Identification of the Rheumatoid Arthritis Shared Epitope Binding Site on Calreticulin

Abstract

BackgroundThe rheumatoid arthritis (RA) shared epitope (SE), a major risk factor for severe disease, is a five amino acid motif in the third allelic hypervariable region of the HLA-DRβ chain. The molecular mechanisms by which the SE affects susceptibility to – and severity of - RA are unknown. We have recently demonstrated that the SE acts as a ligand that interacts with cell surface calreticulin (CRT) and activates innate immune signaling. In order to better understand the molecular basis of SE-RA association, here we have undertaken to map the SE binding site on CRT.Principal FindingsSurface plasmon resonance (SPR) experiments with domain deletion mutants suggested that the SE binding site is located in the P-domain of CRT. The role of this domain as a SE-binding region was further confirmed by a sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azido-benzamido) hexanoamido] ethyl-1,3-dithiopropionate (sulfo-SBED) photoactive cross-linking method. In silico analysis of docking interactions between a conformationally intact SE ligand and the CRT P-domain predicted the region within amino acid residues 217–224 as a potential SE binding site. Site-directed mutagenesis demonstrated involvement of residues Glu217 and Glu223 - and to a lesser extent residue Asp220 - in cell-free SPR-based binding and signal transduction assays.SignificanceWe have characterized here the molecular basis of a novel ligand-receptor interaction between the SE and CRT. The interaction represents a structurally and functionally well-defined example of cross talk between the adaptive and innate immune systems that could advance our understanding of the pathogenesis of autoimmunity.

Amblyomma americanum tick calreticulin binds C1q but does not inhibit activation of the classical complement cascade

“…The expectation would be that an antigen that is highly homologous to the vertebrate protein might be a poor antigen in that it might be recognized as self and thus not stimulate a robust antibody response as is observed with tick and other parasite CRT proteins.. It is also notable that human CRT acts as an auto-antigen in autoimmune diseases such as rheumatoid arthritis, complete congenital heart block, and coeliac disease (Holoshitz et al, 2010, Ling et al, 2010). In mice, cross-reactivity of antibodies to T. cruzi CRT with the mouse CRT protein was advanced as a possible explanation for the observed chagasic autoimmunity (Teixeira et al, 2011, 2012, Machado et al, 2012).…”

MHC molecules in health and disease: At the cusp of a paradigm shift

“…40 The SE binding site on CRT has been recently mapped to a well-defined region in the P-domain of the molecule. 25 Directly relevant to the MHC Cusp theory, our recent data show that the SE ligand activates an important immune regulatory function through its effect on dendritic cells (DCs). DCs have a central role in immune tolerance and T cell regulation.…”

Spontaneous destructive periodontitis and skeletal bone damage in transgenic mice carrying a human shared epitope-codingHLA-DRB1allele

“…14 We have recently identified the SE as a ligand that binds to cell surface calreticulin and activates pro-osteoclastogenic signalling. [15][16][17][18][19][20][21][22][23][24][25][26][27][28] Using cell-free synthetic ligands expressing the SE motif, we have demonstrated that the SE enhanced Th17 polarisation 24 and directly activated OC differentiation. 26 When administered in vivo to mice with collagen-induced arthritis, the SE ligand enhanced joint inflammation, increased the abundance of tissue and bone marrow OCs, and enhanced bone damage in arthritic joints.…”