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De-N-acetyl-gangliosides in Humans

Unusual Subcellular Distribution of a Novel Tumor Antigen¹

Glycobiology Program and Cancer Center, Divisions of Hematology-Oncology and Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093-0687 [R.C., J.L.S., N.E.W., M.G.F., N.M.V., A.V.], and Tokyo Metropolitan Institute, Tokyo 113, Japan [T. T.]

ABSTRACT

The disialoganglioside G_D3 is a major antigen in human melanomas that can undergo 9-O-acetylation of the outer sialic acid (giving 9-OAc-G_D3). Monoclonal antibody SGR37 detects a different modification of the G_D3, de-N-acetylation of the 5-N-acetyl group (giving de-N-Ac-G_D3). We found that conventional immunohistochemistry of the SGR37 antigen is limited by a reduction in reactivity upon fixation with aldehydes (which presumably react with the free amino group) or with organic reagents (which can extract glycolipids). We optimized conditions for detection of this antigen in unfixed frozen tissue sections and studied its distribution in human tissues and tumors. It is expressed at low levels in a few blood vessels, infiltrating mononuclear cells in the skin and colon, and at moderate levels in skin melanocytes. In contrast, the antigen accumulates at high levels in many melanomas and in some lymphomas but not in carcinomas. In positive melanomas, expression is sometimes more intense and widespread than that of G_D3.

Both 9-O-acetylation and de-N-acetylation of G_D3 seem to occur after its initial biosynthesis. Isotype-matched antibodies against G_D3, 9-O-acetyl-G_D3 and de-N-acetyl-G_D3 were used to compare their subcellular localization and trafficking. 9-O-acetyl-G_D3 colocalizes with G_D3 predominantly on the cell surface and partly in lysosomal compartments. In contrast, de-N-acetyl-G_D3 has a diffuse intracellular location. Adsorptive endocytosis of antibodies indicates that whereas G_D3 remains predominantly on the cell surface, de-N-acetyl-G_D3 is efficiently internalized into a compartment that is distinct from lysosomes. Rounding up of melanoma cells occurring during growth in culture is associated with relocation of the internal pool of de-N-acetyl-G_D3 to the cell surface. Thus, a minor modification of the polar head group of a tumor-associated glycosphingolipid can markedly affect the subcellular localization and trafficking of the whole molecule. The high levels of the SGR37 antigen in melanomas and lymphomas, its selective endocytosis from the cell surface, and its relocation to the cell surface of rounded up cells suggest potential uses in diagnostic or therapeutic approaches to these diseases.

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