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

• Published in: PloS one 2014-11, Vol.9 (11), p.e113924-e113924
• Main Authors: Lundström, Susanna L, Fernandes-Cerqueira, Cátia, Ytterberg, A Jimmy, Ossipova, Elena, Hensvold, Aase H, Jakobsson, Per-Johan, Malmström, Vivianne, Catrina, Anca I, Klareskog, Lars, Lundberg, Karin, Zubarev, Roman A
• Format: Article
• Language: English
• Subjects: Abundance; Adult; Aged; Amino Acid Sequence; Antibodies; Antigens; Arthritis; Arthritis, Rheumatoid - blood; Arthritis, Rheumatoid - immunology; Biochemistry; Biology and Life Sciences; Biophysics; Citrulline; Exhibitions; Fc receptors; Female; Glycan; Glycopeptides; Glycopeptides - analysis; Glycopeptides - blood; Glycopeptides - immunology; Glycosylation; Humans; Immunoglobulin Fragments - analysis; Immunoglobulin Fragments - blood; Immunoglobulin Fragments - immunology; Immunoglobulin G; Immunoglobulin G - analysis; Immunoglobulin G - blood; Immunoglobulin G - immunology; Immunoglobulins; Immunologic factors; In vivo methods and tests; Isotypes; Medicin och hälsovetenskap; Medicine; Medicine and Health Sciences; Middle Aged; Molecular Sequence Data; Multivariate analysis; Pathogenesis; Patients; Peptides; Peptides, Cyclic - immunology; Physical Sciences; Physiology; Polysaccharides; Polysaccharides - analysis; Polysaccharides - blood; Protein A; Protein G; Protein purification; Proteins; Proteomics; Purification; Research and Analysis Methods; Rheumatoid arthritis; Rheumatology; Synovial fluid; Synovial Fluid - chemistry; Synovial Fluid - immunology; Young Adult
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0113924

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.