The effect of glycosylation trimming enzyme inhibitors on monoclonal antibody recognition of alpha-sialoglycoprotein epitopes.
The human erythrocyte membrane contains four sialoglycoproteins, denoted alpha, beta, gamma and delta (also known as glycophorins A, C, D and B respectively), of which alpha-sialoglycoprotein (alpha-SGP) is the most predominant species. The extracellular portion of alpha-SGP is heavily glycosylated with approximately 15 O-linked carbohydrate side-chains and a single N-linked group. We have used inhibitors of carbohydrate trimming enzymes to investigate the contribution of this single N-glycan moiety towards the recognition of a range of antibody binding sites on alpha-SGP. Two erythromyeloid cell lines, K562 and HEL, were cultured in the presence of these inhibitors and altered binding of antibodies to epitopes adjacent to the N-glycan was observed. Digoxigenin-coupled lectins were used to stain cytocentrifuge preparations and Western blots of cell lysates in order to confirm that modification of N-linked carbohydrate side-chains had been achieved. We suggest that the N-glycan side chain of alpha-SGP has a role in conferring conformational stability upon epitopes which lie in its vicinity.
Strickland
A
A
University Department of Pathology, General Hospital, Southampton, U.K.
Wilkins
B S
BS
Jones
D B
DB
D017485:1-Deoxynojirimycin / Q000494:pharmacology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions / Q000187:drug effects*; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D004076:Digoxigenin; D000939:Epitopes / Q000187:drug effects / Q000276:immunology*; D006021:Glycophorins / Q000187:drug effects / Q000276:immunology*; D065089:Glycoside Hydrolase Inhibitors; D006031:Glycosylation; D006801:Humans; D007212:Indolizines / Q000494:pharmacology*; D037102:Lectins; D008361:Mannosidases / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D017026:Swainsonine / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D000520:alpha-Glucosidases; D043323:alpha-Mannosidase; D054836:Indolizidines
D017485:1-Deoxynojirimycin; D000911:Antibodies, Monoclonal; D004076:Digoxigenin; D000939:Epitopes; D006021:Glycophorins; D065089:Glycoside Hydrolase Inhibitors; D007212:Indolizines; D037102:Lectins; D008361:Mannosidases; D017026:Swainsonine; D000520:alpha-Glucosidases; D043323:alpha-Mannosidase; C042059:glucosidase I; C037806:castanospermine; D054836:Indolizidines