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Identification of the rheumatoid arthritis shared epitope binding site on calreticulin.

Ling S, Cheng A, Pumpens P, Michalak M, Holoshitz J - PLoS ONE (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, Michigan, United States of America. songlin@med.unich.edu

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.

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Related in: MedlinePlus

Interaction between the SE and domain-deleted CRT.A. A theoretical 3D structure of CRT. Rabbit CRT (Accession No. AAB20096) was computationally modeled based on the known crystal structure of Calnexin (PDB ID: 1JHN), using Modeler 9v7 software (School of Pharmacy, UCSF). CRT N-domain is shown in green, P-domain is in cyan and C-domain is shown in orange. B. Schematic representation of rabbit CRT domain-deletion mutants. C,D. SPR-based interactions between the SE ligand and CRT domain-deletion mutants. WT CRT or its domain-deletion mutants were immobilized on a CM5 biosensor chip surface and the SE peptidic ligands 65-79*0401 (C) or 65-79*0404 (D) were run in the analyte.
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pone-0011703-g001: Interaction between the SE and domain-deleted CRT.A. A theoretical 3D structure of CRT. Rabbit CRT (Accession No. AAB20096) was computationally modeled based on the known crystal structure of Calnexin (PDB ID: 1JHN), using Modeler 9v7 software (School of Pharmacy, UCSF). CRT N-domain is shown in green, P-domain is in cyan and C-domain is shown in orange. B. Schematic representation of rabbit CRT domain-deletion mutants. C,D. SPR-based interactions between the SE ligand and CRT domain-deletion mutants. WT CRT or its domain-deletion mutants were immobilized on a CM5 biosensor chip surface and the SE peptidic ligands 65-79*0401 (C) or 65-79*0404 (D) were run in the analyte.

Mentions: The primary sequence of CRT suggests that similar to calnexin it has three domains [16]. Because the tri-dimensional structure of CRT has not been fully resolved, it is a common practice in the literature to model it based on the known crystal structure of calnexin [17]. According to this model (Fig. 1A) the amino-terminal segment (N-domain) has a globular β-sheet structure. This domain is followed by a proline-rich sequence, called the P-domain that folds into a long hairpin-like structure with two pairs of short anti-parallel β-sheet sequences. The third region of CRT, called the C-domain, forms, together with the N-domain, a globular “head” to the molecule.


Identification of the rheumatoid arthritis shared epitope binding site on calreticulin.

Ling S, Cheng A, Pumpens P, Michalak M, Holoshitz J - PLoS ONE (2010)

Interaction between the SE and domain-deleted CRT.A. A theoretical 3D structure of CRT. Rabbit CRT (Accession No. AAB20096) was computationally modeled based on the known crystal structure of Calnexin (PDB ID: 1JHN), using Modeler 9v7 software (School of Pharmacy, UCSF). CRT N-domain is shown in green, P-domain is in cyan and C-domain is shown in orange. B. Schematic representation of rabbit CRT domain-deletion mutants. C,D. SPR-based interactions between the SE ligand and CRT domain-deletion mutants. WT CRT or its domain-deletion mutants were immobilized on a CM5 biosensor chip surface and the SE peptidic ligands 65-79*0401 (C) or 65-79*0404 (D) were run in the analyte.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908537&req=5

pone-0011703-g001: Interaction between the SE and domain-deleted CRT.A. A theoretical 3D structure of CRT. Rabbit CRT (Accession No. AAB20096) was computationally modeled based on the known crystal structure of Calnexin (PDB ID: 1JHN), using Modeler 9v7 software (School of Pharmacy, UCSF). CRT N-domain is shown in green, P-domain is in cyan and C-domain is shown in orange. B. Schematic representation of rabbit CRT domain-deletion mutants. C,D. SPR-based interactions between the SE ligand and CRT domain-deletion mutants. WT CRT or its domain-deletion mutants were immobilized on a CM5 biosensor chip surface and the SE peptidic ligands 65-79*0401 (C) or 65-79*0404 (D) were run in the analyte.
Mentions: The primary sequence of CRT suggests that similar to calnexin it has three domains [16]. Because the tri-dimensional structure of CRT has not been fully resolved, it is a common practice in the literature to model it based on the known crystal structure of calnexin [17]. According to this model (Fig. 1A) the amino-terminal segment (N-domain) has a globular β-sheet structure. This domain is followed by a proline-rich sequence, called the P-domain that folds into a long hairpin-like structure with two pairs of short anti-parallel β-sheet sequences. The third region of CRT, called the C-domain, forms, together with the N-domain, a globular “head” to the molecule.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, Michigan, United States of America. songlin@med.unich.edu

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.

Show MeSH
Related in: MedlinePlus