Profiling terminal N-acetyllactosamines of glycans on mammalian cells by an immuno-enzymatic assay.
Profiling of carbohydrate structures on cell membranes has been difficult to perform because of the complexity and the variations of such structures on cell surface glycans. This study presents a novel method for rapid profiling of cell surface glycans for terminal N-acetyllactosamines (Galbeta1-(3)4GlcNAc-R) that are uncapped, capped with sialic acid as SA-Galbeta1-(3)4GlcNAc-R, or with alpha1,3galactosyls as the alpha-gal epitope- Galalpha1-3Galbeta1-(3)4GlcNAc-R. This method includes two enzymatic reactions: (1) Terminal sialic acid is removed by neuraminidase, and (2) alpha-gal epitopes are synthesized on the exposed N-acetyllactosamines by alpha1,3galactosyltransferase. Existing and de novo synthesized alpha-gal epitopes on cells are quantified by a modification of radioimmunoassay designated as "ELISA inhibition assay," which measures binding of the monoclonal anti-Gal antibody M86 to alpha-gal epitopes. This binding is proportional to the number of cell surface alpha-gal epitopes. The amount of free M86 antibody molecules remaining in the solution is determined by ELISA using synthetic alpha-gal epitopes linked to albumin as solid phase antigen. The number of alpha-gal epitopes on cells is estimated by comparing binding curves of M86 incubated with the assayed cells, at various concentrations of the cells, with the binding of M86 to rabbit red cells expressing 2 x 10(6) alpha-gal epitopes/cell. We could demonstrate large variations in the number of sialic acid capped N-acetyllactosamines, alpha-gal epitopes and uncapped N-acetyllactosamines on different mammalian red blood cells, and on nucleated cells originating from a given tissue in various species. This method may be useful for rapid identification of changes in glycosylation patterns in cells subjected to various treatments, or in various states of differentiation.
Ogawa
Haruko
H
Division of Hematology/Oncology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Galili
Uri
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D000818:Animals; D019556:COS Cells; D002240:Carbohydrate Sequence; D002417:Cattle; D002460:Cell Line; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000737:chemistry; D005347:Fibroblasts / Q000166:cytology; D006367:HeLa Cells; D006801:Humans; D020014:K562 Cells; D016393:Lymphoma, B-Cell / Q000473:pathology; D008297:Male; D008545:Melanoma / Q000473:pathology; D008546:Melanoma, Experimental / Q000473:pathology; D051379:Mice; D041681:NIH 3T3 Cells; D010190:Pancreatic Neoplasms / Q000473:pathology; D011134:Polysaccharides / Q000737:chemistry*; D011817:Rabbits; D012867:Skin / Q000166:cytology; D013552:Swine
D011134:Polysaccharides