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| STRUCTURE/ACTIVITY ANALYSIS OF HUMAN MONOCYTE CHEMOATTRACTANT PROTEIN-1 (MCP-1) BY MUTAGENESIS - IDENTIFICATION OF A MUTATED PROTEIN THAT INHIBITS MCP-1-MEDIATED MONOCYTE CHEMOTAXIS |
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| Author(s): ZHANG YJ, RUTLEDGE BJ, ROLLINS BJ |
| Source: JOURNAL OF BIOLOGICAL CHEMISTRY Volume: 269 Issue: 22 Pages: 15918-15924 Published: JUN 3 1994 |
| Times Cited: 111 References: 30 |
| Abstract: Monocyte chemoattractant protein-1 (MCP-1) is a monocyte-specific chemoattractant and activator and is a member of the chemokine-beta family of cytokines. To identify regions of MCP-1 which are required for its biological activity, me constructed human MCP-1 mutants that were expressed in eukaryotic cells and tested for their ability to attract monocytes in vitro. Deletion of amino acids 2-8 destroyed activity, suggesting that the amino-terminal region is necessary for activity. Within the deleted region, mutation of aspartate 3 to alanine produced a protein with 9% of wild-type activity, whereas mutation of asparagine 6 to alanine produced a protein with 52.9% of wild-type activity. Mutation of amino acids within the first intercysteine loop yielded variable results. Changing tyrosine 28 to aspartate or arginine 30 to leucine each produced proteins with essentially no monocyte chemoattractant activity. The side chains of these amino acids are predicted to point into a putative receptor binding cleft, and these loss-of-function mutations are consistent with this model. Also consistent is the retention of 60% of wad-type activity after mutation of serine 27 to glutamine, since the side chain of serine 27 is predicted to point away from the binding cleft. However, mutation of arginine 24, which lies outside of this area, to phenylalanine produced a protein with only 5% of wild-type activity, suggesting more complex interactions. Truncations of the carboxyl terminus, as well as mutation of aspartate 68 to leucine, generated proteins with 10-20% of wild-type activity. (Another carboxyl-terminal insertional mutation demonstrated that O-linked carbohydrate in MCP-l alpha may be added to a threonine in the carboxyl-terminal region.) These findings are consistent with a structural model of dimeric MCP-1 which is similar to interleukin-8 in which amino acids that point into a cleft between the two carboxyl-terminal alpha-helices of the subunits are important for receptor binding. In addition, however, amino acids at the amino terminus and others outside of the interhelical cleft are also essential for activity. The carboxyl terminal alpha-helix is not required for signaling per se but is required for maximal specific activity. Finally, four mutant proteins partially inhibited the ability of wild-type MCP-1 to attract monocytes in vitro. In particular, mutant 7ND (deletion of amino acids 2-8) in hibited MCP-1 activity by 50% at a molar ratio of 75:1, displaced MCP-1 from its receptor on monocytes at a similar ratio, and bound to a single class of receptors on human monocytes with a K-d of 2.6 nM. However, 7ND bound only to 10% of the number of receptors to which MCP-1 bound, suggesting that part of its inhibitory activity may be due to binding to MCP-1 itself. |
| Document Type: Article |
| Language: English |
Addresses:
1. HARVARD UNIV, SCH MED, DANA FARBER CANC INST, DEPT MED, BOSTON, MA 02115 USA |
| Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814 |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: NP513 |
| ISSN: 0021-9258 |
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