PMID- 8195247
OWN - NLM
STAT- MEDLINE
DCOM- 19940630
LR  - 20210210
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 269
IP  - 22
DP  - 1994 Jun 3
TI  - 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.
PG  - 15918-24
AB  - 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, we 
      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 wild-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-1 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.(ABSTRACT TRUNCATED AT 400 WORDS)
FAU - Zhang, Y J
AU  - Zhang YJ
AD  - Department of Medicine, Dana-Farber Cancer Institute, Harvard Medical School, 
      Boston, Massachusetts 02115.
FAU - Rutledge, B J
AU  - Rutledge BJ
FAU - Rollins, B J
AU  - Rollins BJ
LA  - eng
GR  - CA53091/CA/NCI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (Chemokine CCL2)
RN  - 0 (Chemotactic Factors)
RN  - 0 (Cytokines)
RN  - 0 (Macromolecular Substances)
RN  - 0 (Recombinant Proteins)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Cell Line
MH  - Chemokine CCL2
MH  - Chemotactic Factors/analysis/*biosynthesis/pharmacology
MH  - *Chemotaxis, Leukocyte/drug effects
MH  - Chlorocebus aethiops
MH  - Cytokines/biosynthesis
MH  - Humans
MH  - Immunoblotting
MH  - Macromolecular Substances
MH  - Models, Structural
MH  - Molecular Sequence Data
MH  - Monocytes/drug effects/*physiology
MH  - Mutagenesis
MH  - Point Mutation
MH  - Polymerase Chain Reaction
MH  - Recombinant Proteins/analysis/biosynthesis/pharmacology
MH  - Sequence Deletion
MH  - Transfection
EDAT- 1994/06/03 00:00
MHDA- 1994/06/03 00:01
CRDT- 1994/06/03 00:00
PHST- 1994/06/03 00:00 [pubmed]
PHST- 1994/06/03 00:01 [medline]
PHST- 1994/06/03 00:00 [entrez]
AID - S0021-9258(17)40768-X [pii]
PST - ppublish
SO  - J Biol Chem. 1994 Jun 3;269(22):15918-24.