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Biosynthesis, Glycosylation and Intracellular Processing of the Neuroglandular Antigen, a Human Melanoma-associated Antigen¹

Oncology Research Group [W. T. D., L. K. J. S., K. O., L. M. J.], and Department of Pathology [D. J. D.], The University of Calgary, and Tom Baker Cancer Centre [L. M. J.], Calgary, Alberta, Canada T2N 4N2, and Department of Biochemistry [K. O.], Tokyo College of Pharmacy, Tokyo, Japan 192-03

Neuroglandular antigen (NGA) was identified as a human melanoma-associated antigen by a panel of murine monoclonal antibodies of both IgG2a (LS62, LS76, LS159) and IgG1 (LS113, LS140, LS152) subclasses, developed in this laboratory (L. Sikora, A. Pinto, D. Demetrick, W. Dixon, S. Urbanski, and L. M. Jerry, Int. J. Cancer, 39: 138–145, 1987). Monoclonal antibody LS62 was used to immunoprecipitate NGA from radiolabeled cultured melanoma cells, and it behaved as a heterogeneous glycoprotein "smear" on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (M_r 29,000–70,000). Radioactive pulse-chase time course experiments using human melanoma cells cultured in the presence or absence of inhibitors of protein glycosylation showed that the antigen consisted of a core protein with a molecular weight of 22,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This molecule was modified by the addition of at least three N-linked oligosaccharide side chains (as revealed by limited N-glycanase digestion) to give a precursor form with a molecular weight of approximately 34,000. Subsequent processing steps yielded a heterogeneous family of glycoproteins with varying amounts of covalently attached carbohydrate. Much of this heterogeneity in both molecular weight and pI (as revealed by two-dimensional electrophoresis) could be removed by treatment of the antigen with neuraminidase, suggesting heavy sialylation of the glycoprotein.

NGA could be detected on the surface of melanoma cells by fluorescence-activated cell sorter analysis, surface radioiodination, and, as previously shown, immunoperoxidase staining. However, there was a larger intracellular pool of the molecule and the antigen was rapidly released into the culture supernatant. The function of NGA remains unknown but its elevated expression in transformed melanocytes have prompted this characterization to understand its biochemical nature and relation to other melanoma-associated antigens.

¹ W. D., L. S., and D. D. acknowledge the generous support of the Alberta Heritage Fund for Medical Research, Medical Research Council of Canada, and the National Cancer Institute of Canada.

² To whom requests for reprints should be addressed, at Tom Baker Cancer Centre, 1331 - 29 Street N.W., Calgary, Alberta, Canada T2N 4N2

Received 2/27/89. Revised 2/ 8/90.

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