AIM: Staphylococcus aureus evades host defense through releasing several virulence proteins, such as chemotaxis inhibitory protein of staphylococcus aureus (CHIPS). It has been shown that extracellular N terminus of C5a receptor (C5aR) forms the binding domain for CHIPS, and tyrosine sulfation is emerging as a key factor in determining protein-protein interaction. The aim of this study was to evaluate the role of tyrosine sulfation of N-terminal of C5aR in its binding with CHIPS. METHODS: Expression plasmids encoding C5aR and its mutants were prepared using PCR and site-directed mutagenesis and were used to transfect HEK 293T cells using calcium phosphate. Recombinant CHIPS protein was purified. Western blotting was used to examine the binding efficiency of CHIPS to C5aR or its mutants. RESULTS: CHIPS exclusively binds to C5aR, but not to C5L2 or C3aR. A nonspecific sulfation inhibitor, sodium chlorate (50 nmol/L), diminishes the binding ability of C5aR with CHIPS. Blocking sulfation by mutation of tyrosine to phenylalanine at positions 11 and 14 of C5aR N terminus, which blocked sulfation, completely abrogates CHIPS binding. When tyrosine 14 alone was mutated to phenylalanine, the binding efficiency of recombinant CHIPS was substantially decreased. CONCLUSION: The results demonstrate a structural basis of C5aR-CHIPS association, in which tyrosine sulfation of N-terminal C5aR plays an important role. Our data may have potential significance in development of novel drugs for therapeutic intervention.
AIM: Staphylococcus aureus evades host defense through releasing several virulence proteins, such as chemotaxis inhibitory protein of staphylococcus aureus (CHIPS). It has been shown that extracellular N terminus of C5a receptor (C5aR) forms the binding domain for CHIPS, and tyrosine sulfation is emerging as a key factor in determining protein-protein interaction. The aim of this study was to evaluate the role of tyrosine sulfation of N-terminal of C5aR in its binding with CHIPS. METHODS: Expression plasmids encoding C5aR and its mutants were prepared using PCR and site-directed mutagenesis and were used to transfect HEK 293T cells using calcium phosphate. Recombinant CHIPS protein was purified. Western blotting was used to examine the binding efficiency of CHIPS to C5aR or its mutants. RESULTS: CHIPS exclusively binds to C5aR, but not to C5L2 or C3aR. A nonspecific sulfation inhibitor, sodium chlorate (50 nmol/L), diminishes the binding ability of C5aR with CHIPS. Blocking sulfation by mutation of tyrosine to phenylalanine at positions 11 and 14 of C5aR N terminus, which blocked sulfation, completely abrogates CHIPS binding. When tyrosine 14 alone was mutated to phenylalanine, the binding efficiency of recombinant CHIPS was substantially decreased. CONCLUSION: The results demonstrate a structural basis of C5aR-CHIPS association, in which tyrosine sulfation of N-terminal C5aR plays an important role. Our data may have potential significance in development of novel drugs for therapeutic intervention.
Authors: Anne-Marie Scola; Adrian Higginbottom; Lynda J Partridge; Robert C Reid; Trent Woodruff; Stephen M Taylor; David P Fairlie; Peter N Monk Journal: J Biol Chem Date: 2006-12-11 Impact factor: 5.157
Authors: Johannes H Ippel; Carla J C de Haas; Anton Bunschoten; Jos A G van Strijp; John A W Kruijtzer; Rob M J Liskamp; Johan Kemmink Journal: J Biol Chem Date: 2009-02-27 Impact factor: 5.157
Authors: Andrew J Wright; Adrian Higginbottom; Didier Philippe; Abhishek Upadhyay; Stefan Bagby; Robert C Read; Peter N Monk; Lynda J Partridge Journal: Mol Immunol Date: 2007-01-26 Impact factor: 4.407
Authors: María Florencia Ercoli; Dee Dee Luu; Ellen Youngsoo Rim; Alexandra Shigenaga; Artur Teixeira de Araujo; Mawsheng Chern; Rashmi Jain; Randy Ruan; Anna Joe; Valley Stewart; Pamela Ronald Journal: Proc Natl Acad Sci U S A Date: 2022-02-22 Impact factor: 11.205