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Crystal structure of a human autoimmune complex between IgM rheumatoid factor RF61 and IgG1 Fc reveals a novel epitope and evidence for affinity maturation.

ABSTRACT: Rheumatoid factors (RF) are autoantibodies that recognize epitopes in the Fc region of immunoglobulin (Ig) G and that correlate with the clinical severity of rheumatoid arthritis (RA). Here we report the X-ray crystallographic structure, at 3 A resolution, of a complex between the Fc region of human IgG1 and the Fab fragment of a monoclonal IgM RF (RF61), derived from an RA patient and with a relatively high affinity for IgG Fc. In the complex, two Fab fragments bind to each Fc at epitopes close to the C terminus, and each epitope comprises residues from both Cgamma3 domains. A central role in the unusually hydrophilic epitope is played by the side-chain of Arg355, accounting for the subclass specificity of RF61, which recognizes IgG1,-2, and -3 in preference to IgG4, in which the corresponding residue is Gln355. Compared with a previously determined complex of a lower affinity RF (RF-AN) bound to IgG4 Fc, in which only residues at the very edge of the antibody combining site were involved in binding, the epitope bound by RF61 is centered in classic fashion on the axis of the V(H):V(L) beta-barrel. The complementarity determining region-H3 loop plays a key role, forming a pocket in which Arg355 is bound by two salt-bridges. The antibody contacts also involve two somatically mutated V(H) residues, reinforcing the suggestion of a process of antigen-driven maturation and selection for IgG Fc during the generation of this RF autoantibody.

SUBMITTER: Duquerroy S

PROVIDER: S-EPMC4625532 | biostudies-literature | 2007 May

REPOSITORIES: biostudies-literature

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Crystal structure of a human autoimmune complex between IgM rheumatoid factor RF61 and IgG1 Fc reveals a novel epitope and evidence for affinity maturation.

Duquerroy Stephane S Stura Enrico A EA Bressanelli Stéphane S Fabiane Stella M SM Vaney Marie C MC Beale Dennis D Hamon Maureen M Casali Paolo P Rey Felix A FA Sutton Brian J BJ Taussig Michael J MJ

Journal of molecular biology 20070306 5

Rheumatoid factors (RF) are autoantibodies that recognize epitopes in the Fc region of immunoglobulin (Ig) G and that correlate with the clinical severity of rheumatoid arthritis (RA). Here we report the X-ray crystallographic structure, at 3 A resolution, of a complex between the Fc region of human IgG1 and the Fab fragment of a monoclonal IgM RF (RF61), derived from an RA patient and with a relatively high affinity for IgG Fc. In the complex, two Fab fragments bind to each Fc at epitopes close ...[more]

PMID: 17395205

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Project description:ObjectiveRheumatoid arthritis (RA) is characterized by the presence of anti-citrullinated protein antibodies (ACPAs); nevertheless, the origin, specificity, and functional properties of ACPAs remain poorly understood. The aim of this study was to characterize the evolution of ACPAs by sequencing the plasmablast antibody repertoire at serial time points in patients with established RA.MethodsBlood samples were obtained at up to 4 serial time points from 8 individuals with established RA who were positive for ACPAs by the anti-cyclic citrullinated peptide test. CD19+CD3-IgD-CD14-CD20-CD27+CD38++ plasmablasts were isolated by single-cell sorting and costained with citrullinated peptide tetramers to identify ACPA-expressing plasmablasts. Cell-specific oligonucleotide barcodes were utilized, followed by large-scale sequencing and bioinformatics analysis, to obtain error-corrected, paired heavy- and light-chain antibody gene sequences for each B cell.ResultsBioinformatics analysis revealed 170 persistent plasmablast lineages in the RA blood, of which 19% included multiple isotypes. Among IgG- and IgA-expressing plasmablasts, significantly more IgA-expressing than IgG-expressing persistent lineages were observed (P < 0.01). Shared complementarity-determining region 3 sequence motifs were identified across subjects. A subset of the plasmablast lineages included members derived from later time points with divergent somatic hypermutations that encoded antibodies that bind an expanded set of citrullinated antigens. Furthermore, these recombinant, differentially mutated plasmablast antibodies formed immune complexes that stimulated higher macrophage production of tumor necrosis factor (TNF) compared to antibodies representing earlier time point-derived lineage members that were less mutated.ConclusionThese findings demonstrate that established RA is characterized by a persistent IgA ACPA response that exhibits ongoing affinity maturation. This observation suggests the presence of a persistent mucosal antigen that continually promotes the production of IgA plasmablasts and their affinity maturation and epitope spreading, thus leading to the generation of ACPAs that bind additional citrullinated antigens and more potently stimulate macrophage production of TNF.

Project description:To analyze the structure and formally ascertain the B-1a cellular origin of IgM rheumatoid factor (RF) autoantibodies, we generated 4 IgM RF mAb-producing cell lines using sorted (surface CD5+) B-1a cells from a patient with active rheumatoid arthritis. The RF mAb111, mAb112, mAb113, and mAb114 were monoreactive and displayed a relatively high affinity for human IgG Fc fragment (Kd, 3.1 x 10(-7) to 6.8 x 10(-7) M). The B-1a origin of the lymphocytes that gave rise to the IgM RF was confirmed by the expression of surface CD5 and specific CD5 mRNA by all mAb-producing cell lines. Analysis of the genes encoding the RF mAb VH and VL regions revealed that members of the VHI and VHIII families were utilized in conjunction with V kappa IIIa, V kappa IIIb, or V lambda I genes. JH3 and JH4 genes were each utilized twice. The H chain CDR3 sequences were divergent and variable in length. The RF mAb VH genes were identical or closely related to those expressed in the "restricted" fetal B cell repertoire and/or were JH-proximal. For instance, mAb111 VH gene likely constituted a mutated variant of the expressed fetal 20P3 which is the second most JH-proximal gene (125 kb from JH). In addition, the expressed VH and VL genes were among those that have been found to encode other RF, different autoantibodies, high affinity antibodies induced by exogenous Ag, and natural autoantibodies in the adult and neonatal B cell repertoires. When compared with those of known germline genes, the expressed V gene sequences displayed a number of differences. By cloning and sequencing DNA from PMN of the same patient whose B lymphocytes were used for the mAb generation, we showed that such differences resulted from somatic hypermutation in the RF mAb112 VH gene. The germline gene (112GL) that presumably gave rise to the RF mAb112 VH segment was identical to the expressed fetal 51P1 gene. The distribution and the high replacement to silent mutation ratio of the nucleotide mutations in RF mAb112 VH segment were highly consistent with their selection by Ag. RF mAb113 was clonally related to RF mAb112, as shown by the utilization of the same sets of VHI-D-JH4 and V kappa IIIb-J kappa 4 genes, displaying identical junctional sequences, and the presence of two identical replacement and one silent mutations.(ABSTRACT TRUNCATED AT 400 WORDS)

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Crystal structure of a human autoimmune complex between IgM rheumatoid factor RF61 and IgG1 Fc reveals a novel epitope and evidence for affinity maturation.

Publications

Crystal structure of a human autoimmune complex between IgM rheumatoid factor RF61 and IgG1 Fc reveals a novel epitope and evidence for affinity maturation.

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