title: IgG antibodies to cyclic citrullinated peptides exhibit profiles specific in terms of IgG subclasses, Fc-glycans and a fab-Peptide sequence.
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.
authors.affiliation: Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Susanna L
authors.initials: SL
authors.lastname: Lundström
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Cátia
authors.initials: C
authors.lastname: Fernandes-Cerqueira
authors.affiliation: Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: A Jimmy
authors.initials: AJ
authors.lastname: Ytterberg
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Elena
authors.initials: E
authors.lastname: Ossipova
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Aase H
authors.initials: AH
authors.lastname: Hensvold
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Per-Johan
authors.initials: PJ
authors.lastname: Jakobsson
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Vivianne
authors.initials: V
authors.lastname: Malmström
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Anca I
authors.initials: AI
authors.lastname: Catrina
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Lars
authors.initials: L
authors.lastname: Klareskog
authors.affiliation: Rheumatology Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Karin
authors.initials: K
authors.lastname: Lundberg
authors.affiliation: Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Roman A
authors.initials: RA
authors.lastname: Zubarev
mesh_terms: D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D001172:Arthritis, Rheumatoid / Q000097:blood / Q000276:immunology*; D005260:Female; D006020:Glycopeptides / Q000032:analysis / Q000097:blood / Q000276:immunology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000032:analysis / Q000097:blood / Q000276:immunology; D007074:Immunoglobulin G / Q000032:analysis / Q000097:blood / Q000276:immunology*; D008875:Middle Aged; D008969:Molecular Sequence Data; D010456:Peptides, Cyclic / Q000276:immunology*; D011134:Polysaccharides / Q000032:analysis / Q000097:blood; D013582:Synovial Fluid / Q000737:chemistry / Q000276:immunology; D055815:Young Adult
chemical_list: D006020:Glycopeptides; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D010456:Peptides, Cyclic; D011134:Polysaccharides; C487763:cyclic citrullinated peptide