Journal
PLOS ONE
Volume 5, Issue 7, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0011703
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Categories
Funding
- US National Institutes of Health [AR55170, UL1RR02498, AI47331, GM088560, AR20557, AR56786, AR48310]
- University of Michigan Global Reach Collaborative Research
- Arthritis Foundation
- American College of Rheumatology Research and Education Foundation
- Canadian Institutes of Health Research [MOP-15291]
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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-DR beta 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.
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