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Identification of the Rheumatoid Arthritis Shared Epitope Binding Site on Calreticulin

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-DRβ chain. The molecular mechanisms by which the SE affects susceptibility to – and severity of - RA are unknown. We...

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Detalles Bibliográficos
Autores principales: Ling, Song, Cheng, Andrew, Pumpens, Paul, Michalak, Marek, Holoshitz, Joseph
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2908537/
https://www.ncbi.nlm.nih.gov/pubmed/20661469
http://dx.doi.org/10.1371/journal.pone.0011703
Descripción
Sumario: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-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 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.