| Tumor invasion and metastasis are the mainly cause of cancer-related death. Cancer metastasis results from several interdependent processes. These include: 1) detachment of cancer cell from their original localization and cancer cell migration. 2) Invasion of cancer cell into the surrounding tissue, requiring adhesion to and subsequent degradation of extracellular matrix (ECM) components. 3) Access of cancer cell to blood and lymphatic vessels. 4) Adhesion to and invasion through the endothelium, allowing colonization at distant sites in the organism. The extracellular matrix (ECM), including the basement membrane and interstitial stroma, are composed of collagens, glycoproteins (such as fibronectin, laminin, and entactin/nidogen), proteoglycans, and glycosaminoglycans. Degradation of extra cellular matrix (ECM) and cellular migration are two prominent steps of tumor Metastasis. Matrix-degrading proteases, including the plasminogen activator-plasmin system of serine proteases, MMP-2 and MMP-9 of the matrix metalloprotease (MMP) family, and cathepsins B (a cysteine protease) and D (an aspartic protease), have been implicated in tumor cell invasion. Degradation of the ECM occurs in close proximity to the cell surface, and that matrix-degrading proteases must be localized on the cell surface to perform their functions.SNC19/ST14 gene was firstly cloned by subtractve hybridization method between normal mucosa cDNA and colorectal cancer tissue mRNA. We obtained 46 positive clones from normal mucosal cDNA library. The sequence data revealed that one of these clones, named SNC19, encodes a novel human gene. Accession number HSU20428 was given by NCBI8genebank in 1995, and was named by Gene Nomenclature Committee as ST14 (suppression of tumorigenicity 14) in 1998. The data from human genome project (HOP) shows that SNC19/ST14 gene contains 19 exons and 18 introns that span 50420 bp. The full-length cDNA is 3142bp, and encodes an 855 amino protein, which contains a serine protease domain. Lin.C.Y et al found a protease, which was purified from T-47D breast cancer cells, has the same sequence of SNC19. He calls this protease as Matriptase because it has matrix-degrading function. Takeuchi et al also found this protease at human PC-3 prostates cancer tumors at same time and named this protease as membrane-type'serine protease 1 (MT-SP1). It can activate urokinase plasminogen activator (uPA) and hepatocyte growth factor (HGF), suggesting its association with tissue remodeling, cancer invasion, and metastasis.Hepatocyte growth factor activator inhibitor type 1 (HAI-1) was initially identified as a potent inhibitor of hepatocyte growth factor (HGF) activator from the conditioned medium of a human stomach carcinoma cell line. It is a Kunitz-type serine protease inhibitor and can inhibit Matriptase (SNC19)'s cleavage activity. A protein complex from human milk contains Matriptase and HAI-1.To study the structure and function of the protein encoded by SNC19 (ST14), we make this study: 1) expression of SNC19 gene ORF in E.coli and purification of the GST fusion protein. 2) Preparation of the monoclonal antibody against SNC19 protein and confirmed by western bolt. 3) Analysis structure of SNC19 protein and predict its function using bioinformatics methods. 4) Characterization of SNC19 protein and its related protein HAI-1 expression in human normal and tumor tissues. 5)Detected the potential mutation in SNC19 gene between normal colorectal tissues and colorectal cancer tissues.1) Expression of SNC19 gene ORF in E.coli and purification of the GST fusion protein:To obtain enough amount of SNC19 protein for further studies, SNC19 gene part-length ORF, amplified by PCR, was cloned into expression vector pGEX-4T-2, and then expressed in E.coli. BamH I and Not I restriction analysis and DNA sequencing showed the SNC19 part-length ORF/ pGEX-4T-2 recombinant plasmid was constructed successfully with correct sequence. SDS-PAGE indicated that SNC19-GST fusion protein was expressed in E.coli as expected at SSkda...
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