Subtractive immunization using highly metastatic human tumor cells identifies SIMA135 CDCP1, a 135 kDa cell surface phosphorylated glycoprotein antigen

• Published in: Oncogene Vol. 22; no. 12; pp. 1783 - 1794
• Main Authors: HOOPER, John D, ZIJLSTRA, Andries, AIMES, Ronald T, HONGYAN LIANG, CLAASSEN, Gisela F, TARIN, David, TESTA, Jacqueline E, QUIGLEY, James P
• Format: Journal Article
• Language: English
• Published: Basingstoke Nature Publishing 27-03-2003; Nature Publishing Group
• Subjects: Amino Acid Sequence; Antibodies, Monoclonal - immunology; Antigens; Antigens, CD; Base Sequence; Biological and medical sciences; Cancer; Cell Adhesion Molecules - chemistry; Cell Adhesion Molecules - immunology; Cell Adhesion Molecules - metabolism; Cell surface; Cells; Classical genetics, quantitative genetics, hybrids; Colon; Colorectal cancer; DNA Primers; Endothelial cells; Epithelial cells; Fibroblasts; Fundamental and applied biological sciences. Psychology; Gene expression; Genetics of eukaryotes. Biological and molecular evolution; Glycoproteins; Glycosylation; Human; Humans; Immunization; Immunocytochemistry; Immunohistochemistry; Kinases; Lysates; Metastases; Metastasis; Molecular Sequence Data; Molecular weight; Monoclonal antibodies; mRNA; Neoplasm Metastasis; Neoplasm Proteins - chemistry; Neoplasm Proteins - immunology; Neoplasm Proteins - metabolism; Phosphorylation; Proteins; Proteomics; Sequence analysis; Skeletal muscle; Subtraction Technique; Tumor cell lines; Tumor cells; Tumor Cells, Cultured; Tyrosine; La Jolla California; United States > US; Human; Immunization; proteomics; colon cancer; Glycoprotein; Identification; Cell surface; SIMA135/ CDCP1; Antigen; CUB; subtractive immunization; metastasis; Tumor cell
• ISSN: 0950-9232; 1476-5594
• DOI: 10.1038/sj.onc.1206220

We have previously used a subtractive immunization (SI) approach to generate monoclonal antibodies (mAbs) against proteins preferentially expressed by the highly metastatic human epidermoid carcinoma cell line, M(+)HEp3. Here we report the immunopurification, identification and characterization of SIMA135/CDCP1 (subtractive immunization M(+)HEp3 associated 135 kDa protein/CUB domain containing protein 1) using one of these mAbs designated 41-2. Protein expression levels of SIMA135/CDCP1 correlated with the metastatic ability of variant HEp3 cell lines. Protein sequence analysis predicted a cell surface location and type I orientation of SIMA135/CDCP1, which was confirmed directly by immunocytochemistry. Analysis of deglycosylated cell lysates indicated that up to 40 kDa of the apparent molecular weight of SIMA135/CDCP1 is because of N-glycosylation. Western blot analysis using a antiphosphotyrosine antibody demonstrated that SIMA135/CDCP1 from HEp3 cells is tyrosine phosphorylated. Selective inhibitor studies indicated that an Src kinase family member is involved in the tyrosine phosphorylation of the protein. In addition to high expression in M(+)HEp3 cells, the SIMA135/CDCP1 protein is expressed to varying levels in 13 other human tumor cell lines, manifesting only a weak correlation with the reported metastatic ability of these tumor cell lines. The protein is not detected in normal human fibroblasts and endothelial cells. Northern blot analysis indicated that SIMA135/CDCP1 mRNA has a restricted expression pattern in normal human tissues with highest levels of expression in skeletal muscle and colon. Immunohistochemical analysis indicated apical and basal plasma membrane expression of SIMA135/CDCP1 in epithelial cells in normal colon. In colon tumor, SIMA135/CDCP1 expression appeared dysregulated showing extensive cell surface as well as cytoplasmic expression. Consistent with in vitro shedding experiments on HEp3 cells, SIMA135/CDCP1 was also detected within the lumen of normal and cancerous colon crypts, suggesting that protein shedding may occur in vivo. Thus, specific immunodetection followed by proteomic analysis allows for the identification and partial characterization of a heretofore uncharacterized human cell surface antigen.