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Mapping of MCP‐1 functional domains by peptide analysis and site‐directed mutagenesis
Author(s) -
Steitz Susan A,
Hasegawa Ko,
Chiang Shiu-Lan,
Cobb Ronald R,
Castro Mary A,
Lobl Thomas J,
Yamada Masaki,
Lazarides Elias,
Cardarelli Pina M
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)00637-1
Subject(s) - chemotaxis , amino acid , biochemistry , peptide , mutagenesis , alanine , peptide sequence , n terminus , chemistry , site directed mutagenesis , receptor , biology , microbiology and biotechnology , mutation , gene , mutant
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.