SITE-DIRECTED MUTAGENESIS OF MONOCYTE CHEMOATTRACTANT PROTEIN-1 IDENTIFIES 2 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 in vestigated structure-activity relationships for human MCP-1. Mutations were introduced based upon differences between MCP-1 and the structur ally 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 chemotaxi s 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-C ys-Cys-Tyr (amino acids 10-13). Point mutations of Thr-10 to Arg and T yr-13 to Ile greatly lowered MCP-1 activity. The second functionally i mportant region is formed by Ser-34 and Lys-35. Insertion of a Pro bet ween these two residues, or their substitution by the sequence Gly-Pro -His, caused nearly complete loss of MCP-1 activity. Competition bindi ng experiments showed that the mutations that affected activity also l owered the ability to compete with wild-type MCP-1 for receptors on TH P-1 cells. Point mutations at positions 8, 15, 30, 37, 38 and 68 had l ittle effect on MCP-1 activity. The important regions that we have ide ntified in MCP-1 correspond with previously identified functionally im portant regions of IL-8, suggesting that the two molecules bind to the ir respective receptors by similar contacts.