Studies of aglycosylated chimeric mouse-human IgG. Role of carbohydrate in the structure and effector functions mediated by the human IgG constant region.

TLDR

The study used chimeric mouse‑human IgG to investigate how carbohydrate in the CH2 domain of human IgG affects structure and function. The N‑linked carbohydrate at Asn‑297 was removed by site‑directed mutagenesis, replacing it with Gln or His for IgG1 or Lys for IgG3. Aglycosylated IgG assemble and bind antigen and protein A but are more protease‑sensitive, fail to bind FcγRI, lose or reduce C1q binding, and show altered half‑lives, demonstrating that carbohydrate in the CH2 domain is essential for maintaining structure and effector functions.

Abstract

Chimeric mouse-human IgG was used to study the structural and functional roles of the carbohydrate present in the CH2 domain of human IgG molecules. To remove this N-linked carbohydrate, Asn-297, the oligosaccharide attachment residue, was changed to either Gln (a conservative replacement) or His for IgG1 or Lys for IgG3 (nonconservative replacements) by site-directed mutagenesis. Carbohydrate-deficient antibodies are properly assembled and secreted and bind Ag and protein A. However, aglycosylated IgG are more sensitive to most proteases than their corresponding wild-type IgG, indicating some conformational changes have occurred. Aglycosylated IgG do not bind to the human Fc gamma RI and do not activate C; depending on the isotype, C1q binding ability is either completely lost (IgG1) or dramatically decreased (IgG3). The serum half-life in mice of aglycosylated IgG1-Gln remains the same as wild-type IgG1, 6.5 +/- 0.5 days, whereas aglycosylated IgG3-Gln has a shorter half-life, 3.5 +/- 0.2 days, compared to that of wild-type IgG3, 5.1 +/- 0.4 days. These results indicate the carbohydrate interposed between CH2 domain of human IgG is necessary to maintain the appropriate structure for the maintenance of many of the effector functions dependent on the CH2 domain.