Structural Characterization of Anti-Inflammatory Immunoglobulin G Fc Proteins

Abstract

Immunoglobulin G (IgG) is a central mediator of host defense due to its ability to recognize and eliminate pathogens. The recognition and effector responses are encoded on distinct regions of IgGs. The diversity of the antigen recognition Fab domains accounts for IgG's ability to bind with high specificity to essentially any antigen. Recent studies have indicated that the Fc effector domain also displays considerable heterogeneity, accounting for its complex effector functions of inflammation, modulation and immune suppression. Therapeutic anti-tumor antibodies, for example, require the pro-inflammatory properties of the IgG Fc to eliminate tumor cells, while the anti-inflammatory activity of Intravenous Immunoglobulin G (IVIG) requires specific Fc glycans for activity. In particular, the anti-inflammatory activity of IVIG is ascribed to a small population of IgGs in which the Asn297-linked complex N-glycans attached to each Fc CH2 domain include terminal α2,6-linked sialic acids. We used chemoenzymatic glycoengineering to prepare fully di-sialylated IgG Fc and solved its crystal structure. Comparison of the structures of asialylated Fc, sialylated Fc, and F241A Fc, a mutant that displays increased glycan sialylation, suggests that increased conformational flexibility of the CH2 domain is associated with the switch from pro- to anti-inflammatory activity of the Fc.

Protection in antibody- and T cell-mediated autoimmune diseases by antiinflammatory IgG Fcs requires type II FcRs

“…This switch in receptor specificity coincides with a switch in effector function in vivo, as sialylated IgG suppresses inflammation in mouse models of autoimmunity. Recent structural data support an earlier computational model that sialylation increases the conformational flexibility of the C H 2 domain, sampling conformations compatible with type II FcR binding (8,20,36).…”

“…A complex, N-linked glycan attached to the C H 2 domain of IgG Fc regulates the interaction with these classes of FcRs by modulating the structure of the Fc to alternate between type I and type II binding conformations (8,11,20). The presence of an α2,6-linked sialic acid in this Fc-associated glycan results in reduced affinity for type I FcRs and enhanced binding to type II FcRs, such as DC-SIGN and CD23 (8,11,15).…”

“…modification, may potentially simplify the development of antiinflammatory IgG Fc for therapeutic use (20). We succeeded in identifying a mutation (F241A) predicted to increase mobility of the α1,3-arm, and which replicates the antiinflammatory activity of sialylated Fc even in the absence of sialylation.…”

“…Furthermore, both sFc as well as F241A stimulate the production of IL-33 that in turn activates T reg cells through the ST2 receptor, contributing to the suppression of T cell-mediated autoimmune responses. (20) enabling it to bind to type II Fc receptors (11). As shown in Fig.…”

“…Sialylation of the complex, biantennary glycan of the IgG Fc results in increased conformational flexibility of the C H 2 domain (20), thereby sampling the closed conformations of the C H 2 domain required for type II FcR binding (11). In contrast, asialylated Fc structures uniformly result in open Fc conformations, consistent with their binding specificity for type I FcRs (21).…”