Tyrosine sulfation in N-terminal domain of human C5a receptor is necessary for binding of chemotaxis inhibitory protein of Staphylococcus aureus  

作　　者：Zhen-jia LIU Yan-juan YANG Lei JIANG Ying-chun XU Ai-xia WANG Guan-hua DU Jin-ming GAO 

机构地区：[1]Department of Respiratory Diseases, Peking Union Medical College Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100730, China [2]Department of Respiratory Diseases, Affiliated Hospital of Ningxia Medical University, Yinchuan 750004, China [3]Department of Medical Microbiology, Peking Union Medical College Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100730, China [4]Department of Infectious Diseases, Peking Union Medical College Hospital, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100730, China [5]National Center for Drug Screening, institute of Materia Medica, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100050, China

出　　处：《Acta Pharmacologica Sinica》2011年第8期1038-1044,共7页中国药理学报（英文版）

基　　金：Acknowledgements This work was in part supported by grants from National Natural Science Foundation of China （No 30470767）, Beijing Natural Sciences Foundation （No 7072063）, Education Ministry of China New Century Excellent Talent （NCET 06-0156） and Janssen Research Council China （All grants were to Jin- ming GAO）. We are grateful for Dr Hyeryun CHOE＇s helpful encouragement and for providing the plasmids. We thank Prof Wen- hui LI for kind help in designing the primers. We particularly thank Prof Kian Fan CHUNG for his helpful suggestions, discussion, and excellent English editing of the text.

摘　　要：Aim： Staphylococcus auteus 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 auteus 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.

关 键 词：Staphylococcus aureus chemotaxis inhibitory protein tyrosine sulfation post-translational modification chemotactic receptor C5aR 

分 类 号：Q26[生物学—细胞生物学] TS207.4[轻工技术与工程—食品科学]