Effect of altered CH2-associated carbohydrate structure on the functional properties and in vivo fate of chimeric mouse-human immunoglobulin G1.
Immunoglobulin G (IgG) molecules are glycosylated in CH2 at Asn297; the N-linked carbohydrates attached there have been shown to contribute to antibody (Ab) stability and various effector functions. The carbohydrate attached to the IgG constant region is a complex biantennary structure. Alterations in the structure of oligosaccharide have been associated with human diseases such as rheumatoid arthritis and osteoarthritis. To study the effects of altered carbohydrate structure on Ab effector function, we have used gene transfection techniques to produce mouse-human chimeric IgG1 Abs in the Chinese hamster ovary (CHO) cell line Lec 1, which is incapable of processing the high-mannose intermediate through the terminal glycosylation steps. We also produced IgG1 Abs in Pro-5, the wild-type CHO cell line that is the parent of Lec 1. The Pro-5-produced Ab (IgG1-Pro-5) was similar to IgG1-My 1, a myeloma-produced IgG1 Ab of the same specificity, in its biologic properties such as serum half-life, ability to effect complement-mediated cytolysis, and affinity for Fc gamma RI. Although the Lec 1-produced Ab, IgG1-Lec 1, was properly assembled and retained antigen specificity, it was incapable of complement-mediated hemolysis and was substantially deficient in complement consumption, C1q binding, and C1 activation. IgG1-Lec 1 also showed reduced but significant affinity for Fc gamma R1 receptors. The in vivo half-life of IgG1-Lec 1 was shorter than that of either the myeloma- or Pro-5-produced counterpart, with more being cleared during the alpha-phase and with more rapid clearance during the beta-phase. Clearance of IgG1-Lec 1 could be inhibited by the administration of yeast-derived mannan. Thus the uptake of IgG1-Lec 1 appears to be accelerated by the presence of terminally mannosylated oligosaccharide. Therefore, certain Ab functions as well as the in vivo fate of the protein are dramatically affected by altered carbohydrate structure. Expression of Igs in cell lines with defined glycosylation mutations is shown to be a useful technique for investigating the contribution of carbohydrate structure to Ab function.
Wright
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Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024.
Morrison
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D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry*; D003167:Complement Activation; D006224:Cricetinae; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000737:chemistry / Q000502:physiology*; D007074:Immunoglobulin G / Q000737:chemistry / Q000502:physiology*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000502:physiology*; D037181:Lectins, C-Type; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000502:physiology; D017452:Receptors, IgG / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000502:physiology*; D013329:Structure-Activity Relationship
D002241:Carbohydrates; D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D037181:Lectins, C-Type; D000090323:Mannose Receptor; D037241:Mannose-Binding Lectins; D011956:Receptors, Cell Surface; D017452:Receptors, IgG; D011993:Recombinant Fusion Proteins