PT  - JOURNAL ARTICLE
AU  - Zhenjia Liu
AU  - Aixia Wang
AU  - Guanhua Du
AU  - Jinming Gao
TI  - Tyrosine sulfation in the N-terminal domain of human C5aR is necessary for high-affinity binding of chemotaxis inhibitory protein of staphylococcus aureus
DP  - 2011 Sep 01
TA  - European Respiratory Journal
PG  - p3493
VI  - 38
IP  - Suppl 55
4099  - http://erj.ersjournals.com/content/38/Suppl_55/p3493.short
4100  - http://erj.ersjournals.com/content/38/Suppl_55/p3493.full
SO  - Eur Respir J2011 Sep 01; 38
AB  - Background: Staphylococcus aureus evades host defense through releasing several virulence proteins, such as chemotaxis inhibitory protein of staphylococcus aureus (CHIPS). Previous studies have shown that extracellular N terminus of C5a receptor (C5aR) forms the binding domain for CHIPS. Tyrosine sulfation is emerging as a key factor in determining protein-protein interaction. The goal of this study was to evaluate the role of tyrosine sulfation of N-terminal C5aR in binding to CHIPS.Methods: Expression plasmids encoding C5aR and its mutants were made by PCR and site-directed mutagenesis. HEK 293T cells were transfected with plasmids encoding C5aR using calcium phosphate. Recombinant CHIPS protein was purified. Western blotting was used to assay the binding of CHIPS to C5aR or its mutants and p-38 phosphorylation.Results: We report that CHIPS exclusively binds to C5aR, but not to C5L2 or C3aR. A nonspecific sulfation inhibitor, sodium chlorate, diminishes the binding ability of C5aR to CHIPS. Blocking sulfation by mutation of tyrosine to phenylalanine at positions 11 and 14 of C5aR N terminus completely abrogates CHIPS binding. When tyrosine 14 alone was mutated to phenylalanine, the binding affinity of recombinant CHIPS was substantially decreased. CHIPS fails to induce p-38 phosphorylation in cells overexpressing wild-type C5aR or its mutants.Conclusion: This study defines a structural basis of C5aR-CHIPS association, in which tyrosine sulfation of N-terminal C5aR plays an important role in CHIPS binding. Our data would make it possible to develop potent drugs for therapeutic intervention.