Site-directed mutagenesis of monocyte chemoattractant protein-1 identifies two regions of the polypeptide essential for biological activity.
Monocyte chemoattractant protein-1 (MCP-1) mediates monocyte migration into tissues in inflammatory diseases and atherosclerosis. We have investigated structure-activity relationships for human MCP-1. Mutations were introduced based upon differences between MCP-1 and the structurally related but functionally distinct molecule interleukin-8 (IL-8). Mutant proteins produced using the baculovirus/insect cell expression system were purified and their ability to stimulate monocyte chemotaxis and elevation of intracellular calcium in THP-1 monocytic leukaemia cells was measured. Two regions in MCP-1 were identified as important for its biological activity. One region consists of the sequence Thr-Cys-Cys-Tyr (amino acids 10-13). Point mutations of Thr-10 to Arg and Tyr-13 to Ile greatly lowered MCP-1 activity. The second functionally important region is formed by Ser-34 and Lys-35. Insertion of a Pro between these two residues, or their substitution by the sequence Gly-Pro-His, caused nearly complete loss of MCP-1 activity. Competition binding experiments showed that the mutations that affected activity also lowered the ability to compete with wild-type MCP-1 for receptors on THP-1 cells. Point mutations at positions 8, 15, 30, 37, 38 and 68 had little effect on MCP-1 activity. The important regions that we have identified in MCP-1 correspond with previously identified functionally important regions of IL-8, suggesting that the two molecules bind to their respective receptors by similar contacts.
Beall
C J
CJ
Neurobiotechnology Center, Ohio State University, Columbus 43210, USA.
Mahajan
S
S
Kuhn
D E
DE
Kolattukudy
P E
PE
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D018932:Chemokine CCL2 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
D018932:Chemokine CCL2; D017931:DNA Primers; D011994:Recombinant Proteins