Sialylation of human IgG-Fc carbohydrate by transfected rat alpha2,6-sialyltransferase.
A recombinant IgG3 antibody with Phe-243 replaced by Ala (FA243) was expressed in a CHO-K1 parental cell line. The resulting IgG-Fc-linked carbohydrate was significantly alpha2,3-sialylated (53% of glycans), as indicated by normal- and reverse-phase HPLC analyses. Following transfection of a rat alpha2,6-sialyltransferase gene into this parental cell line, IgG-Fc-linked glycans were sialylated (60% of glycans) such that the ratio of alpha2,6- to alpha2,3-linked sialic acid was 0.9:1.0. By comparison, the wild-type IgG3 (F243) is minimally sialylated (2-3% alpha2,3-linked), thus suggesting that sialylation is controlled primarily by the protein structure local to the carbohydrate and that the two sialyltransferases compete to sialylate the nascent oligosaccharide. The additional alpha2,6-sialylation affected the function of the recombinant antibody. FA243 IgG3 having both alpha2,6 and alpha2,3-sialylation restored recognition to wild-type IgG3 levels for human FcgammaRI, FcgammaRII, and target cell lysis by complement. We discuss how sialylation linkage could modulate IgG function.
Jassal
R
R
Department of Biological Sciences, De Montfort University, Leicester, LE1 9BH, United Kingdom.
Jenkins
N
N
Charlwood
J
J
Camilleri
P
P
Jefferis
R
R
Lund
J
J
D000818:Animals; D016466:CHO Cells; D002241:Carbohydrates / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D003167:Complement Activation; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D020014:K562 Cells; D009154:Mutation; D019158:N-Acetylneuraminic Acid / Q000032:analysis; D009592:Nitrohydroxyiodophenylacetate / Q000276:immunology; D009844:Oligosaccharides / Q000032:analysis; D051381:Rats; D012799:Sialyltransferases / Q000235:genetics*; D013481:Superoxides / Q000378:metabolism; D014162:Transfection; D020298:U937 Cells; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D002241:Carbohydrates; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D009844:Oligosaccharides; D013481:Superoxides; D009592:Nitrohydroxyiodophenylacetate; D012799:Sialyltransferases; D019158:N-Acetylneuraminic Acid; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase