathed trichloroacetic acid, and the precipitates are washed with acetone. The aggregated proteins were dissolved in SDS loading buffer under reducing conditions and the proteins were separated using SDS-PAGE gels according to standard methods. Separated proteins were electrotransferred to Hybond filters and then immunoblotted with rabbit antiserum against full-length PDGF-C, which was prepared as described above. Bound antibodies can be detected using enhanced chemiluminescence (ECL, Amersham Inc.). Figure 15 shows the results of this immunoblotting. The sample is only partially reduced, the monomer of human PDGF-C is 55 kDa in size (lower band) and the dimer is 100 kDa in size (upper band). This result indicates that the protein is secreted in intact form and that there is no major proteolytic process during secretion in mammalian cells.

Example 3 Expression of full-length and truncated human PDGF-C in baculovirus-infected Sf9 cells

The full-length coding portion of the cDNA (970bp) of human PDGF-C can be amplified by PCR method using Deep Vent DNA polymerase (Biolabs) according to standard conditions and procedures. Full-length PDGF-C was amplified for 30 cycles, each cycle including denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, and extension at 72°C for 2 minutes. The 5' end primer is 5'CGGGATCCCGAATCCAACCTGAGTAG3' (SEQ ID NO: 33), which includes a BamHI site (underlined) for cloning. The 3' end primer was 5' GGAATTCCTAATGGTGATGGTGATGATGTTTGTCATCGTCATCTCCTCCTGTGCTCCCTCT3' (SEQ ID NO: 34). This primer includes an EcoRI site (underlined) and the sequence of the C-terminal 6XHis tag generated by the enterokinase site. In addition, residues 230-345 of the PDGF/VEGF homology domain (PVHD) of human PDGF-C can be amplified by PCR method using Deep Vent DNA polymerase (Biolabs) according to standard conditions and methods. Residues 230-345 of PVHD of PDGF-C were amplified for 25 cycles, each cycle including denaturation at 94°C for 1 minute, annealing at 56°C for 4 minutes, and extension at 72°C for 4 minutes. The 5' end primer is 5'CGGATCCCG.GAAGAAAATCCAGAGTGGTG3' (SEQ ID NO: 35). This primer includes a BamHI site (underlined) for cloning. The 3' end primer is 5' GGAATTCCTAATGGTGATGGTGATGATGTTTGTCATCGTCATCTCCTCCTGTGCTCCCTCT-3' (SEQ ID NO: 36), which includes an EcoRI site (underlined) and the sequence encoding the C-terminal 6X His tag produced by the enterokinase site. The PCR product was digested with BamHI and EcoRI, and then cloned into the baculovirus expression vector pAcGP67A. Confirmation that the correct sequence of the PCR product is cloned into the vector can be verified by nucleic acid sequencing. The linearized baculovirus DNA expression vector was then co-transfected into Sf9 insect cells Sf9 according to the manual (Pharminogen). Recombinant baculoviruses were amplified several times according to the manual (Pharminogen) before starting large-scale protein production and purification.

Sf9 cells (multiplicity of infection: ~7) cultured in serum-free medium were infected with recombinant baculovirus. Four days after infection, the medium containing the recombinant protein was collected and incubated with Ni-NTA-agarose beads (Qiagen). The beads are collected into the column, using high-strength elution conditions: 50mM sodium phosphate buffer, pH8, containing 300mM NaCl (elution buffer), the bound protein can be increased by increasing the concentration of imidazole in the elution buffer (from 100mM to 500mM) eluted. The eluted proteins were analyzed by SDS-PAGE on a 12.5% polyacrylamide gel under reducing and non-reducing conditions. For immunoblot analysis, proteins were electrotransferred to Hybond filters for 45 minutes.

Figures 16A-C show the isolation process of full-length human PDGF-C protein and its partial characterization. In Figure 16A, the recombinant full-length protein can be visualized by hybridization using an anti-peptide antibody directed against the N-terminal peptide (as above). In Figure 16B, the band of the recombinant full-length protein was visualized by hybridization using an anti-peptide antibody against the internal peptide (as above). The separated protein bands can be visualized by staining with Coomassie brilliant blue. The isolated proteins were visualized by staining with Coomassie blue (Fig. 16C). The numbers at the bottom of Figures 16A-C refer to the concentration of imidazole used to elute the protein from the Ni-NTA column, expressed in moles. Figures 16A-C show that the full-length protein migrates at a size of 99 kDa under non-reducing conditions and at 55 kDa under reducing conditions. This indicates that the full-length protein is a disulfide-crosslinked dimer.

Figures 17A-C show the isolation and partial characterization of a truncated form of human PDGF-C containing only the PDGF/VEGF homology domain. In Fig. 17A, immunoblot analysis of the fragments eluted from the Ni-agarose column showed that the protein could be eluted depending on the concentration of imidazole (range 100-500 mM). The eluted fragments were analyzed under non-reducing conditions and truncated human PDGF-C was visualized by hybridization using an anti-peptide antibody against the internal peptide (as above). Figure 17B shows the same fragment stained with Coomassie brilliant blue as in Figure 17A. This indicates that the process produced a highly purified material that migrated at a size of 36 kDa. Figure 17C shows the bands of truncated human PDGF-C protein stained with Coomassie brilliant blue under reducing and non-reducing conditions. The data indicate that the protein is a disulfide-linked secretable dimer, with monomers migrating at a size of 24 kDa.

Example 4: Receptor binding properties of full-length and truncated PDGF-C

By investigating the binding ability of full-length and truncated PDGF-C and soluble Ig-fusion protein (wherein the Ig-fusion protein includes the extracellular domain of VEGF-1, VEGF-2 or VEGF-3), thereby The state of interaction between full-length and truncated PDGF-C and VEGF receptor can be known (Olofsson et al., Proc. Natl. Acad. Sci. USA, 1998 9511709-11714). Here the fusion protein refers to VEGFR-1-Ig, VEGFR-2-Ig or VEGFR-3-Ig, which are expressed in human 293EBNA cells. All Ig fusion proteins are human VEGFRs. After transfection, the cells were incubated for 24 hours, washed with DMEM (Dulbecco's Modified Eagle Medium) containing 0.2% bovine serum albumin, and cultured for another 24 hours. The addition of Protein A-Sepharose beads can cause fusion proteins to aggregate from the medium. These beads were combined with 100 μl of 10X binding buffer (5% bovine serum albumin, 0.2% Tween 20, and 10 μg/ml heparin) and 900 μl of conditioned medium for culturing 293 cells that had been treated with Mammalian expression plasmids encoding full-length or truncated PDGF-C or control vectors were transfected and then metabolically labeled with ³⁵ S-cysteine and methionine for 4-6 hours. After 2.5 hours, Sepharose beads were washed 3 times at room temperature with binding buffer containing phosphate-buffered saline at 4°C and boiled in SDS-PAGE buffer. Tagged proteins bound to Ig-fusion proteins can be analyzed by SDS-PAGE under reducing conditions. Radioactively labeled proteins can be detected using a phosphorescence analyzer. In all of these analyses, radiolabeled PDGF-C did not appear to interact with any VEGF receptors.

In the next step, the ability of full-length and truncated PDGF-C to bind to human PDGF-C receptor α and β was tested by analyzing their ability to compete with PDGF-BB for binding to PDGF receptor. Binding experiments were performed on porcine aortic endothelial-1 cells stably expressing human PDGF-C receptors α and β (Erikson et al., EMBO J, 1992, 11, 543-550). For the specific process of the binding experiment, refer to Heldin et al., EMBO J, 1988, 71387-1393. Different concentrations of full-length and truncated human PDGF-C or human PDGF-BB were mixed with 5 ng/ml of ¹²⁵ I-PDGF-BB in binding buffer (PBS containing 1 mg/ml bovine serum albumin). The liquid and the receptor-expressing PAE-1 cells placed in a 24-well culture plate were placed on ice for 90 minutes. After washing three times with binding buffer, ¹²⁵ I-PDGF-BB attached to cells was extracted by lysing cells in 20 mM Tris-HCl, pH 7.5, 10% glycerol, 1% Triton X-100. The radioactivity carried by the cells was detected with a β counter. Figure 18 shows the standard curve ^of125I -labeled PDGF-BB homodimer binding to PDGFα-receptor expressing PAE-1 cells. Unlabeled excess protein added to the incubation will compete efficiently with the cellular binding of the radiolabeled tracer.

Figure 19 shows that truncated PDGF-C efficiently competes for binding to the PDGF α-receptor, whereas full-length PDGF does not. Neither the full-length nor the truncated protein competes for binding to the PDGF β-receptor.

Example 5: PDGF α-receptor phosphorylation

To verify whether PDGF-C caused increased phosphorylation of PDGF α-receptors, the ability of full-length and truncated PDGF-C to bind to PDGF α-receptors and stimulate increased phosphorylation was examined. Serum-free porcine aortic endothelial cells stably expressing human PDGF α-receptor were placed on ice for 90 minutes in PBS supplemented with 1 mg/ml BSA and 10 ng/ml PDGF-AA, 100 ng/ml full-length Human PDGF-CC homodimer (flPDGF-CC), 100ng/ml truncated PDGF-CC homodimer (cPDGF-CC), 10ng/ml PDGF-AA and 100ng/ml truncated PDGF- A mixture of CC. Full-length and truncated PDGF-CC homodimers were prepared as described above. 60 minutes after adding the polypeptide, cells were lysed with cell lysate (20mM tris-HCl, pH7.5, 0.5% Triton-X100, 0.5% deoxycholic acid, 10mM EDTA, 1mM orthvanadate, 1mM PMSF, 1% Trasylol). PDGF α-receptors can be immunoprecipitated from clarified lysates with rabbit antiserum directed against human PDGF α-receptors (Eriksson et al., EMBO J, 1992 11 543-550). The aggregated receptors were subjected to SDS-PAGE. After SDS gel electrophoresis, coacervated receptors were transferred to nitrocellulose membranes, which were detected with anti-phosphotyrosine antibody PY-20 (Transduction Laboratories). The membrane was then incubated with horseradish peroxidase-conjugated anti-mouse antibody. Bound antibodies were detected by enhanced chemiluminescence (ECL, Amersham Inc.). Then the material on the membrane was washed away, and then checked with PDGF α-receptor rabbit antiserum, and the amount of receptor was determined by incubation with anti-rabbit antibody linked to horseradish peroxidase, and then detected. Bound antibodies were detected by enhanced chemiluminescence (ECL, Amersham Inc.). Detection of PDGF α-receptor antibody on the membrane showed that all lanes contained an equal amount of receptor. In the experiment, PDGF-AA was used as the control. Figure 20 shows that truncated rather than full-length PDGF-CC can efficiently induce PDGFα-receptor tyrosine phosphorylation. This suggests that truncated PDGF-CC is a potential PDGF α-receptor agonist.

Example 6: Mitogenic effect of PDGF-C on fibroblasts

Figure 21 shows the mitogenic activity of truncated and full-length PDGF-CC on fibroblasts. For the assay method, see Moil et al., J.Biol.Chem., 1991 266 21158-21164. Serum-free cultured human foreskin fibroblasts were incubated with 1ml serum-free medium containing 0.2umCi [3H] thymidine, and added 1mg/ml BSA and 3ng/ml, 10ng/ml for 24 hours , 30ng/ml full-length PDGF-CC (flPDGF-CC), truncated PDGF-CC (cPDGF-CC) or PDGF-AA. The extent to which [3H]thymidine infiltrates DNA after trichloroacetic acid coagulation is determined by a β-counter. The results indicated that it is not the full-length PDGF-CC, but the truncated PDGF-CC that is a potential fibroblast mitogenic agent. In the experiment, PDGF-AA was used as the control.

PDGF-C does not bind to any known VEGF receptors. PDGF-C is the only known member of the VEGF family that can bind to the PDGF α-receptor and increase its phosphorylation. PDGF-C is also the only member of the VEGF family known to be potentially responsible for fibroblast division. These features suggest that the truncated form of PDGF-C may not be a VEGF family member, but a novel PDGF. In addition, the full-length protein may be a potential growth factor, which needs to be activated by proteolysis to release the active PDGF/VEGF homology domain. The known proteolytic site is located in a binary motif at residues 231-234 (-R-K-S-R-) of the full-length protein. The structure of this site was shown to be conserved by comparing mouse and human PDGF-Cs (Fig. 7). Preferred proteases include, but are not limited to, Factor X and enterokinase. The N-terminal CUB domain can act as an inhibitory domain and can be used to localize potential growth factors in certain extracellular compartments (e.g. the extracellular skeleton), which can be removed by specific proteolysis if desired, e.g. during embryonic development , during tissue regeneration and tissue remodeling processes, including bone remodeling, active angiogenesis, tumor progression, tumor invasion, metastasis formation and/or wound healing.

Example 7: PDGF receptors bound to truncated PDGF-C

Truncated PDGF-C and PDGF α and β receptors were assessed by assaying their ability to bind to porcine aortic endothelial-1 (PAE-1) cells expressing PDGF α or β receptors, respectively (Eriksson et al., EMBO J, 1992, 11, 543-550). For the specific binding steps in the experiment, please refer to Heldin et al., EMBOJ, 1988, 7 1687-1393. Truncated PDGF-C protein (5 μg) was radiolabeled with Bolton-Hunter reagent (Amersham) in 10 μl of sodium borate buffer to achieve a radioactivity of 4×10 5 cpm/ng. Add the binding buffer (1 mg/ml bovine serum albumin PBS) containing different concentrations of radioactively labeled truncated PDGF-C to a 24-well culture plate, and each well of the culture plate contains cells that can express the receptor PAE -1, place on ice for 90 minutes, or add unlabeled protein and place on ice for 90 minutes. The binding buffer was washed three times, and the cells were lysed with a lysis solution (20 mM Tris-HCl, pH 7.5, 10% glycerol, 1% Triton-100) to extract ¹²⁵ I-labeled PDGF-C bound to the cells. Cell-bound radioactive doses were determined by a gamma-counter. In some experiments a 100-fold excess of truncated PDGF-C was added, and no non-specific binding was detected. All the binding data are the average of three experiments, and the experimental error in the experiment is 10-15%. As shown in Figure 22, truncated PDGF-C only bound to cells expressing PDGF alpha receptors, but not to cells expressing PDGF beta receptors. When quantitatively replacing radiolabeled PDGF-C with a 100-fold excess of unlabeled protein, the binding was shown to be specific.

Embodiment 8: Effect of proteolytic enzyme on full-length PDGF-C

To demonstrate whether full-length PDGF-C can be activated by specific proteolysis and release the PDGF/VEGF homologous domain from the CUB domain, the full-length protein was treated with different proteases. For example, plasmin (Sigma) was used to treat full _- length _PDGF- C, incubate at 37 degrees Celsius for 1.5-4.5 hours. The released domain was essentially identical in size to the truncated PDGF-C produced in the transfected cells described above. Plasmin-digested PDGF-C and undigested full-length PDGF-C were subjected to SDS-PAGE gels in the reducing state. After SDS-PAGE gel electrophoresis, the separated proteins were transferred to nitrocellulose filter membranes, and the filter membranes were labeled with rabbit anti-peptide antiserum probes at residues 230-250 of the full-length protein (residue GRKSRVVDLNLLTEEVRLYSC (SEQ ID NO: 37) is located just N-terminal to the PDGF/VEGF homology domain). Antibody binding can be detected by enhanced chemiluminescence (ECL, Amersham Inc). Figure 23 shows the results of immunoblotting of the 26-28 kDa protein with the 55 kDa undigested full-length protein and the plasmin-digested full-length protein.

Example 9: PDGF alpha receptor binds plasmin digested PDGF-C

To determine the interaction of PDGF α receptor with plasmin-digested PDGF-C, plasmin-digested PDGF-C was bound to PDGF α receptor expressed by porcine aortic endothelial-1 cells to detect its binding ability (Eriksson et al., EMBO J, 1992, 11 543-550). The receptor binding experiment was performed according to the steps in Example 7, and 30 ng/ml I ¹²⁵ -labeled cut-off PDGF-C was used as a tracer. As shown in Figure 24, increasing concentrations of plasmin-digested PDGF-C can compete with PDGFα receptor binding more efficiently than undigested full-length PDGF-C. These experiments showed that full-length PDGF-C is a potential growth factor that cannot interact with the PDGF α receptor, but is restricted to proteolysis, which releases the C-terminal PDGF/VEGF homology domain, which is important for A ligand/agonist for the active PDGF alpha receptor is required.

Embodiment 10: clone and express human PDGF-C CUB domain

There is a cDNA fragment of 430 bases in the human PDGF-C gene encoding the CUB domain (amino acid residues 23-159 in the full-length PDGF-C), which can be obtained using Deep VentDNA polymerase (Biolabs) under standard conditions and steps Carry out PCR amplification under, this coding sequence 5' end primer uses 5'cgggatcccgaatccaacctgagtag3' (SEQ ID NO:38). This primer contains a BamHI site (underlined) for cloning. The coding sequence 3' end primer uses 5' ccggaattcctaatggtgatggtgatgatgtttgtcatcgtcgtcgacaatgttgtagtg3' (SEQ ID NO: 39). This primer contains an EcoRI site (underlined) and the C-terminus of this sequence encodes a 6x histidine tag joined by an enterokinase site. The amplified PCR fragment was then cloned into the pACgp67A transformation vector. The correct sequence of the CUB-pACgp67A expression cassette was verified by an automatic nucleic acid sequencer. The expression vector was then co-transformed with Baculogold linearized baculovirus DNA into insect cells Sf9 according to the prepared protocol (Pharmingen). Recombinant baculoviruses were amplified several times prior to large-scale protein production, and protein purification steps were performed according to the manual (Pharmingen).

Sf9 cells (multiplicity of infection: ~7) cultured in serum-free medium were infected with recombinant baculovirus. After 72 hours of infection, the medium containing the recombinant protein was collected and incubated with Ni-NTA-agarose beads (Qiagen). The beads are collected into the column, using high-strength elution conditions: 50mM sodium phosphate buffer, pH8, containing 300mM NaCl (elution buffer), the bound protein can be increased by increasing the concentration of imidazole in the elution buffer (from 100mM to 500mM) eluted. The eluted proteins were analyzed by SDS-PAGE on a polyacrylamide gel under reducing and non-reducing conditions.

Figure 25 shows the results after staining the gel with Coomassie brilliant blue. The human PDGF-CUB domain is a disulfide-linked homodimer with a molecular weight of about 55kD in the non-reduced state, while two monomers of about 25 and 30kD exist in the reduced state, respectively. The reason for this difference may be the different N-linked glucoamidation reactions at the two glucoamidation sites of amino acids 25 and 55 in the CUB domain. The left side of the figure is the lane with standard protein added.

Example 11: Localization of PDGF-C Transcripts in Developing Mouse Embryos

In order to learn more about the biological function of PDGF-C, the expression of PDGF-C was observed by non-radioactive tissue section in situ hybridization in the head (Fig. 26A-26S) and urogenital tract (Fig. Express status. For non-radioactive tissue section in situ hybridization procedures and PDGF-A and PDGFR-α probes, please refer to Bostrom et al., Cell, 1996 85 863-873. The PDGF-C probe was derived from mouse PDGF-C cDNA. The hybridization model shown in Figures 26A-26V is embryo E16.5, but similar models can also be seen at E14.5, E-15.5, E-17.5. The detection probe served as a control, which did not show the same pattern in the hybridized segment.

Figure 26A shows the tooth base (t) passing through the frontal section of the oral cavity. Arrows indicate sites of PDGF-C expression in the oral ectoderm. Tongue (to) is also indicated. Figures 26B-26D show expression of PDGF-C in epithelial cells of developing dental tubes. As with the developing palatal ectoderm (Fig. 26C right arrow), individual cells showed strong labeling in this region (Fig. 26D arrow). Figure 26E shows the frontal segment through the eye, where PDGF-C expression was found at the hair follicle (double arrow) and the developing eyelid. The retina (r) is also indicated. In Figures 26F and 26G, expression of PDGF-C was found in the outer epithelium of the developing hair follicle root sheath. In Figure 26H, expression of PDGF-C in the developing eyelid is shown. Individual PDGF-C positive cells with a strong marker signal appeared at the beginning of development. The lens (1) is also indicated. Figure 26I, PDGF-C expression in developing lacrimal glands is indicated by arrows. Figure 26J, PDGF-C expression in the developing outer ear. Expression is seen in the external auditory canal (left arrow) and in the early pinna at epidermal clefts (e). Figures 26K and 26L show the expression of PDGF-C in the cochlea. Expression is seen in semicircular canals (26K arrows). Epithelial cells adjacent to developing hair cells had a polarized distribution of PDGF-C mRNA (Fig. 26L arrows). Figures 26M and 26N show the expression of PDGF-C in the oral cavity. Horizontal sections demonstrate the expression of PDGF-C in the buccal epithelium (arrow in FIG. 26M ) and in the cleft forming between the lower lip and buccal and gingival epithelium (arrow in FIG. 26N ). Tooth primordia (t) and tongue (to) are shown simultaneously. Figures 26O and 26P show expression of PDGF-C in developing nares in horizontal sections. PDGF-C expression was strongest prior to epithelial stratification and normal duct formation (Fig. 26O and 26P arrows). The developing nostrils are also noted (n). Figures 26Q-26S show expression of PDGF-C in developing salivary gland ducts. Figure 26Q is the sublingual gland. Figures 26R and 26S show maxillary glands, salivary glands (sg) and salivary ducts (sd). Figures 26T-26V show the expression of PDGF-C in the urogenital tract. Figure 26T shows the expression of PDGF-C in the metanephric mesoderm of the developing kidney. Figure 26U shows the expression of PDGF-C around the epithelium of the urethra (ua) and developing penis. Figure 26V shows PDGF-C expression in the developing ureter (u).

Example 12: Expression of PDGF-C, PDGF-A and PDGFR-α in the developing kidney

One of the strongest sites of PDGF-C expression is in the developing kidney, so expression of PDGF-C, PDGF-A and PDGFR-α is also seen in the developing kidney. Figures 27A-27F show the results of non-radioactive in situ hybridization, (blue stained with DIC optical detection on an unstained background) showing their mRNA expression in kidneys at E16.5, PDGF-C (Fig. 27A and 27B), PDGF-A (FIGS. 27C and 27D) and PDGFR-α (FIGS. 27E and 27F). The white dashed lines in Figures 27B, 27D and 27F delineate the outline of the cortical boundaries. The dashed line in Figures 27A, 27C and 27E represents 250 μM, and in Figures 27B, 27D and 27F represents 50 μM.

The expression of PDGF-C is found in the metanephric stroma (Fig. 27Amm), and its expression level is enhanced in the fused stroma (arrow in Fig. 27B). The fused stroma can undergo epithelial transformation and further undergo tubular development. It is located at the two ends of the ureteric bud (ub). side. PDGF-C maintains a low level of expression in early nephron epithelial aggregation (arrow B), and does not express in mature glomeruli (gl) to tube structures.

PDGF-A expression was not found in these early aggregates, but was strongly expressed at later stages of tube development (Figures 24C and 24D). PDGF-A is expressed in epithelial aggregates in early nephrons (arrows in Figure 27D), but once nephrons develop further, PDGF-A expression becomes restricted to the developing microtubules (arrows in Figure 27D). The strongest expression was seen in the microtubules of the developing bone marrow (arrows in Figure 27C). PDGF-A was not seen in branch ureters (u) and ureteral buds (ub).

Thus, PDGF-C and PDGF-A expression patterns differ spatially and temporally in developing nephrons. PDGF-C is expressed at the earliest stages of nephron development (interstitial aggregates), whereas PDGF-A is expressed at the latest stages (ureteric studs).

PDGFR-[alpha] is expressed throughout the interstitium of the developing kidney (Figures 27E and 27F), and thus can be a target of PDGF-C and PDGF-A. PDGF-B is also expressed in the developing kidney, but only in vascular endothelial cells. The expression of PDGFR-β occurs in the peripheral vascular interstitium, and the activation of PDGFR-β by PDGF-B is a necessary condition for the reorganization of glomerular cells in glomeruli.

These results demonstrate that PDGF-C expression occurs near sites of PDGFR-α expression, but not near those of PDGF-A or PDGF-B. This shows that PDGF-C may act through PDGFR-α in vivo without the help of PDGF-A or PDGF-B.

Since the unique expression pattern of PDGF-C in the developing kidney suggests that it functions as a PDGFR-α agonist and is independent of PDGF-A and PDGF-B, we performed a histological analysis of embryonic day 16.5 kidneys. Comparison: Comparing the kidneys of PDGFR-α knockout mice (Figure 28B and 28F) and wild type mice (Figure 28A and 28C), and PDGF-A knockout mice (Figure 28D) and PDGF-A/PDGF-B Kidneys of all knockout mice (Fig. 28E). The dashes represent 250 mm in Figures 28A and 28B and 50 μm in Figures 28C-F.

Using heterozygous mutants of PDGF-A, PDGF-B and PDGFR-α (Bostron et al., Cell, 1996 85 863-873; Leveen et al., Genes Dev., 1994 8 1875-1887; Soriano et al., Development, 1997 124 2691-70) construct C57B16/129sv hybrid line, carry out cross again to produce homozygous mutant embryo. PDGF-A/PDGF-B heterozygous mutants were crossed to generate PDGF-A/PDGF-B knockout embryos. Due to the high lethality of PDGF-A-/- before E10 (Bostron et al., Cell, 1996 85 863-873), the proportion of E16.5 embryos with double gene knockout is less than 1/40 in hybrid lines . Except for one PDGF-A/PDGF-B double-knockout embryo, at least two homozygous renal phenotype embryos were verified for each genotype.

Interestingly, interstitium was absent in PDGFR-α-/- renal cortex (arrows in FIG. 28A and asterisk in FIG. 28F ), whereas interstitium was present in all other genotypes (asterisk in FIG. 28C-E ). Branching ureters (u) and metanephric mesenchyme (mm) and its epithelial derivatives appeared normal in all mutants. Abnormal glomeruli in PDGF-A/PDGF-B double knockout embryos reflect the failure of glomerular cells to recombine in glomeruli due to the absence of PDGF-B.

These results demonstrate a renal phenotype in PDGFR-α knockout that is not present in PDGF-A or PDGF-A/PDGF-B knockout individuals, thus reflecting an underlying loss of PDGF-C-derived signaling. The phenotype consisted of interstitial mesenchymal loss of markers in the developing renal cortex. In PDGFR-α-/- kidneys, the absence of these cells was due to the fact that normal PDGFR-α-positive cells were adjacent to PDGF-C expression sites.

Bioanalytical methods for testing PDGF-C function

The experiments were performed to assess whether PDGF-C has similar effects as PDGF-A, PDGF-B, VEGF, VEGF-B, VEGF-C or VEGF-D in affecting connective tissue cells, fibroblasts, myofibroblasts and Glial cell growth and motility, alter endothelial cell function, regulate angiogenesis, and promote wound healing. Depending on the study of the receptor binding profile, further experiments can be performed. I. Mitogenicity of PDGF-C on Endothelial Cells

In order to detect the mitogenic ability of PDGF-C on endothelial cells, PDGF-C polypeptide was introduced into the cell culture medium containing 5% serum, and the medium containing 10% serum was used to propagate bovine aortic endothelial cells (BAEs). BAEs were first seeded in 24-well culture plates, and the cell density in each well reached 10,000 one day before PDGF-C was added. Three days after the addition of the peptides, trypsin was used to separate and count them. Purified VEGF served as a positive control in this experiment.

II. Endothelial cell function assay

A) Endothelial cell proliferation

Endothelial cell growth assays employ well known techniques such as Ferrara and Henzel, Nature, 1989 380 439-443, Gospodarowics et al., Proc.Natl.Acad.Sci.USA, 1989 86 7311-7315, and Claffey et al., Biochem . Biophys. Acts, 1995 1246 1-9.

B) Cell attachment assay

The effect of PDGF-C on the adhesion of polymorphonuclear granulocytes to endothelial cells was detected.

C) Chemotaxis

The effect of PDGF-C on chemotaxis was detected by standard Boyden chamber chemotaxis assay.

D) Plasminogen activation assay

Endothelial cells were used to examine the effect of PDGF-C on the production of plasminogen activity inhibitors using the method of Pepper et al., Biochem. Biophy. Res. Commun., 1991 181902-906.

E) Endothelial cell migration assay

The ability of PDGF-C to stimulate endothelial cell migration and tube formation is described in the following experiment: Montesano et al., Proc. Natl. Acad. Sci. USA, 1986 83 7297-7301. Alternatively, three-dimensional collagen gel experiments are described in Joukov et al., EMBO J., 1996 15 290-298, or gelled membranes can be used in modified Boyden chambers (Glaser et al., Nature, 1980 288 483 -484).

III. Angiogenesis Experiment

The ability of PDGF-C to induce an angiogenic response on the chorioallantoic membrane is described experimentally in: Leung et al., Science, 1989 246 1306-1309. Or use the rat cornea experiment Rastinejad et al., Cell, 1989 56 345-355; this is an acceptable method for in vivo angiogenesis experiments, and the results can be easily transferred to other in vivo systems.

IV. Wound Healing

The ability of PDGF-C to stimulate wound healing can be tested in most clinically relevant visible models as described in Schilling et al., Surgery, 1995 46 702-710 or using Hunt et al., Surgery, 1967 114 302-307.

V. Erythropoiesis-stimulating system

Various in vivo and in vitro experiments were performed using specialized cell populations of the erythropoietin system. In the experiment of in vitro stem cell analysis in mice, the cells were purified using a fluorescent cell sorter, and the results were satisfactory.

A) Re-implantation of stem cells

These cells can re-implant into the bone marrow of lethally irradiated mice, and have several phenotypes of Lin ^- , Rh ^hl , Ly-6A/E ⁺ , and c-kit ⁺ . In these cells, PDGF-C was added alone, or along with other factors, and the proliferation of the cells was measured by ³ H-thymidine incorporation.

B) Advanced Stem Cells

These cells have relatively little bone marrow re-engraftment capacity, but are able to produce D13 CFU-S. These cells also have Lin ^- , Rh ^hl , Ly-6A/E ⁺ , c-kit ⁺ several phenotypes. These cells were spiked with PDGF-C and then injected into lethally irradiated recipients, and spleen colonies were counted on D13.

C) Progenitor-Enriched cells

These cells respond to the stimulation of a single growth factor in vitro, and have several phenotypes of Lin ^- , Rh ^hl , Ly-6A/E ⁺ , and c-kit ⁺ . The purpose of this experiment is to verify whether PDGF-C can directly act on the progenitor cells of erythropoietin. On agar medium, PDGF-C was incubated with these cells, and surviving colonies were counted after 7-14 days.

VI. Atherosclerosis

Smooth muscle cells play an important role in development or atherosclerosis, requiring their phenotype to change from a contractile to a synthetic state. Macrophages, endothelial cells, T lymphocytes, and platelets all play a role in the atherosclerotic process by affecting the growth and phenotypic changes of smooth muscle cells. Using a modified Rose chamber, in which different cell types are seeded on opposite sides, an in vitro assay can dynamically measure the proliferation rate and phenotype adaptation of smooth muscle cells in a multicellular environment, whereby it was used to examine the effect of PDGF-C on smooth muscle cells Impact.

VII. Transfer

Use Lewis' lung cancer model to test the ability of PDGF-C to inhibit metastasis, for example, using the method of Cao et al., J.Exp.Med., 1995 182 2069-2077.

VIII. Migration of Smooth Muscle Cells

The effect of PDGF-C on the migration of smooth muscle cells and other cell types can be verified by the method described by Koyama et al., J. Biol. Chem., 1992 267 22806-22812.

IX Chemotaxis

The effect of PDGF-C on fibroblasts, monocytes, granulocytes and other cells can be verified by the method described by Siegbahn et al., J.Clin.Invest., 1990 85 916-920.

X. PDGF-C in other cell types

The effect of PDGF-C on the proliferation, differentiation and function of other cell types, such as hepatocytes, cardiomyocytes and other cells, endocrine cells and osteoblasts, etc., can be verified by known technical methods, such as by uptake of ³ H in in vitro culture medium - Thymidine. PDGF-C expression in these and other tissues can be measured by Northern dot or in situ hybridization techniques.

XI. Construction of PDGF-C variants and analogs

PDGF-C is a member of the PDGF growth factor family, and it maintains a high degree of homology with other members of the PDGF-C family. PDGF-C contains eight conserved cysteine residues, which are characteristic of this family of growth factors. These conserved cysteine residues form intrachain disulfide bonds, resulting in cysteine knot structures, and protein dimers formed by internal chain disulfide bonds are also characteristic of members of the PDGF growth factor family. Interaction of PDGF-C and protein tyrosine kinase growth factor receptor.

Little is known about the structure of this protein, the active site necessary for binding to the receptor, and its associated activity. According to the active site and important amino acid residues in many known members of the PDGF family growth factor, the activity of PDGF-C is designed Mutants could greatly advance our understanding in this area.

There are many published articles that clarify the relationship between the structure and activity of PDGF growth factor family members, among which the contents of PDGF are: Oestman et al., J.Biol.Chem., 1991266 10073-10077; Andersson et al., J.Biol .Chem., 1992 267 11260-11266; Oefner et al., EMBO J., 1992 11 3921-3926; Flemming et al., Molecular and Cell Biol., 1993 13 4066-4076 and Anderson et al., GrowthFactors, 1995 12 159-164; About VEGF include: Kim et al., GrowthFactors, 1992 7 53-64; Potgens et al., J.Biol.Chem., 1994 269 32879-328885 and Claffey et al., Biochem.Biophys. Acta, 1995 1246 1-9. These papers show that members of the PDGF growth factor family exhibit a characteristic knot-like fold structure and dimerization due to eight conserved cysteine residues, so that three triads are formed at each end of the dimer molecule. An exposed circular region where the active receptor binding site is predicted to be located.

Based on the above information, existing biotechnology can be used to artificially design PDGF-C mutants, that is, to retain 8 cysteine residues to form a knot-like folding arrangement and dimer, which may highly preserve PDGF-C activity. At the same time, possible receptor sequences in loop 1, loop 2, and loop 3 regions of the protein structure are detected only by retaining or replacing conservative amino acid residues.

Mutations to form specific target sites on protein structures are standard techniques in the research methods of protein chemists (Kunkel et al., Methods in Enzymol., 1987 154 367-382). Examples of direct mutagenesis of these sites on VEGF can be found in Potgens et al., J.Biol.Chem., 1994 269 32879-32885 and Claffey et al., Biochem.Biophys.Acta, 1995 1246 1-9. In fact, direct site mutation is extremely common, and commercial kits can easily implement these steps (for example, Promega 1994-1995 catalog 142-145 pages).

For PDGF-C mutants, to test their effects on the growth and motility of connective tissue cells, fibroblasts, myofibroblasts and glial cells, endothelial cell proliferation activity, angiogenesis activity and wound healing, can be used Established screening procedures are rapidly validated. For example, Claffey et al. have described (Biochem. Biophys. Acta, 1995 1246 1-9) similar procedures for endothelial cell mitosis experiments, which can be used directly. Similarly, the effects of PDGF-C on the proliferation of other cell types, cell differentiation, and human metabolism can be tested using currently available techniques.

The above description of the present invention and the enumerated specific examples are only for the purpose of explaining and illustrating, not limiting, that is, it does not mean that the present invention only includes the above content. Professionals in the field can make certain changes and modifications according to their own needs on the basis of the basic inventive concept of the present invention, so the expansions derived from the present invention involved in the claims also belong to the category of the present invention .

Sequence Listing <110> Ulf Eriksson

Karin Orser

Li Xuri

Annika Ponten

Marco Yutla

Cary Alitalo

Arne Osterman

Carl-Henrik Helding

Christ Bettsholz <120> Platelet-derived growth factor C, its coding DNA and its application <130> Sequence listing <140>60/102, 461<141>1998-09-30<150>60/ 108, 109<151>1998-11-12<150>60/110, 749<151>1998-12-03<150>60/113, 002<151>1998-12-18<150>60/135, 426<151>1999-05-21<150>60/144, 022<151>1999-07-15<160>39<170>PatentIn Ver.2.0<210>1<211>16<212>PRT<213 >Homo sapiens<400>1Pro Xaa Cys Leu Leu Val Xaa Arg Cys Gly Gly Xaa Cys Xaa Cys Cys1               5                  10                  15<210>2<211>2108<212>DNA<213>Homo sapiens<400>2ccccgccgtg agtgagctct caccccagtc agccaaatga gcctcttcgg gcttctcctg 60gtgacatctg ccctggccgg ccagagacga gggactcagg cggaatccaa cctgagtagt 120aaattccagt tttccagcaa caaggaacag aacggagtac aagatcctca gcatgagaga 180attattactg tgtctactaa tggaagtatt cacagcccaa ggtttcctca tacttatcca 240agaaatacgg tcttggtatg gagattagta gcagtagagg aaaatgtatg gatacaactt 300acgtttgatg aaagatttgg gcttgaagac ccagaagatg acatatgcaa gtatgatttt 360gtagaagttg aggaacccag tgatggaact atattagggc gctggtgtgg ttctggtact 420gtaccaggaa aacagatttc taaaggaaat caaattagga taagatttgt atctgatgaa 480tattttcctt ctgaaccagg gttctgcatc cactacaaca ttgtcatgcc acaattcaca 540gaagctgtga gtccttcagt gctaccccct tcagctttgc cactggacct gcttaataat 600gctataactg cctttagtac cttggaagac cttattcgat atcttgaacc agagagatgg 660cagttggact tagaagatct atataggcca acttggcaac ttcttggcaa ggcttttgtt 720tttggaagaa aatccagagt ggtggatctg aaccttctaa cagaggaggt aagattatac 780agctgcacac ctcgtaactt ctcagtgtcc ataagggaag aactaaagag aaccgatacc 840attttctggc caggttgtct cctggttaaa cgctgtggtg ggaactgtgc ctgttgtctc 900cacaattgca atgaatgtca atgtgtccca agcaaagtta ctaaaaaata ccacgaggtc 960cttcagttga gaccaaagac cggtgtcagg ggattgcaca aatcactcac cgacgtggcc 1020ctggagcacc atgaggagtg tgactgtgtg tgcagaggga gcacaggagg atagccgcat 1080caccaccagc agctcttgcc cagagctgtg cagtgcagtg gctgattcta ttagagaacg 1140tatgcgttat ctccatcctt aatctcagtt gtttgcttca aggacctttc atcttcagga 1200tttacagtgc attctgaaag aggagacatc aaacagaatt aggagttgtg caacagctct 1260tttgagagga ggcctaaagg acaggagaaa aggtcttcaa tcgtggaaag aaaattaaat 1320gttgtattaa atagatcacc agctagtttc agagttacca tgtacgtatt ccactagctg 1380ggttctgtat ttcagttctt tcgatacggc ttagggtaat gtcagtacag gaaaaaaact 1440gtgcaagtga gcacctgatt ccgttgcctt gcttaactct aaagctccat gtcctgggcc 1500taaaatcgta taaaatctgg attttttttt ttttttttgc tcatattcac atatgtaaac 1560cagaacattc tatgtactac aaacctggtt tttaaaaagg aactatgttg ctatgaatta 1620aacttgtgtc rtgctgatag gacagactgg atttttcata tttcttatta aaatttctgc 1680catttagaag aagagaacta cattcatggt ttggaagaga taaacctgaa aagaagagtg 1740gccttatctt cactttatcg ataagtcagt ttatttgttt cattgtgtac atttttatat 1800tctccttttg acattataac tgttggcttt tctaatcttg ttaaatatat ctatttttac 1860caaaggtatt taatattctt ttttatgaca acttagatca actattttta gcttggtaaa 1920tttttctaaa cacaattgtt atagccagag gaacaaagat ggatataaaa atattgttgc 1980cctggacaaa aatacatgta tntccatccc ggaatggtgc3tagagttgga ttaaacctgc 2040attttaaaaa acctgaattg ggaanggaan ttggtaaggt tggccaaanc ttttttgaaa 2100ataattaa                                                          2108<210>3<211>345<212>PRT<213>Homo sapiens<400>3Met Ser Leu Phe Gly Leu Leu Leu Val Thr Ser Ala Leu Ala Gly Gln1               5                  10                  15Arg Arg Gly Thr Gln Ala Glu Ser Asn Leu Ser Set Lys Phe Gln Phe

20 25 30Ser Ser Asn Lys Glu Gln Asn Gly Val Gln Asp Pro Gln His Glu Arg

35 40 45Ile Ile Thr Val Ser Thr Asn Gly Ser Ile His Ser Pro Arg Phe Pro

50 55 60His Thr Tyr Pro ARG Asn Thr Val Leu Val Trp ARG Leu Val Ala Val65 70 75 80GLU Glu Asn Val Trn Leu ThR PHE ARG PHLY Leu Gly Leu

85 90 95Glu Asp Pro Glu Asp Asp Ile Cys Lys Tyr Asp Phe Val Glu Val Glu

100 105 110Glu Pro Ser Asp Gly Thr Ile Leu Gly Arg Trp Cys Gly Ser Gly Thr

115 120 125Val Pro Gly Lys Gln Ile Ser Lys Gly Asn Gln Ile Arg Ile Arg Phe

135 135 140VAL Ser ASP GLU TYR PHE PRO SET GLU Pro Gly PHE CYS ILE HIS Tyr145 150 155 160s Ile Val Met Pro Gln PHR Glu Ala Val Leu Ser Val Leu

                                                      

180 185 190Phe Ser Thr Leu Glu Asp Leu Ile Arg Tyr Leu Glu Pro Glu Arg Trp

195 200 205Gln Leu Asp Leu Glu Asp Leu Tyr Arg Pro Thr Trp Gln Leu Leu Gly

210 215 220LYS ALA PHE VAL PHE GLY ARG LYS Serg Val Val Val ASN Leu225 230 235 240Leu Thr Glu Val ARG Leu Tyr PRO ARG Asn Phe Serg Asn Phe Ser

245 250 255Val Ser Ile Arg Glu Glu Leu Lys Arg Thr Asp Thr Ile Phe Trp Pro

260 265 270Gly Cys Leu Leu Val Lys Arg Cys Gly Gly Asn Cys Ala Cys Cys Leu

275 280 285His Asn Cys Ash Glu Cys Gln Cys Val Pro Ser Lys Val Thr Lys Lys

290 295 300tyr His Glu Val Leu Gln Leu ARG Pro Lys Thr Gly Val ARG GLY Leu305 310 320HIS LEU THR ASP Val Ala Leu His GLU GLU CYS ASP

325 330 335Cys Val Cys Arg Gly Ser Thr Gly Gly

340                 345<210>4<211>1536<212>DNA<213>Homo sapiens<400>4cgggtaaatt ccagttttcc agcaacaagg aacagaacgg agtacaagat cctcagcatg 60agagaattat tactgtgtct actaatggaa gtattcacag cccaaggttt cctcatactt 120atccaagaaa tacggtcttg gtatggagat tagtagcagt agaggaaaat gtatggatac 180aacttacgtt tgatgaaaga tttgggcttg aagacccaga agatgacata tgcaagtatg 240attttgtaga agttgaggaa cccagtgatg gaactatatt agggcgctgg tgtggttctg 300gtactgtacc aggaaaacag atttctaaag gaaatcaaat taggataaga tttgtatctg 360atgaatattt tccttctgaa ccagggttct gcatccacta caacattgtc atgccacaat 420tcacagaagc tgtgagtcct tcagtgctac ccccttcagc tttgccactg gacctgctta 480ataatgctat aactgccttt agtaccttgg aagaccttat tcgatatctt gaaccagaga 540gatggcagtt ggacttagaa gatctatata ggccaacttg gcaacttctt ggcaaggctt 600ttgtttttgg aagaaaatcc agagtggtgg atctgaacct tctaacagag gaggtaagat 660tatacagctg cacacctcgt aacttctcag tgtccataag ggaagaacta aagagaaccg 720ataccatttt ctggccaggt tgtctcctgg ttaaacgctg tggtgggaac tgtgcctgtt 780gtctccacaa ttgcaatgaa tgtcaatgtg tcccaagcaa agttactaaa aaataccacg 840aggtccttca gttgagacca aasaccggtg tcaggggatt gcacaaatca ctcaccgacg 900tggccctgga gcaccatgag gagtgtgact gtgtgtgcag agggagcaca ggaggatagc 960cgcatcacca ccagcagctc ttgcccagag ctgtgcagtg cagtggctga ttctattaga 1020gaacgtatgc gttatctcca tccttaatct cagttgtttg cttcaaggac ctttcatctt 1080caggatttac agtgcattct gaaagaggag acatcaaaca gaattaggag ttgtgcaaca 1140gctcttttga gaggaggcct aaaggacagg agaaaaggtc ttcaatcgtg gaaagaaaat 1200taaatgttgt attaaataga tcaccagcta gtttcagagt taccatgtac gtattccact 1260agctgggttc tgtatttcag ttctttcgat acggcttagg gtaatgtcag tacaggaaaa 1320aaactgtgca agtgagcacc tgattccgtt gccttgctta actctaaagc tccatgtcct 1380gggcctaaaa tcgtataaaa tctggatttt tttttttttt tttgctcata ttcacatatg 1440taaaccagaa cattctatgt actacaaacc tggtttttaa aaaggaacta tgttgctatg 1500aattaaactt gtgtcatgct gataggacag actgga                           1536<210>5<211>318<212>PRT<213>Homo sapiens<400>5Gly Lys Phe Gln Phe Ser Ser Asn Lys Glu Gln Asn Gly Val Gln Asp1 5 As 10 I is er le 15Pro Gln His Glu Arg Ile H Ile ThrVal Ser

20 25 25 30Ser Pro Arg Phe Pro His Thr Tyr Pro Arg Asn Thr Val Leu Val Trp

35 40 45Arg Leu Val Ala Val Glu Asn Val Trp Ile Gln Leu Thr Phe Asp

50 55 60Glu ARG PHLY Leu Glu Glu ASP Pro Glu ASP ASP ILE CYS LYS TYR ASP65 70 75 80PHE Val Glu Glu Pro Serle Leu GLY ARG TRP

85 90 95Cys Gly Ser Gly Thr Val Pro Gly Lys Gln Ile Ser Lys Gly Asn Gln

100 105 110Ile Arg Ile Arg Phe Val Ser Asp Glu Tyr Phe Pro Ser Glu Pro Gly

115 120 125Phe Cys Ile His Tyr Asn Ile Val Met Pro Gln Phe Thr Glu Ala Val

130 135 140ser Pro Ser Val Leu Pro Pro Seru Prou Leu Leu Leu Leu asn145 155 160ASH ALA Ile THR Ala Phe Seru ASP Leu Ile ARG Tyr Leu

165 170 175Glu Pro Glu Arg Trp Gln Leu Asp Leu Glu Asp Leu Tyr Arg Pro Thr

180 185 190Trp Gln Leu Leu Gly Lys Ala Phe Val Phe Gly Arg Lys Ser Arg Val

195 200 205Val Asp Leu Asn Leu Leu Thr Glu Glu Val Arg Leu Tyr Ser Cys Thr

210 215 220Pro ARG ASN PHE Ser Val Ile ARG Glu Leu Leu Lys ARG THR ASP2225 235 240thr Ile PHE TRP Pro GLY CYS Leu Val Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly ASN

245 250 255Cys Ala Cys Cys Leu His Asn Cys Asn Glu Cys Gln Cys Val Pro Ser

260 265 270Lys Val Thr Lys Lys Tyr His Glu Val Leu Gln Leu Arg Pro Lys Thr

275 280 285Gly Val Arg Gly Leu His Lys Ser Leu Thr Asp Val Ala Leu Glu His

290                 295                 300His Glu Glu Cys Asp Cys Val Cys Arg Gly Ser Thr Gly Gly305                 310                 315<210>6<211>1474<212>DNA<213>Murinae gen.sp.<400>6cacctggaga cacagaagag ggctctagga aaaattttgg atggggatta tgtggaaact 60accctgcgat tctctgctgc cagagccggc caggcgcttc caccgcagcg cagcctttcc 120ccgggctggg ctgagccttg gagtcgtcgc ttccccagtg cccgccgcga gtgagccctc 180gccccagtca gccaaatgct cctcctcggc ctcctcctgc tgacatctgc cctggccggc 240caaagaacgg ggactcgggc tgagtccaac ctgagcagca agttgcagct ctccagcgac 300aaggaacaga acggagtgca agatccccgg catgagagag ttgtcactat atctggtaat 360gggagcatcc acagcccgaa gtttcctcat acgtacccaa gaaatatggt gctggtgtgg 420agattagttg cagtagatga aaatgtgcgg atccagctga catttgatga gagatttggg 480ctggaagatc cagaagacga tatatgcaag tatgattttg tagaagttga ggagcccagt 540gatggaagtg ttttaggacg ctggtgtggt tctgggactg tgccaggaaa gcagacttct 600aaaggaaatc atatcaggat aagatttgta tctgatgagt attttccatc tgaacccgga 660ttctgcatcc actacagtat tatcatgcca caagtcacag aaaccacgag tccttcggtg 720ttgccccgtt catctttgtc attggacctg ctcaacaatg ctgtgactgc cttcagtacc 780ttggaagagc tgattcggta cctagagcca gatcgatggc aggtggactt ggacagcctc 840tacaagccaa catggcagct tttgggcaag gctttcctgt atgggaaaaa aagcaaagtg 900gtgaatctga atctcctcaa ggaagaggta aaactctaca gctgcacacc ccggaacttc 960tcagtgtcca tacgggaaga gctaaagagg acagatacca tattctggcc aggttgtctc 1020ctggtcaagc gctgtggagg aaattgtgcc tgttgtctcc ataattgcaa tgaatgtcag 1080tgtgtcccac gtaaagttac aaaaaagtac catgaggtcc ttcagttgag accaaaaact 1140ggagtcaagg gattgcataa gtcactcact gatgtggctc tggaacacca cgaggaatgt 1200gactgtgtgt gtagaggaaa cgcaggaggg taactgcagc cttcgtagca gcacacgtga 1260gcactggcat tctgtgtacc cccacaagca accttcatcc ccaccagcgt tggccgcagg 1320gctctcagct gctgatgctg gctatggtaa agatcttact cgtctccaac caaattctca 1380gttgtttgct tcaatagcct tcccctgcag gacttcaagt gtcttctaaa agaccagagg 1440caccaanagg agtcaatcac aaagcactgc accg                             1474<210>7<211>345<212 > PRT <213> Murinae Gen.sp. <400> 7MET Leu Leu Leu Gly Leu Leu Leu Thr Seru Ala GLY GLN1 5 10 15ARG THR ARG Ala Glu Serite

20 25 25 30Ser Ser Asp Lys Glu Gln Asn Gly Val Gln Asp Pro Arg His Glu Arg

35 40 45Val Val Thr Ile Ser Gly Asn Gly Ser Ile His Ser Pro Lys Phe Pro

50 55 60his Thr Tyr Pro ARG Asn Met Val Leu Val Trp ARG Leu Val Ala Val65 70 80asp Glu Asn Val ARG Ile Gln Leu Thr PHE ARG PHE GLY Leu Gly Leu

85 90 95Glu Asp Pro Glu Asp Asp Ile Cys Lys Tyr Asp Phe Val Glu Val Glu

100 105 110Glu Pro Ser Asp Gly Ser Val Leu Gly Arg Trp Cys Gly Ser Gly Thr

115 120 125Val Pro Gly Lys Gln Thr Ser Lys Gly Asn His Ile Arg Ile Arg Phe

130 135 140VAL Ser ASP GLU TYR PRO PRO Ser Glu GLY PHE CYS Ile His Tyr145 150 160 160SER Ile Met Pro Gln Val THR THR THR Val Leu

165 170 175Pro Pro Ser Ser Leu Ser Leu Asp Leu Leu Asn Asn Ala Val Thr Ala

180 185 190Phe Ser Thr Leu Glu Glu Leu Ile Arg Tyr Leu Glu Pro Asp Arg Trp

195 200 205Gln Val Asp Leu Asp Ser Leu Tyr Lys Pro Thr Trp Gln Leu Leu Gly

210                 215                 220Lys Ala Phe Leu Tyr Gly Lys Lys Ser Lys Val Val Asn Leu Asn Leu225                 230                 235                 240Leu Lys Glu Glu Val Lys Leu Tyr Ser Cys Thr Pro Arg Asn Phe Ser

245 250 255Val Ser Ile Arg Glu Glu Leu Lys Arg Thr Asp Thr Ile Phe Trp Pro

260 265 270Gly Cys Leu Leu Val Lys Arg Cys Gly Gly Asn Cys Ala Cys Cys Leu

275 280 285His Asn Cys Asn Glu Cys Gln Cys Val Pro Arg Lys Val Thr Lys Lys

290 295 300tyr His Glu Val Leu Gln Leu ARG Pro Lys Thr Gly Val Lys Gly Leu305 315 320HIS LEU THR ASP Val AIA Leu His Glu GLU CYS ASP

325 330 335Cys Val Cys Arg Gly Asn Ala Gly Gly

340 345 <210> 8 <2111> 192 <212> PRT <213> Homo Sapiens <400> 8MET ASN PHE Leu Leu Serp Val His TRP Serp Seru Leu Leu1 5 10 15tyr Leu His Ala Lys Trp Sern Ala Ala Pro Met Ala Glu Gly

20 25 25 30Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln

35 40 45Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu

50 55 60tyr Pro ASP GLU Ile Glu Tyr Ile PHE LYS Pro Ser Cys Val Prou65 70 75 80MET ARG CYS GLY GYS CYS CYS Asn ASP GLU GLU CYS Val Val Val Val Val Val Valiws

85 90 95Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His

100 105 110Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys

115 120 125Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly

130 135 140pro Cys Serg ARG LYS HIS Leu PHE VAL GLN ASP Pro Gln145 155 160thr Cys Cys Cys Lys Lys ARG CYS LYS LYS ALA ARG

165 170 175Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg

180 185 190 <210> <211> 170 <212> PRT <213> Homo Sapiens <400> 9MET Pro Val Met ARG Leu PHE PRO CYS PHE Leu Leu Leu Leu Gly1 5 10 15le Ala Ala Val Pro Gln Gln Trp Ala Leu Ser Ala Gly

20 25 25 30Asn Gly Ser Ser Glu Val Glu Val Val Pro Phe Gln Glu Val Trp Gly

35 40 45Arg Ser Tyr Cys Arg Ala Leu Glu Arg Leu Val Asp Val Val Ser Glu

50 55 60tyr Pro Ser Glu Val Glu His MET PHE Ser Pro Ser Cys Val Seru65 75 80leu ARG CYS CYS GLS GLY ASP Leu His Cys Val Val Val Val Val Val Val Val Pro

85 90 95Val Glu Thr Ala Asn Val Thr Met Gln Leu Leu Lys Ile Arg Ser Gly

100 105 110Asp Arg Pro Ser Tyr Val Glu Leu Thr Phe Ser Gln His Val Arg Cys

115 120 125Glu Cys Arg Pro Leu Arg Glu Lys Met Lys Pro Glu Arg Arg Arg Pro

130 135 140lys Gly Lys ARG ARG GLU Asn Gln ARG Pro ThR ASP CYS145 150 160HIS Leu Cys Gly Ala Val Pro ARG ARG

165 170 <210> 10 <111> 188 <212> PRT <213> Homo Sapiens <400> 10MET Serg ARG Leu Leu Leu Ala Ala Ala Leu Leu Gln Leu1 5 10 15ALA GLN ALA PRO Val Ser Gln Pro Asp Ala Pro Gly His Gln

20 25 30Arg Lys Val Val Ser Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys Gln

35 40 45Pro Arg Glu Val Val Pro Leu Thr Val Glu Leu Met Gly Thr Val

50 55 60ALA LYS GLN Leu Val Pro Ser Cys Val THR Val Gln ARG CYS GLY GLY65 70 75 80CYS Pro ASP ASP GLU GLU GLS Val VR Gln His Gln Gln Gln Gln Gln Gln Gln Gln

85 90 95Val Arg Met Gln Ile Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly

100 105 110Glu Met Ser Leu Glu Glu His Ser Gln Cys Glu Cys Arg Pro Lys Lys

115 120 125Lys Asp Ser Ala Val Lys Pro Asp Ser Pro Arg Pro Leu Cys Pro Arg

130 135 140CYS Thr Gln His Gln ARG Pro ASP Pro ARG THR CYS ARG CYS ARG145 150 160 16CY ARG Serg Serg Cys Gln GLY Leu GLY Leu Leu Leu

165 170 175Asn Pro Asp Thr Cys Arg Cys Arg Lys Leu Arg Arg

180 185 <210> 11 <111> 133 <212> PRT <213> Homo Sapiens <400> 11MET LEU Leu Val Gly Gly Ileu Val Ala Val Cys Leu His Gln Tyr1 5 10 15leu asn THR LYS GLY Trp Ser Glu Val Leu Lys

20 25 25 30Gly Ser Glu Cys Lys Pro Arg Pro Ile Val Val Pro Val Ser Glu Thr

35 40 45His Pro Glu Leu Thr Ser Gln Arg Phe Asn Pro Pro Cys Val Thr Leu

50 55 60MET ARG CYS GLY GLY CYS CYS ASN ASP GLU Seru Glu Cys Val Val PRO65 70 75 80thr Glu Val Seru Leu Leu Gly Ala Ser Gly Ser

85 90 95Gly Ser Asn Gly Met Gln Arg Leu Ser Phe Val Glu His Lys Lys Cys

100 105 110Asp Cys Arg Pro Arg Phe Thr Thr Thr Pro Pro Thr Thr Thr Arg Pro

115 120 125Pro Arg Arg Arg Arg

130<210>12<211>419<212>PRT<213>Homo sapiens<400>12Met His Leu Leu Gly Phe Phe Ser Val Ala Cys Ser Leu Leu Ala Ala1               5                  10                  15Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala Ala Ala Ala Ala Phe

20 25 25 30Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro Asp Ala Gly Glu Ala

35 40 45Thr Ala Tyr Ala Ser Lys Lys Asp Leu Glu Glu Gln Leu Arg Ser Val Ser

50 55 60Ser Val ASP GLU Leu Met ThR Val Leu Tyr Pro Glu Tyr Trp Lys Met65 70 75 80tyr LYS GLN Leu ARG LYS GLY GLN His ARG Glu Gln Glu Gln

85 90 95Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala

100 105 110His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys

115 120 125Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe

130 135 140Gly Val Ala THR Asn Thr Phe PHE PRO Pro Cys Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Val Tyr145 155 160ALG GLS GLY GLY CYS ASN SER GLN CYS MET Asn Thr.

165 170 175Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu

180 185 190Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser

195 200 205 Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile

210 215 220ile ARG ARG Sero Pro Ala THR Leu Pro Gln Cys Gln Ala Ala ALA ASN225 235 THR CYS Pro Thr ASN His Ile Cys Ile Cys Ile Cys ARG Cys

245 250 255Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser

260 265 270Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu Asp Glu

275 280 285Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro Ala Ser Cys

290 295 300Gly Pro His LYS GLU Leu ASP ARG Asn Ser Cys GLN CYS Val Cys Lys305 315 320asn LYS Leu Pro Sergs GLY ARG GLU PHE ASP GLU GLU

325 330 335Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro

340 345 350Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys

355 360 365Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys Tyr

370 375 380ARG ARG Pro CYS THR ARG GLN LYS ALA CYS GLU PRO GLY PHE Ser385 395 400tyr Serg Cys ARG CYS Val Pro Serg Pro LYS ARG Pro

405 410 415GLN MET Ser <210> 13 <211> 358 <212> PRT <213> Homo Sapiens <400> 13MET TYR GLY GLU TRP GLY MET GLY Asn Ile Leu Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Met Mal1 15thr Leu Val Gln Gln Gln Gln Ser Glu His Gly Pro Val Lys Asp Phe

20 25 30Ser Phe Glu Arg Ser Ser Arg Ser Met Leu Glu Arg Ser Glu Gln Gln

35 40 45Ile Arg Ala Ala Ser Ser Leu Glu Glu Leu Leu Gln Ile Ala His Ser

50 55 60GLU ASP TRP LYS Leu TRP ARG CYS ARG Leu Lys Leu Ly Leu Ala65 70 75 80r Met ARA SER HIS ARG PHR Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

85 90 95Thr Phe Tyr Asp Thr Glu Thr Leu Lys Val Ile Asp Glu Glu Trp Gln

100 105 110Arg Thr Gln Cys Ser Pro Arg Glu Thr Cys Val Glu Val Ala Ser Glu

115 120 125Leu Gly Lys Thr Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Asn Val

130 135 140phe ARG CYS GLY GLY GLY CYS CYS ASN GLU GLU GLY VAL MET CYS MET ASN145 150 155 160thr Serle Serle Serlas Gln Leu Phe Glu Ile Ser Val Val Val Val Val Val Val Val Pro

165 170 175 Leu Thr Ser Val Pro Glu Leu Val Pro Val Lys Ile Ala Asn His Thr

180 185 190Gly Cys Lys Cys Leu Pro Thr Gly Pro Arg His Pro Tyr Ser Ile Ile

195 200 205Arg Arg Ser Ile Gln Thr Pro Glu Glu Asp Glu Cys Pro His Ser Lys

210 215 220LYS Leu Cys Pro Ile Asp Met Leu TRP ASN ThR LYS LYS LYS CYS 225 235 240VAL Leu Gln Asp Glu Leu Leu GLY THR GLU Aser Tyr Tyr Tyr Tyr Tyr Tyr Tyr

245 250 255Leu Gln Glu Pro Thr Leu Cys Gly Pro His Met Thr Phe Asp Glu Asp

260 265 270Arg Cys Glu Cys Val Cys Lys Ala Pro Cys Pro Gly Asp Leu Ile Gln

275 280 285His Pro Glu Asn Cys Ser Cys Phe Glu Cys Lys Glu Ser Leu Glu Ser

290 295 300CYS CYS GLN LYS HIS LYS ILE PHE HIS PRO ASP THR CYS SES GLU30 310 320asp ARG CYS Pro PHR ARG THR CYS ARG LYS Pro Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

325 330 335Cys Gly Lys His Trp Arg Phe Pro Lys Glu Thr Arg Ala Gln Gly Leu

340 345 350Tyr Ser Gln Glu Asn Pro

355 <210> 14 <211> 211 <212> PRT <213> Homo Sapiens <400> 14MET ARG THR Leu Ala Cys Leu Leu Leu GLY CYS GLY TYR Leu Ala Glu Glu Glu Glu Ile Pro ARG Glu Val Ile Glu Arg

20 25 30Leu Ala Arg Ser Gln Ile His Ser Ile Arg Asp Leu Gln Arg Leu Leu

35 40 45Glu Ile Asp Ser Val Gly Ser Glu Asp Ser Leu Asp Thr Ser Leu Arg

50 55 60ALA HIS GLY VAL HIS ALA THR LYS HIS Val Pro Glu LYS ARG Pro 65 70 75 80Pro Ile ARG LYS ARG LYS Ile Glu Glu Val Pro Ala Val Cys

85 90 95Lys Thr Arg Thr Val Ile Tyr Glu Ile Pro Arg Ser Gln Val Asp Pro

100 105 110Thr Ser Ala Asn Phe Leu Ile Trp Pro Pro Cys Val Glu Val Lys Arg

115 120 125Cys Thr Gly Cys Cys Asn Thr Ser Ser Val Lys Cys Gln Pro Ser Arg

130 135 140VAL HIS ARG Ser Val Val Val Ala Lys Val Glu Tyr Val ARG LYS145 150 155LYS PRO LYS Leu Val Gln Val ARG Leu His Leu Glu Glu Glu

165 170 175Cys Ala Cys Ala Thr Thr Ser Leu Asn Pro Asp Tyr Arg Glu Glu Asp

180 185 190Thr Gly Arg Pro Arg Glu Ser Gly Lys Lys Arg Lys Arg Lys Arg Leu

195 200 205Lys Pro Thr

210 <210> 15 <211> 241 <212> PRT <213> Homo Sapiens <400> 15MET Asn ARG CYS TRP Ala Leu Phe Leu Seru Cys Cys Tyr Leu ARG1 15le Val Ala Gly Asp Pro Ile Pro Glu Leu Tyr Glu Met

20 25 25 30Leu Ser Asp His Ser Ile Arg Ser Phe Asp Asp Leu Gln Arg Leu Leu

35 40 45His Gly Asp Pro Gly Glu Glu Asp Gly Ala Glu Leu Asp Leu Asn Met

50 55 60thr arg ser his gly glu leu j leu seleu ala arg gly trog65 75 80arg seer sehr Ile Ala Glu plas Ile Ala Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu

85 90 95Cys Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp

100 105 110Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln

115 120 125Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr

130 135 140GLN Val Gln Leu ARG Pro Val Gln Val ARG LYS Ile Glu iLe Val ARG145 150LYS LYS Pro Ile PHE LYS LYS ALA THR VR Leu Glu ASP His Leu

165 170 175Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser

180 185 190Pro Gly Gly Ser Gln Glu Gln Arg Ala Lys Thr Pro Gln Thr Arg Val

195 200 205Thr Ile Arg Thr Val Arg Val Arg Arg Pro Pro Lys Gly Lys His Arg

210 215 220LYS PHR HIS THR HIS Asp Lys Thr Ala Leu Lys Glu ThR Leu GLY225 235 240ALA <211> 182 <212> PRT <213> Homo Sapient <400> 16MET Pro GLN PHR ASP CYSP CYSP CYS PHR ASP CXR ASP CYS Arg Gly Ser Thr Gly Gly Glu1 5 10 15Ala Val Ser Pro Ser Val Leu Pro Pro Ser Ala Leu Pro Leu Asp Leu

20 25 25 30Leu Asn Asn Ala Ile Thr Ala Phe Ser Thr Leu Glu Asp Leu Ile Arg

35 40 45Tyr Leu Glu Pro Glu Arg Trp Gln Leu Asp Leu Glu Asp Leu Tyr Arg

50 55 60pro ThR TRP GLN Leu Leu Gly Lys Ala Phe Val Phe Gly ARG LYS Ser65 70 75 80arg Val Val Val Val Val ASN Leu Leu THR Glu Val ARG Leu Tyr Ser

85 90 95Cys Thr Pro Arg Asn Phe Ser Val Ser Ile Arg Glu Glu Leu Lys Arg

100 105 110Thr Asp Thr Ile Phe Trp Pro Gly Cys Leu Leu Val Lys Arg Cys Gly

115 120 125Gly Asn Cys Ala Cys Cys Leu His Asn Cys Asn Glu Cys Gln Cys Val

130 135 140Pro Ser Lys Val THR LYS LYS TYR HIS GLU VAL Leu Gln Le ARG PRO145 150 155LYS Thr Gly Val Leu His LEU Thr Ala Leuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu Leuus

165 170 175Glu His His Glu Glu Cys

180 <210> 17 <211> 182 <212> PRT <213> Murinae Gen.SP. <400> 17MET Pro Gln Val THR THR THR THR Serial Leu Prou Pro Ser1 5 10 15ser Leu aser Leu Leu Asn Asn Ala Val Thr Ala Phe Ser Thr

20 25 25 30Leu Glu Glu Leu Ile Arg Tyr Leu Glu Pro Asp Arg Trp Gln Val Asp

35 40 45Leu Asp Ser Leu Tyr Lys Pro Thr Trp Gln Leu Asp Cys Val Cys Arg

50 55 60Gly Asn Ala GLY Leu Gly Lys Ala Phe Leu Tyr Gly Lys Lys Lys Ser65 70 80lys Val Val Val ASN Leu Leu Leu Glu Val Lys Leu Tyr Ser

85 90 95Cys Thr Pro Arg Asn Phe Ser Val Ser Ile Arg Glu Glu Leu Lys Arg

100 105 110Thr Asp Thr Ile Phe Trp Pro Gly Cys Leu Leu Val Lys Arg Cys Gly

115 120 125Gly Asn Cys Ala Cys Cys Leu His Asn Cys Asn Glu Cys Gln Cys Val

130 135 140Pro ARG LYS VAL THR LYS LYS TYR HIS GLU VAL Leu Gln Le ARG Pro145 150 155LYS Thr Gly Val Leu His LEU ThR Ala Leuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu Leuu

165 170 175Glu His His Glu Glu Cys

180 <210> 18 <211> 117 <212> PRT <213> Murinae Gen.sp. <400> 18GLU ARG Val Val THR ILE Serle Sern Gly Serle His Serly Met Val Leu Val Trp Arg Leu Val

20 25 25 30Ala Val Asp Glu Asn Val Arg Ile Gln Leu Thr Phe Asp Glu Arg Phe

35 40 45Gly Leu Glu Asp Pro Glu Asp Asp Ile Cys Lys Tyr Asp Phe Val Glu

50 55 60VAL GLU GLU Pro Ser ASP GLY SER VAL Leu Gly ARG TRP CYS GLY Ser65 70 75 80GLY ThR Val Pro Gln Thr Lysh ARG ILE ARG Ile

85 90 95Arg Phe Val Ser Asp Glu Tyr Phe Pro Ser Glu Pro Gly Phe Cys Ile

100 105 110His Tyr Ser Ile Ile

115 <210> 19 <211> 117 <212> PRT <213> Homo Sapiens <400> 19GLU ARG Ile Ile THR Val Ser THR ALY Serle His Serg1 5 10 15phe Pro His Tyr THR Val Leuuuuu Val Trp Arg Leu Val

20 25 25 30Ala Val Glu Glu Asn Val Trp Ile Gln Leu Thr Phe Asp Glu Arg Phe

35 40 45Gly Leu Glu Asp Pro Glu Asp Asp Ile Cys Lys Tyr Asp Phe Val Glu

50 55 60VAL GLU GLU Pro Ser ASP GLY THR ILE Leu Gly ARG TRP CYS GLY Ser65 70 75 80Gly Thr Val Pro Gln Ile Serite Asn Gln Ile ARG Ile

85 90 95Arg Phe Val Ser Asp Glu Tyr Phe Pro Ser Glu Pro Gly Phe Cys Ile

100 105 110His Tyr Asn Ile Val

115 <210> 20 <211> 113 <212> PRT <213> Homo Sapiens <400> 20CYS GLY GLU Thr Leu Gln Asr Gly Asn PHE Ser, 5 10 15TYR Pro His Met His Met His Cys Cys Val Trp Arg Ile Ser

20 25 25 30Val Thr Pro Gly Glu Lys Ile Ile Leu Asn Phe Thr Ser Leu Asp Leu

35 40 45Tyr Arg Ser Arg Leu Cys Trp Tyr Asp Tyr Val Glu Val Arg Asp Gly

50 55 60phe trp arg LYS ALA Pro Leu ARG GLY ARG PHE CYS GLY Ser LYS Leu65 70 75 80pro Glu Pro Ile Val Ser ARG Leu TRP Val Glu Phe Arg

85 90 95Ser Ser Ser Asn Trp Val Gly Lys Gly Phe Phe Ala Val Tyr Glu Ala

100 105 110ILE <210> 21 <211> 112 <212> PRT <213> Homo Sapiens <400> 21CYS GLE GLY ASP Val LYS LYS LYS Asp Tyr Gln Serle Gln Sern1 5 10 15TYR ARG Pro Sergs Lys Val Cys Ile Trp Arg Ile Gln

20 25 25 30Val Ser Glu Gly Phe His Val Gly Leu Thr Phe Gln Ser Phe Glu Ile

35 40 45Glu Arg Met Asp Ser Cys Ala Tyr Asp Tyr Leu Glu Val Arg Asp Gly

50 55 60His Ser Glu Sering Leu Ile Gly ARG TYR CYS GLY TYR GLU LYS65 75 80pro ASP ILE LYS Serg Leu TRP Leu Lys Phes Phe Val Val

85 90 95Ser Asp Gly Ser Ile Asn Lys Ala Gly Phe Ala Val Asn Phe Phe Lys

100                 105                 110<210>22<211>113<212>PRT<213>Homo sapiens<400>22Cys Gly Gly Phe Leu Thr Lys Leu Asn Gly Ser Ile Thr Ser Pro Gly1               5                  10                  15Trp Pro Lys Glu Tyr Pro Pro Asn Lys Asn Cys Ile Trp Gln Leu Val

20 25 25 30Ala Pro Thr Gln Tyr Arg Ile Ser Leu Gln Phe Asp Phe Phe Glu Thr

35 40 45Glu Gly Asn Asp Val Cys Lys Tyr Asp Phe Val Glu Val Arg Ser Gly

50 55 60leu Thr Ala ASP Ser Lys Leu His Gly LYS PHE CYS GLY Ser Glu Lys65 75 80pro Glu Val Ile Thrn Tyr Asn Met ARG Val Glu Pro Lysn

85 90 95Ser Asp Asn Thr Val Ser Lys Lys Gly Phe Lys Ala His Phe Phe Ser

100                 105                 110Glu<210>23<211>113<212>PRT<213>Homo sapiens<400>23Gly Asp Thr Ile Lys Ile Glu Ser Pro Gly Tyr Leu Thr Ser Pro Gly1               5                  10                  15Tyr Pro His Ser Tyr His Pro Ser Glu Lys Cys Glu Trp Leu Ile Gln

20 25 25 30Ala Pro Asp Pro Tyr Gln Arg Ile Met Ile Asn Phe Asn Pro His Phe

35 40 45Asp Leu Glu Asp Arg Asp Cys Lys Tyr Asp Tyr Val Glu Val Phe Asp

50 55 60Gly Glu Asn Glu asn Gly His PHE ARG GLY LYS PHE CYS GLY LYS ILE65 75 80ALA Pro Pro Val Val Val Val Val Val Gly PHE Leu PHE Ile LYS PHE

85 90 95Val Ser Asp Tyr Glu Thr His Gly Ala Gly Phe Ser Ile Arg Tyr Glu

100 105 110ILE <210> 24 <211> 119 <212> PRT <213> Homo Sapiens <400> 24CYS SERN TYR THR THR PRO Serge Val Ile LYS Serle Lyser1 5 10 15phe GLU LYR Pro Asn Seruuuu Glu Cys Thr Tyr Ile Val Phe

20 25 30Ala Pro Lys Met Ser Glu Ile Ile Leu Glu Phe Glu Ser Phe Asp Leu

35 40 45Glu Pro Asp Ser Asn Pro Pro Gly Gly Met Phe Cys Arg Tyr Asp Arg

50 55 60leu ILE TALU TOLY PHE PRO ASP Val Gly Pro His Ile Gly ARG65 70 75 80ty GLN LYS THR Pro Gly ARG Serg Serg Serg Serg Serge Ile

85 90 95Leu Ser Met Val Phe Tyr Thr Asp Ser Ala Ile Ala Lys Glu Gly Phe

100 105 110Ser Ala Asn Tyr Ser Val Leu

115 <210> 25 <211 <212> DNA <213> Homo Sapiens <400> 25GAAGTTGAGG AACCCCAGTG 19 <210> 26 <211> 20 <212> DNA <213> Homo Sapiens <400> 26CTTGCAAGCAAGCAAG 20 <210> 27 <211> 19 <212> DNA <213> Murinae Gen.sp. <400> 27CTTCAGTACC TTGGAGAG 19 <2L0> 28 <212> DNA <213> Murinae Gen.Sp. <400> 28CGCTGACAGACAC 19 < 210>29<211>30<212>DNA<213>Murinae gen.sp.<400>29acgtgaattc agcaagttca gcctggttaa 30<210>30<211>30<212>DNA<213>Murinae gen.sp.<400>30acgtggat TGAGTATTTC TTCCCAGGTA 30 <210> 31 <211> 22 <212> PRT <213> Homo Sapiens <400> 31CYS PHE GLN PHE SER Asn Lysn Gln GLN GLN ASP1 5 10 15Pro Gln His Glu ARG CYS

20 <210> 32 <211> 21 <212> PRT <213> Homo Sapiens <400> 32Gly ARG LYS Serg Val Val Val ASN Leu Leu Leu THR Glu Glu Val1 5arg Leu Tyr Ser Cys

20<210>33<211>26<212>DNA<213>Homo sapiens<400>33cgggatcccg aatccaacct gagtag                                      26<210>34<211>61<212>DNA<213>Homo sapiens<400>34ggaattccta atggtgatgg tgatgatgtt tgtcatcgtc atctcctcct gtgctccctc 60t                                                                 61<210>35<211>29<212>DNA<213>Homo sapiens<400>35cggatcccgg aagaaaatcc agagtggtg<210>36<211>61<212>DNA<213>Homo sapiens<400>36ggaattccta atggtgatgg tgatgatgtt tgtcatcgtc ATCTCCCCT GTGCCCCTC 60T 61 <210> 37 <211> 21 <212> PRT <213> Homo SapIens <400> 37Gly ARG LYS Serg Val Val Val ASN Leu Leu THR GLU Val1 5arg Leu Tyr Sercs Cyser

     20<210>38<211>26<212>DNA<213>Homo sapiens<220><223>from human PDGF—C 430bp cDNA encoding CUB domain

Forward PCR primers for fragments including - BamHI site <400>38cgggatcccg aatccaacct gagtag<210>39<211>60<212>DNA<213>Homo sapiens<220><223>from human PDGF encoding CUB domain —C 430bp cDNA

The reverse PCR primers of the fragment, which include—EcoRI site and coding

The sequence of the C-terminal 6XHis formerly the enterokinase site

site<400>39ccggaattcc taatggtgat ggtgatgatg tttgtcatcg tcgtcgacaa tgttgtagtg 60

Claims (74)

1. An isolated nucleic acid molecule comprising a polynucleotide sequence having at least 85% identity to the sequence of Figure 1, Figure 3 or Figure 5 (SEQ ID NO: 2, 4 and 6, respectively). 2. The isolated nucleic acid molecule of claim 1, wherein the sequence identity is at least 90%. 3. The isolated nucleic acid molecule of claim 1, wherein the sequence identity is at least 95%. 4. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid is cDNA. 5. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid is a mammalian polynucleotide. 6. The isolated nucleic acid molecule of claim 5, wherein the nucleic acid is a murine polynucleotide. 7. The nucleic acid molecule of separation according to claim 6, comprising the sequence of Figure 5 (SEQ ID NO: 6). 8. The isolated nucleic acid molecule of claim 5, wherein the nucleic acid is a human polynucleotide. 9. The isolated nucleic acid molecule of claim 8, wherein said nucleic acid comprises the sequence of Figure 1 or Figure 3 (SEQ ID NO: 2 and 4, respectively). 10. An isolated nucleic acid molecule, which encodes a polypeptide molecule or its analog or fragment with PDGF-C biological activity, the amino acid sequence of the polypeptide molecule is the same as that of Fig. 2 (SEQ ID NO: 3), Fig. 4 ( The amino acid sequences in SEQ ID NO: 5) have at least 85% identity. 11. The isolated nucleic acid molecule of claim 10, wherein the amino acid sequence identity is at least 90%. 12. The isolated nucleic acid molecule of claim 10, wherein the amino acid sequence identity is at least 95%. 13. An isolated nucleic acid molecule, the amino acid sequence of the polypeptide molecule it encodes comprises the following amino acid sequence: PXCXXVXRCGGXXXCC (SEQ ID NO: 1). 14. A vector comprising the nucleic acid of claim 1 operably linked to a promoter sequence. 15. The vector according to claim 14, which is a eukaryotic vector. 16. The vector of claim 14, which is a prokaryotic vector. 17. The vector according to claim 14, which is a plasmid. 18. The vector of claim 14, which is a baculovirus vector. 19. A method for constructing a carrier expressing a polypeptide or a polypeptide having PDGF-C biologically active analogs or fragments, wherein the amino acid sequence of the polypeptide is the same as that of Fig. 2 (SEQ ID NO: 3) or Fig. 6 (SEQ ID NO 7) has at least 85% identity to the amino acid sequence, and the method comprises inserting the isolated nucleic acid of claim 1, 10 or 13 into the vector in an operably linked manner with a promoter. 20. A host cell transformed or transfected with the vector of claim 14. 21. The host cell according to claim 20, which is a eukaryotic cell. 22. The host cell according to claim 20, which is a COS cell. 23. The host cell according to claim 20, which is a prokaryotic cell. 24. The host cell according to claim 20, which is a 293EBNA cell. 25. The host cell according to claim 20, which is an insect cell. 26. A host cell transformed or transfected with a vector, the vector comprising the nucleic acid sequence of claim 1 operably linked to a promoter, so that the host cell expresses a polypeptide or has a PDGF-C A biologically active analogue or fragment, the amino acid sequence of the polypeptide has at least 85% identity with the amino acid sequence in Figure 2 (SEQ ID NO: 3) or Figure 6 (SEQ ID NO: 7). 27. An isolated polypeptide or an analog or fragment thereof with PDGF-C biological activity, the polypeptide has at least 85% of the amino acid sequence of Fig. 2 (SEQ ID NO: 3) or Fig. 6 (SEQ ID NO: 7) % identity amino acid sequence. 28. The isolated polypeptide of claim 27 which is a murine polypeptide. 29. The isolated polypeptide of claim 27 which is a human polypeptide. 30. The isolated polypeptide according to claim 27, which has the ability to stimulate and/or enhance the proliferation and/or differentiation and/or growth and/or motility of cells expressing the PDGF-C receptor. 31. An isolated polypeptide produced by expression of a polynucleotide comprising at least 85% identity to the sequence of Figure 1 , Figure 3 or Figure 5 (SEQ ID NO: 2, 4 or 6) Sexual polynucleotide sequences, or the polynucleotides are polynucleotides that hybridize to at least one of the above DNA sequences under stringent conditions. 32. An isolated polypeptide comprising the following characteristic sequence: PXCXXVXRCGGXXXCC (SEQ ID NO: 1). 33. An isolated polypeptide dimer comprising the polypeptide of claim 27. 34. The polypeptide dimer of claim 33, which is a homodimer of said polypeptide. 35. The polypeptide dimer according to claim 33, which is a heterodimer formed between said polypeptide and VEGF, VEGF-B, VEGF-C, VEGF-D, PDGF-A, PDGF-B or PlGF. 36. The polypeptide dimer of claim 33, which is a disulfide-linked dimer. 37. A pharmaceutical composition, comprising the polypeptide described in 27, 31 or 32 in an effective amount for promoting cell proliferation, and a polypeptide selected from VEGF, VEGF-B, VEGF-C, VEGF-D, PDGF-A, PDGF-B or At least one other growth factor in PlGF. 38. The pharmaceutical composition according to claim 37, further comprising heparin. 39. A pharmaceutical composition comprising the isolated polypeptide of 27, 31 or 32 in an effective amount for promoting cell proliferation, and at least one pharmaceutical carrier or diluent. 40. A pharmaceutical composition comprising a PDGF receptor stimulating amount of the isolated polypeptide of 27, 31 or 32, and at least one pharmaceutical carrier or diluent. 41. A pharmaceutical composition comprising an effective amount of the isolated polypeptide of claim 27, 31 or 32 to stimulate connective tissue or wound healing, and at least one pharmaceutical carrier or diluent. 42. A device for amplifying the polynucleotide of claim 1 in a test sample, said device comprising at least one pair of primers complementary to the nucleic acid of claim 1. 43. A device for amplifying the polynucleotide of claim 1 in a test sample, said device comprising a polymerase and at least one pair of primers complementary to the nucleic acid of claim 1 for use by polymerization Enzyme chain reaction amplifies the polynucleotide to facilitate comparison of the polynucleotide with the nucleic acid of claim 1 . 44. An antibody specifically reactive with the polypeptide of claim 27, 31 or 32. 45. The antibody of claim 44, which is a polyclonal antibody. 46. The antibody of claim 44, which is a monoclonal antibody, or a F(ab') ₂ , F(ab'), F(ab) fragment or chimeric antibody. 47. The antibody of claim 45 or 46, which is labeled with a detectable label. 48. The antibody of claim 47, wherein the detectable label is a radioisotope. 49. A method of preparing the polypeptide of claim 27, 31 or 32, the method comprising the steps of: culturing host cells transformed or transfected with a vector comprising a polynucleotide encoding said polypeptide operably linked to a promoter sequence so that the nucleic acid sequence encoding said polypeptide is expressed; The polypeptide is isolated from the host cell or the medium in which the host cell is grown. 50. A method of stimulating connective tissue growth or wound healing in a mammal comprising administering to the mammal an effective amount of the polypeptide of claim 27, 31 or 32. 51. A method for producing an active truncated form of PDGF-C, comprising the step of expressing the expression vector of claim 69 comprising a polynucleotide encoding a polypeptide. 52. A method for regulating the receptor binding specificity of PDGF-C, comprising the steps of: expressing an expression vector comprising a polynucleotide encoding a polypeptide as claimed in claims 27, 31 and 32; and providing at least the amount of proteolysis An enzyme used to process the expressed polypeptide to obtain an active truncated form of PDGF-C. 53. A method for selectively activating a polypeptide with growth factor activity, comprising the steps of: expressing an expression vector, the expression vector comprising a polynucleotide encoding a polypeptide with growth factor activity, CUB structure domain and a protease cleavage site between the polypeptide and the CUB domain; and at least one enzyme in a proteolytic amount is provided to process the expressed polypeptide to obtain an active polypeptide with growth factor activity. 54. The isolated polypeptide of claims 27, 31 and 32, comprising a proteolytic cleavage site having the amino acid sequence RKSR or a structurally conserved amino acid sequence thereof. 55. The nucleic acid molecule according to claim 10, the polypeptide encoded by the nucleic acid molecule comprises a protease cleavage site whose amino acid sequence is RKSR or its structurally conserved amino acid sequence. 56. An isolated heterodimer comprising active monomers of VEGF, VEGF-B, VEGF-C, VEGF-D, PDGF-C, PDGF-A, PDGF-B or PlGF and a CUB domain Linked PDGF-C active monomer. 57. An isolated heterodimer comprising PDGF-C active monomers and VEGF, VEGF-B, VEGF-C, VEGF-D, PDGF-C, PDGF-A, PDGF linked to a CUB domain - active monomer of B or PlGF. 58. The heterodimer of claim 56, further comprising a proteolytic cleavage site between the active monomer and the CUB domain. 59. The heterodimer of claim 57, further comprising a proteolytic cleavage site between the active monomer and the CUB domain. 60. An isolated polynucleotide comprising a polynucleotide sequence with at least 85% identity to the sequence of Figure 1 , Figure 3 or Figure 5 (SEQ ID NO: 2, 4 or 6), or with at least one A polynucleotide to which the DNA sequence hybridizes under stringent conditions. 61. A method for enhancing mitosis of mammalian fibroblasts, comprising administering to the mammal a mitogenically effective amount of the polypeptide of claim 27, 31 or 32. 62. A method of inducing activation of PDGF-alpha receptors, comprising adding the polypeptide of claim 27, 31 or 32 in an amount that stimulates PDGF-alpha receptors. 63. A method of inhibiting tumor growth of a PDGF-C expressing tumor in a mammal comprising administering to said mammal a PDGF-C antagonist in an amount that inhibits PDGF-C. 64. A method of identifying a specific type of human tumor comprising the steps of obtaining a sample of the tumor and detecting the expression of PDGF-C. 65. The method of claim 64, wherein the particular tumor type is: choriocarcinoma, Wilms' tumor, megakaryocytic leukemia, lung cancer, and erythroleukemia. 66. A method of identifying a PDGF-C antagonist comprising the steps of: admixing a substantially purified preparation of an activated truncated form of PDGF-C with a detection reagent; and Inhibition of PDGF-C biological activity is monitored by any suitable means. 67. A method of identifying a PDGF-C antagonist comprising the steps of: mixing the substantially purified full-length PDGF-C preparation with a detection reagent; and Inhibition of cleavage of the CUB domain from PDGF-C is monitored by any suitable means. 68. The isolated polypeptide of claim 27, wherein said cells are endothelial cells, connective tissue cells, myofibroblasts, or glial cells. 69. A method for constructing a vector, the vector expresses a polypeptide comprising at least 230 to 345 amino acid residues in Figure 2 (SEQ ID NO: 3) or Figure 6 (SEQ ID NO: 7) 85% identical amino acid sequence, said method comprising inserting an isolated nucleic acid molecule encoding said amino acid residue into said vector in operably linked manner with a promoter. 70. The antibody of claim 46, wherein said monoclonal antibody is a human antibody. 71. A method for preparing an activated truncated form of PDGF-C, the method comprising expressing an expression vector comprising a polynucleotide encoding a polypeptide as claimed in claims 27, 31 and 32; and providing at least the amount of proteolysis An enzyme used to process the expressed polypeptide to obtain an activated truncated form of PDGF-C. 72. A method of inhibiting tissue transformation during tumor cell invasion of a normal cell population comprising administering to a mammal a PDGF-C antagonist in an amount that inhibits PDGF-C. 73. A method of treating a condition of fibrosis in a mammal comprising administering to the mammal a PDGF-C antagonist in an amount that inhibits PDGF-C. 74. The method of claim 73, wherein the fibrotic symptoms are found in the lungs, kidneys or liver.

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