Mapping of MCP‐1 functional domains by peptide analysis and site‐directed mutagenesis

FEBS Letters

Castro, Mary A¹  Cardarelli, Pina M¹  Cobb, Ronald R¹  Lazarides, Elias¹  Chiang, Shiu-Lan¹  Steitz, Susan A¹  Hasegawa, Ko²  Lobl, Thomas J¹  Yamada, Masaki² 

[1] Tanabe Research Laboratories USA, Inc., 4540 Towne Centre Court, San Diego, CA 92121, USA;Lead Generation Research Laboratory, Tanabe Seiyaku Co., Ltd., Osaka, Japan

关键词: Monocyte chemoattractant protein;    CC chemokine receptor 2;    Chemokine;    MCP;    monocyte chemoattractant protein;    IC50;    inhibitory concentration required for 50% reduction in activity;    CCR2;    CC chemokine receptor 2;    MIP;    monocyte inflammatory protein;    C5a;    complement factor 5a;    RANTES;    regulated on activation;    normal T cell expressed;    and secreted;   

DOI  :  10.1016/S0014-5793(98)00637-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Monocyte chemoattractant protein-1 (MCP-1) is a member of the β chemokine family which acts through specific seven transmembrane receptors to recruit monocytes, basophils, and T lymphocytes to sites of inflammation. To identify regions of the human MCP-1 protein which are important for its biological activity, we have synthesized domain-specific peptides and tested their ability to antagonize MCP-1 binding and chemotaxis in THP-1 cells. We have found that an intercysteine first loop peptide encompassing amino acids 13–35 inhibits MCP-1 binding and chemotactic activity, while peptides representing the amino-terminus (amino acids 1–10), second loop (amino acids 37–51), and carboxy-terminus (amino acids 56–71) of MCP-1 have no effect. In addition, we have found that cyclization of the first loop peptide by disulfide linkage and blocking the C-terminus of the peptide by amidation increases the activity of this peptide to block MCP-1 binding and chemotaxis. In order to specifically identify amino acid residues within the first loop that are crucial for MCP-1 functional activity, we have substituted alanine for tyrosine (Y13A) or arginine (R18A) in MCP-1 recombinant proteins. While baculovirus produced wild type and R18A MCP-1 proteins are indistinguishable in their ability to induce THP-1 chemotaxis and show modest effects in binding activity compared to commercially available recombinant MCP-1 protein, the Y13A point mutation causes a dramatic loss in function. The identification of functional domains of MCP-1 will assist in the design of MCP-1 receptor antagonists which may be clinically beneficial in a number of inflammatory diseases.

【 授权许可】

Unknown