Recognition of Glycine Versus Nonglycine Citrulline Motifs Dictating the HLA Class II Association of Anticitrullinated Protein Antibodies: Insights From Autoantibody Profiling of 6,900 Scandinavian Patients With Rheumatoid Arthritis

Abstract

Objective

Rheumatoid arthritis (RA)–associated anticitrullinated protein antibodies (ACPAs) target various antigens by binding short citrulline amino acid motifs, resulting in heterogeneous ACPA profiles among patients. Here we analyzed ACPA patterns by recognized citrulline motifs in relation to the RA risk factors HLA-DRB1 shared epitope (SE) alleles and history of smoking.

Methods

Rheumatoid factor (RF) and anticyclic citrullinated peptide (CCP2) isotypes, 15 anti-Cit- and four anti-Carb/Acet-peptide-IgG, were centrally measured in 6,907 patients from five Scandinavian RA cohorts using fluoroenzyme immunoassay and a custom-made multiplex solid-phase microarray. HLA-DRB1 SE alleles were imputed from single-nucleotide polymorphism genotyping data.

Results

Single-citrulline peptides derived from four multicitrulline peptides (Cit Fibα_36-50, Cit Fibβ_60-74, Cit TNC5, and Cit Vim_60-75) showed differential binding patterns, supporting recognition of citrulline motifs rather than long peptides. Four citrulline peptides (Cit Fibβ_36-52, Cit Fibβ_60-74-Cit3, Cit Fil_307-324, and Cit Vim_60-75-Cit1) captured 97% of IgG anti-CCP2+ patients. Patient subsets based on ACPA, anti-Carb/Acet, and RF displayed differences in ACPA composition and disease activity but not comorbidities. Different ACPAs overlapped, but when dichotomizing patients based on high reactivity to peptide citrulline motifs, only ACPA to nonglycine citrulline motif associated with HLA SE alleles. In IgG anti-CCP2+ patients, 90% of those with only high nonglycine ACPA were HLA SE allele carriers compared with 67% in the group with glycine motif–only ACPA (odds ratio 4.5). Smoking status associated with IgA and glycine motif ACPA.

Conclusion

Although citrulline-glycine motifs are prevalent ACPA targets, our data reveal that HLA SE alleles are primarily associated with ACPA to nonglycine citrulline motifs, providing insight in ACPA T cell dependance. Yet, the etiologic significance of ACPA targeting different protein structures remains unknown.