Mapping of MCP-1 functional domains by peptide analysis and site-directed mutagenesis.
Monocyte chemoattractant protein-1 (MCP-1) is a member of the beta 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.
Steitz
S A
SA
Tanabe Research Laboratories USA, Inc., San Diego, CA 92121, USA.
Hasegawa
K
K
Chiang
S L
SL
Cobb
R R
RR
Castro
M A
MA
Lobl
T J
TJ
Yamada
M
M
Lazarides
E
E
Cardarelli
P M
PM
D000595:Amino Acid Sequence; D001120:Arginine / Q000502:physiology; D001667:Binding, Competitive; D002460:Cell Line; D018932:Chemokine CCL2 / Q000737:chemistry* / Q000235:genetics; D002634:Chemotaxis, Leukocyte / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology*; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000494:pharmacology; D011993:Recombinant Fusion Proteins; D014443:Tyrosine / Q000502:physiology
D018932:Chemokine CCL2; D010455:Peptides; D010456:Peptides, Cyclic; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D001120:Arginine