keratan-sulfate and Carcinoma--Embryonal

Human embryonal carcinoma (EC) cells are the stem cells of teratocarcinomas, and they are key components of germ cell tumors (GCTs). They express several high molecular weight glycoprotein antigens that are down-regulated upon differentiation. One of these antigens, defined by monoclonal antibody TRA-1-60, can be detected in the serum of GCT patients and provides a useful complement to the established serum markers human chorionic gonadotropin and alpha-fetoprotein, especially in those patients without elevated serum human chorionic gonadotropin or alpha-fetoprotein. To examine the relationship of the TRA-1-60-defined antigen to similar antigens defined by other monoclonal antibodies, we have carried out comparative Western blot and immunoprecipitation analyses of human GCT-derived cell lines with monoclonal antibodies TRA-1-60, TRA-1-81, GCTM2, and K21. The TRA-1-60 antigen was detected by Western blot analysis in extracts of all human EC cell lines and in clinical specimens of GCT tested as a diffuse band with a molecular weight of >200,000. A similar but noticeably fainter band was detected in GCT composed of seminoma only. The antigen was not expressed by GCT-derived lines without an EC phenotype. Affinity bead-purified TRA-1-60, TRA-1-81, GCTM2 and K21 antigens reacted in Western blot analysis with each of the other antibodies tested, indicating that the epitopes recognized by each antibody are carried by the same molecular species. This molecule could be metabolically labeled with inorganic [35S]sulfate and was degraded by keratanase. Glycopeptides produced from affinity-purified TRA-1-60 antigen by extensive digestion with Pronase exhibited a molecular weight in excess of 10,000 and were degraded by keratanase. The TRA-1-60 epitope was destroyed by digestion with neuraminidase, but the epitopes defined by TRA-1-81, GCTM2, and K21 were not. Our results indicate that human EC cells generally express a cell surface sialylated keratan sulfate proteoglycan that is subject to modification to yield a variety of epitopes, one of which is recognized by the monoclonal antibody TRA-1-60. Sensitivity to milk alkaline digestion suggests that the oligosaccharides of this proteoglycan are O-linked to a core polypeptide.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Surface; Biomarkers, Tumor; Blotting, Western; Carcinoma, Embryonal; Chondroitin Sulfate Proteoglycans; Glycopeptides; Glycoproteins; Humans; Keratan Sulfate; Lumican; N-Acetylneuraminic Acid; Proteoglycans; Sulfates; Tumor Cells, Cultured

We recently purified a 200 kD peanut agglutinin-binding glycoprotein from the embryonal carcinoma cell line HT-E (833K) that is expressed selectively in nonseminomatous germ cell tumors. We now further characterize gp200 using Western blot analysis to compare normal and malignant testicular germ cells for reactivity to peanut agglutinin, a plant lectin that recognizes terminal D-galactosyl residues, and GCTM-2, a monoclonal antibody that recognizes a 200 kD keratan-sulfate proteoglycan on human embryonal carcinoma. The results indicate that normal germ cells express gp200 as a membrane-bound 230 kD glycoform that expresses terminal galactose residues. This 230 kD glycoprotein is absent on spermatozoa but present on seminomatous germ cell tumors and somatic tissue and does not express terminal galactose on its carbohydrate side chains. In contrast, nonseminomatous germ cell tumors express a heavily sialylated glycoform of gp200 that does express terminal galactose residues. These results describe a glycoprotein that exists in several glycoforms in normal and malignant testicular germ cells. The differential expression of these glycoforms may help in understanding the ontogeny of germ cell tumors.

Topics: Blotting, Western; Carcinoma, Embryonal; Galactose; Germinoma; Glycoproteins; Humans; Keratan Sulfate; Male; Membrane Glycoproteins; Molecular Weight; Receptors, Mitogen; Seminoma; Sialoglycoproteins; Spermatozoa; Testicular Neoplasms; Testis; Tumor Cells, Cultured