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-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.


RESULTS
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.


CONCLUSIONS
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.
Ling
Song
S
Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, Michigan, United States of America. songlin@med.unich.edu
Cheng
Andrew
A
Pumpens
Paul
P
Michalak
Marek
M
Holoshitz
Joseph
J
D000818:Animals; D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001665:Binding Sites; D037282:Calreticulin / Q000737:chemistry* / Q000378:metabolism*; D002478:Cells, Cultured; D002942:Circular Dichroism; D003198:Computer Simulation; D000939:Epitopes / Q000378:metabolism*; D007113:Immunity, Innate / Q000235:genetics / Q000502:physiology; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017433:Protein Structure, Secondary; D015398:Signal Transduction; D020349:Surface Plasmon Resonance
D037282:Calreticulin; D000939:Epitopes