IgG antibodies to cyclic citrullinated peptides exhibit profiles specific in terms of IgG subclasses, Fc-glycans and a fab-Peptide sequence

PLoS One. 2014 Nov 26;9(11):e113924. doi: 10.1371/journal.pone.0113924. eCollection 2014.

Abstract

The Fc-glycan profile of IgG1 anti-citrullinated peptide antibodies (ACPA) in rheumatoid arthritis (RA) patients has recently been reported to be different from non-ACPA IgG1, a phenomenon which likely plays a role in RA pathogenesis. Herein we investigate the Fc-glycosylation pattern of all ACPA-IgG isotypes and simultaneously investigate in detail the IgG protein-chain sequence repertoire. IgG from serum or plasma (S/P, n = 14) and synovial fluid (SF, n = 4) from 18 ACPA-positive RA-patients was enriched using Protein G columns followed by ACPA-purification on cyclic citrullinated peptide-2 (CCP2)-coupled columns. Paired ACPA (anti-CCP2 eluted IgG) and IgG flow through (FT) fractions were analyzed by LC-MS/MS-proteomics. IgG peptides, isotypes and corresponding Fc-glycopeptides were quantified and interrogated using uni- and multivariate statistics. The Fc-glycans from the IgG4 peptide EEQFNSTYR was validated using protein A column purification. Relative to FT-IgG4, the ACPA-IgG4 Fc-glycan-profile contained lower amounts (p = 0.002) of the agalacto and asialylated core-fucosylated biantennary form (FA2) and higher content (p = 0.001) of sialylated glycans. Novel differences in the Fc-glycan-profile of ACPA-IgG1 compared to FT-IgG1 were observed in the distribution of bisected forms (n = 5, p = 0.0001, decrease) and mono-antennnary forms (n = 3, p = 0.02, increase). Our study also confirmed higher abundance of FA2 (p = 0.002) and lower abundance of afucosylated forms (n = 4, p = 0.001) in ACPA-IgG1 relative to FT-IgG1 as well as lower content of IgG2 (p = 0.0000001) and elevated content of IgG4 (p = 0.004) in ACPA compared to FT. One λ-variable peptide sequence was significantly increased in ACPA (p = 0.0001). In conclusion, the Fc-glycan profile of both ACPA-IgG1 and ACPA-IgG4 are distinct. Given that IgG1 and IgG4 have different Fc-receptor and complement binding affinities, this phenomenon likely affects ACPA effector- and immune-regulatory functions in an IgG isotype-specific manner. These findings further highlight the importance of antibody characterization in relation to functional in vivo and in vitro studies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Amino Acid Sequence
  • Arthritis, Rheumatoid / blood
  • Arthritis, Rheumatoid / immunology*
  • Female
  • Glycopeptides / analysis
  • Glycopeptides / blood
  • Glycopeptides / immunology
  • Humans
  • Immunoglobulin Fragments / analysis
  • Immunoglobulin Fragments / blood
  • Immunoglobulin Fragments / immunology
  • Immunoglobulin G / analysis
  • Immunoglobulin G / blood
  • Immunoglobulin G / immunology*
  • Middle Aged
  • Molecular Sequence Data
  • Peptides, Cyclic / immunology*
  • Polysaccharides / analysis
  • Polysaccharides / blood
  • Synovial Fluid / chemistry
  • Synovial Fluid / immunology
  • Young Adult

Substances

  • Glycopeptides
  • Immunoglobulin Fragments
  • Immunoglobulin G
  • Peptides, Cyclic
  • Polysaccharides
  • cyclic citrullinated peptide

Associated data

  • GENBANK/P01857
  • GENBANK/P01859
  • GENBANK/P01860
  • GENBANK/P01861

Grants and funding

This study was supported by funds from the Swedish Strategic Research Funds (SSF), from the Swedish Research Council (VR), from the European Research Council (ERC), the EU projects Gums&Joints (contract No. 261460) and Trigger (contract No. 306029) as well as Innovative Medicine Initiative BTCure (115142–2) and EU project FP7-HEALTH-2012-INNOVATION-1 Euro-TEAM (305549–2). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.