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The Human Embryonal Carcinoma Marker Antigen TRA-1-60 Is a Sialylated Keratan Sulfate Proteoglycan

Departments of Biomedical Science [G. B., C. P., P. W. A.] and Pathology [J. G.], University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom

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 -fetoprotein, especially in those patients without elevated serum human chorionic gonadotropin or -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.

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