De-N-acetyl-gangliosides in humans: Unusual subcellular distribution of a novel tumor antigen

The disialoganglioside G(D3) is a major antigen in human melanomas that can undergo 9-O-acetytation of the outer sialic acid (giving 9-OAc-G(D3)), Mon oclonal 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 c onventional immunohistochemistry of the SGR37 antigen is limited by a reduc tion in reactivity upon fixation with aldehydes (which presumably react wit h the free amino group) or with organic reagents (which can extract glycoli pids), We optimized conditions for detection of this antigen in unfixed fro zen tissue sections and studied its distribution in human tissues and tumor s. It is expressed at low levels in a few blood vessels, infiltrating monon uclear cells in the skin and colon, and at moderate levels in skin melanocy tes. In contrast, the antigen accumulates at high levels in many melanomas and in some lymphomas but not in carcinomas, In positive melanomas, express ion 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 localiza tion 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 ce ll surface, de-N-acetyl-G(D3) is efficiently internalized into a compartmen t 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 th e polar head group of a tumor-associated glycosphingolipid can markedly aff ect 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 app roaches to these diseases.