Structural Difference in the Complement Activation Site of Human IgG1 and IgG3

Abstract

The C1q binding epicentre on IgG molecules involves residues Asp270, Lys322, Pro329 and Pro331 in the CH2 domain. IgG1 and IgG3 are usually the most efficient of the four human IgG subclasses in activating complement and they both share all these residues. To reveal possible differences in the structural requirement for complement activation, we created a number of NIP (5‐iodo‐4‐hydroxy‐3‐nitro‐phenacetyl) specific IgG1 and IgG3 antibodies with parallel mutations in or near the putative C1q binding site. The mutants were tested simultaneously for antibody induced, antibody‐dependent complement‐mediated lysis (ADCML) at high and low antigen concentration on the target cells using sera of human, rabbit and guinea pig as complement source. In addition, we tested the antibodies against target cells decorated with the NP hapten, which has 10‐fold lower affinity for the antibodies compared to the NIP hapten. We also used ELISA methods to measure complement activation. We observed a clear difference between IgG1 and IgG3 localized to residues Asp270, Leu334, Leu335. For all these residues, and especially for Asp270, IgG1 was heavily reduced in complement activation, while IgG3 was only moderated reduced, by alanine substitution. This difference was independent of the long hinge region of IgG3, demonstrated by hinge region truncation of this isotype such that it resembles that of IgG1. This report indicates the presence of structural differences between human IgG1 and IgG3 in the C1q binding site, and points to a specialization of the two isotypes with respect to complement activation.

Combined Roles of Human IgG Subclass, Alternative Complement Pathway Activation, and Epitope Density in the Bactericidal Activity of Antibodies to Meningococcal Factor H Binding Protein

“…Human IgG3 has an extended hinge region (63 amino acids, as opposed to 15 amino acids for IgG1) (26), which might allow for greater flexibility of the Fc portion of the IgG3 molecule and more efficient engagement of C1q when fHbp is sparsely exposed. This hypothesis, however, is not supported by studies of mutant human IgG3 molecules with truncated hinge regions where the greater ability of IgG3 than IgG1 to activate complement-mediated lysis of red cells with low hapten density was independent of the hinge region (29). Based upon data from mutant IgG1 and IgG3 molecules with substitutions of single amino acid residues contributing to C1q binding (29), subtle structural differences in the CH2 domain appear to be responsible for the greater ability of IgG3 to engage C1q and activate the classical complement pathway when the target antigen is sparse.…”

Human IgG1, IgG3, and IgG3 Hinge-Truncated Mutants Show Different Protection Capabilities against Meningococci Depending on the Target Antigen and Epitope Specificity

“…However, IgG1 is much more dependent on Asp270 than IgG3, and thus, the IgG3D270A mutant (which carries a mutation from aspartic acid to alanine at position 270) maintains most of the complement activation activity, while the IgG1D270A mutant is devoid of complement activation activity, and this difference is independent of the long hinge region of IgG3 (57).…”

“…Human IgG1 and IgG3 share the amino acid residues that make up the epicenter of the complement activation site on the Fc part of the molecules (Asp270, Lys322, Pro329, and Pro331), but parallel mutations in other common surrounding and shared amino acids in IgG1 and IgG3 indicate that there are subtle differences in the complement activation site of human IgG1 and IgG3 (57). However, IgG1 is much more dependent on Asp270 than IgG3, and thus, the IgG3D270A mutant (which carries a mutation from aspartic acid to alanine at position 270) maintains most of the complement activation activity, while the IgG1D270A mutant is devoid of complement activation activity, and this difference is independent of the long hinge region of IgG3 (57).…”

Fc Engineering of Human IgG1 for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions

“…7D, 7E). As controls, we included hIgG1 with a single point mutation (P329A) within the C H 2 domain at the core of the C1q binding site (47), which completely eliminated hC1q binding, as well as hIgG1 with mutation of either of two leucine residues in the lower hinge (L234A and L235A), crucial for FcgR binding (54), which moderately reduced binding (Supplemental Fig. 3E-H).…”

“…The vector contains the constant H chain gene from hIgG1 with specificity for the hapten 5-iodo-4-hydroxy-3-nitro-phenacetyl (NIP), and it was used as a template for subcloning of DNA fragments encoding (synthesized by GenScript) a panel of Fc mutants using the restriction sites AgeI and SfiI (C H 2 mutations) or SfiI and BamHI (C H 3 mutations): M252Y/ S254T/T256E (MST), M428L/N434S (MN), H433K/N434F (HN), and M252Y/S254T/T256E/H433K/N434F (MST/HN). Vectors encoding the constant H chain gene from hIgG1 with the mutations P329A, L234A, and L235A have previously been described (25,47). The Abs were produced using HEK293E cells by transient cotransfection of the vectors together with a vector encoding the mouse l L chain with NIP specificity (pLNOH2-NIPlLC-oriP) using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions.…”