The polypeptide of immunoglobulin G influences its galactosylation in vivo.
To examine the nature of the factors influencing the galactosylation pattern of the heavy chain of murine immunoglobulin G (IgG), cell fusion was performed between a myeloma (P3x63Ag8) and a hybridoma (Sp2HL/Bu) cell line which secrete different IgGs possessing structurally distinct CH2-linked oligosaccharide moieties. The glycosylation patterns of the IgGs of the parental and fused cells were studied. Pronase digestion of the purified heavy chains and subsequent end labeling with fluorescein isothiocyanate produced fluoresceinated glycopeptides which were detected and purified by polyacrylamide gel electrophoresis. Structural information was obtained by enzymatic digestion, lectin affinity chromatography, and methylation analysis. IgGs from both parental lines possessed oligosaccharide units displaying microheterogeneity based upon a common symmetrical biantennary structure terminating in beta-GlcNAc. The structures of both IgGs, however, differed in the pattern of the mono- and digalactosylated components. Clones, selected following the fusion of the parental cells, were expanded; and the individual IgGs were purified. All clones produced homodimeric IgG1 and IgG2b as well as heterodimeric IgG possessing both the gamma 1 and gamma 2b heavy chains. Analysis of the carbohydrate moieties of the gamma 1 chain from the homodimeric and heterodimeric IgGs and of the gamma 2b chain from the heterodimeric molecule demonstrates that the polypeptide structure of the heavy chain influences the terminal galactosylation of the glycan unit at the conserved site of glycosylation of IgGs.
Lee
S O
SO
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855-1059.
Connolly
J M
JM
Ramirez-Soto
D
D
Poretz
R D
RD
D000118:Acetylglucosaminidase / Q000378:metabolism; D000818:Animals; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D005690:Galactose / Q000378:metabolism*; D006020:Glycopeptides / Q000302:isolation & purification / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006825:Hybridomas / Q000276:immunology; D007074:Immunoglobulin G / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism*; D007142:Immunoglobulin gamma-Chains / Q000032:analysis / Q000378:metabolism; D046911:Macromolecular Substances; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D011402:Pronase / Q000378:metabolism; D014407:Tumor Cells, Cultured
D006020:Glycopeptides; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007142:Immunoglobulin gamma-Chains; D046911:Macromolecular Substances; D006026:Glycoside Hydrolases; D000118:Acetylglucosaminidase; D011402:Pronase; D005690:Galactose