Specific cell-surface labeling of polyglycosyl chains in human erythrocytes and HL-60 cells using endo-beta-galactosidase and galactosyltransferase.
In order to identify the molecule components carrying polyglycosyl chains on cell surfaces a two-step enzymatic method was developed. In the first step, the cells were incubated with endo-beta-galactosidase to selectively expose terminal N-acetylglucosamine residues of the lactosamine backbone to the chains. In the second step these residues were glycosylated by incubation with galactosyltransferase and radioactive UDP-galactose. As many as 2.5-3.0 X 10(6) residues per cell could be transferred to human erythrocytes. Negligible amounts of labeling occurred if either of the enzymes was omitted from the incubations. Of the label 80% was found in glycoproteins. In accordance with previous observations, bands 3 and 4.5 were found to be the main carriers of polyglycosyl chains. In human promyelotic HL-60 leukemia cells, a major band of apparent molecular weight of 110000-140000 was labeled. In addition, bands of lower molecular weight which appear to have escaped detection by previous methods were also labeled. The novel labeling method was found to be simple to perform, uses commercially available reagents, and leads to the efficient and highly specific labeling of cell surface molecules carrying polyglycosyl chains.
D002460:Cell Line; D004910:Erythrocyte Membrane / Q000032:analysis*; D005696:Galactosidases; D005700:Galactosyltransferases; D006017:Glycolipids / Q000097:blood*; D006023:Glycoproteins / Q000097:blood*; D006026:Glycoside Hydrolases; D006801:Humans; D015470:Leukemia, Myeloid, Acute; D011134:Polysaccharides / Q000097:blood*; D001616:beta-Galactosidase
D006017:Glycolipids; D006023:Glycoproteins; D011134:Polysaccharides; D005700:Galactosyltransferases; D005696:Galactosidases; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D001616:beta-Galactosidase