Effect of C2-associated carbohydrate structure on Ig effector function: studies with chimeric mouse-human IgG1 antibodies in glycosylation mutants of Chinese hamster ovary cells.
The complex biantennary oligosaccharide at Asn297 of IgG is essential for some effector functions. To investigate the effect of carbohydrate structure on Ab function, we have now expressed mouse-human chimeric IgG1 Abs in Chinese hamster ovary (CHO) cells with defined defects in carbohydrate biosynthesis. We had previously shown that IgG1 Abs produced in the cell line Lec 1, which attaches a high-mannose intermediate carbohydrate, were severely deficient in complement activation, showed a slightly reduced affinity for Fc gammaRI, and had a reduced in vivo half-life. We have extended these studies by producing the same dansyl-specific IgG1 in cell lines deficient in attachment of sialic acid (Lec 2) and galactose (Lec 8). IgG1-Lec 1, IgG1-Lec 2, and IgG1-Lec 8 all showed varying reactivity with a mAb specific for an epitope in the amino terminal region of C(H)2, suggesting that the conformations of these proteins were altered by the different carbohydrate structures. Functionally, IgG1-Lec 2 and IgG1-Lec 8 were comparable to wild type with respect to in vivo half-life, affinity for Fc gammaRI, and capacity for complement-mediated hemolysis. While IgG1-Lec 2 was essentially identical to wild type in its capacity to interact with individual components of the classical complement activation pathway, IgG1-Lec 8 demonstrated equivalent maximal binding at lower concentrations and was preferentially bound by mannose-binding protein. Although IgG1-Lec 1 was deficient in activation of the classical pathway, it had a superior capacity to activate the alternative pathway. These studies demonstrate that Abs bearing C(H)2-linked carbohydrate of differing structures have different functional properties.
Wright
A
A
Department of Microbiology and Molecular Genetics, Molecular Biology Institute, University of California, Los Angeles 90095, USA.
Morrison
S L
SL
D000818:Animals; D001666:Binding Sites, Antibody / Q000235:genetics; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002352:Carrier Proteins / Q000378:metabolism; D003170:Complement Pathway, Alternative; D003171:Complement Pathway, Classical; D003165:Complement System Proteins / Q000378:metabolism; D006224:Cricetinae; D003602:Cytotoxicity, Immunologic / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005822:Genetic Vectors / Q000276:immunology / Q000378:metabolism; D006031:Glycosylation; D006207:Half-Life; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000235:genetics / Q000378:metabolism*; D007074:Immunoglobulin G / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism*; D007132:Immunoglobulin Isotypes / Q000378:metabolism; D007274:Injections, Intraperitoneal; D037102:Lectins / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D037241:Mannose-Binding Lectins; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017452:Receptors, IgG / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000276:immunology* / Q000378:metabolism / Q000493:pharmacokinetics; D014162:Transfection
D002352:Carrier Proteins; D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D007132:Immunoglobulin Isotypes; D037102:Lectins; D037241:Mannose-Binding Lectins; D009844:Oligosaccharides; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins; D003165:Complement System Proteins; D008358:Mannose