Profiling terminal N-acetyllactosamines of glycans on mammalian cells by an immuno-enzymatic assay |
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Authors: | Haruko Ogawa Uri Galili |
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Institution: | (1) Division of Hematology/Oncology, Department of Medicine, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA |
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Abstract: | 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 (Galβ1-(3)4GlcNAc-R) that are uncapped, capped with sialic acid as SA-Galβ1-(3)4GlcNAc-R, or with α1,3galactosyls
as the α-gal epitope- Galα1-3Galβ1-(3)4GlcNAc-R. This method includes two enzymatic reactions: (1) Terminal sialic acid is
removed by neuraminidase, and (2) α-gal epitopes are synthesized on the exposed N-acetyllactosamines by α1,3galactosyltransferase. Existing and de novo synthesized α-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 α-gal epitopes. This binding is proportional to
the number of cell surface α-gal epitopes. The amount of free M86 antibody molecules remaining in the solution is determined
by ELISA using synthetic α-gal epitopes linked to albumin as solid phase antigen. The number of α-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 × 106 α-gal epitopes/cell. We could demonstrate large variations in the number of sialic acid capped N-acetyllactosamines, α-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. |
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Keywords: | Glycoproteins α -gal epitope Carbohydrate profiling α 1 3galactosyltransferase |
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