Bovine milk galactosyltransferase has been used, in conjunction with UDP-[3H]galactose, as an impermeant probe for accessible GlcNAc residues on the surfaces of lymphocytes. Galactosylation of living thymic lymphocytes is dependent upon cell number, enzyme concentration, UDP-galactose concentration, and Mn2+ concentration. Kinetics of labeling are biphasic, leveling off at approximately 30 min. The data strongly indicate vectorial surface labeling and covalent attachment of galactose. Thymocytes, T-lymphocytes, and B-lymphocytes have approximately 106, 3 x 106, and 5 x 106 galactosylatable sites on their cell surfaces, respectively. Numerous proteins are exogalactosylated that differ quantitatively among the major functional subsets of lymphocytes. Negligible radioactivity is found in lipid. In thymocytes, 49% of the exogalactosylated oligosaccharides are alkali labile, whereas 80 and 90% of that derived from T-lymphocytes and B-lymphocytes can be β-eliminated, respectively. Sensitivity of the intact proteins or tryptic peptides to the peptide:N-glycosidase also confirms the relative amounts of cell surface, N-linked and O-linked oligosaccharides which are exogalactosylated. Composition, size, and high performance liquid chromatography on two types of high resolution columns establish that the bulk of the exogalactosylated, β-eliminated oligosaccharides are Galβ1-4GlcNAcitol. These data suggest the presence of O-glycosidically linked GlcNA monosaccharide on many lymphocyte cell-surface proteins. However, additional experiments indicate that the majority of these moieties appear to be cryptic or inside the cell. Thus, these studies not only describe dramatic differences in the amounts and distribution of terminal GlcNAc residues on phenotypically different lymphocyte populations, but they also describe the presence of a novel protein-saccharide linkage, which is present on numerous lymphocyte proteins.
|Original language||English (US)|
|Number of pages||10|
|Journal||Journal of Biological Chemistry|
|State||Published - 1984|
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