Identification of the rheumatoid arthritis shared epitope binding site on calreticulin

PLoS One. 2010 Jul 22;5(7):e11703. doi: 10.1371/journal.pone.0011703.

Abstract

Background: The 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-DRbeta 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 findings: Surface 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 Glu(217) and Glu(223)--and to a lesser extent residue Asp(220)--in cell-free SPR-based binding and signal transduction assays.

Significance: We 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arthritis, Rheumatoid / immunology*
  • Binding Sites
  • Calreticulin / chemistry*
  • Calreticulin / metabolism*
  • Cells, Cultured
  • Circular Dichroism
  • Computer Simulation
  • Epitopes / metabolism*
  • Immunity, Innate / genetics
  • Immunity, Innate / physiology
  • Mice
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Protein Structure, Secondary
  • Signal Transduction
  • Surface Plasmon Resonance

Substances

  • Calreticulin
  • Epitopes