title: Tyrosine sulfation in N-terminal domain of human C5a receptor is necessary for binding of chemotaxis inhibitory protein of Staphylococcus aureus.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.affiliation: Department of Respiratory Diseases, Peking Union Medical College Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing, China.
authors.forename: Zhen-jia
authors.initials: ZJ
authors.lastname: Liu
authors.forename: Yan-juan
authors.initials: YJ
authors.lastname: Yang
authors.forename: Lei
authors.initials: L
authors.lastname: Jiang
authors.forename: Ying-chun
authors.initials: YC
authors.lastname: Xu
authors.forename: Ai-xia
authors.initials: AX
authors.lastname: Wang
authors.forename: Guan-hua
authors.initials: GH
authors.lastname: Du
authors.forename: Jin-ming
authors.initials: JM
authors.lastname: Gao
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002461:Cell Line, Transformed; D057809:HEK293 Cells; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000166:cytology / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C488033:C5AR1 protein, human; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; C493134:chemotaxis inhibitory protein, Staphylococcus aureus; D014443:Tyrosine