title: Identification and functional characterization of polymorphisms in human cyclooxygenase-1 (PTGS1).
abstract: OBJECTIVE
  Cyclooxygenase-1 (COX-1, PTGS1) catalyzes the conversion of arachidonic acid to prostaglandin H2, which is subsequently metabolized to various biologically active prostaglandins. We sought to identify and characterize the functional relevance of genetic polymorphisms in PTGS1.
  
  
  METHODS
  Sequence variations in human PTGS1 were identified by resequencing 92 healthy individuals (24 African, 24 Asian, 24 European/Caucasian, and 20 anonymous). Using site-directed mutagenesis and a baculovirus/insect cell expression system, recombinant wild-type COX-1 and the R8W, P17L, R53H, R78W, K185T, G230S, L237M, and V481I variant proteins were expressed. COX-1 metabolic activity was evaluated in vitro using an oxygen consumption assay under basal conditions and in the presence of indomethacin.
  
  
  RESULTS
  Forty-five variants were identified, including seven nonsynonymous polymorphisms encoding amino acid substitutions in the COX-1 protein. The R53H (35+/-5%), R78W (36+/-4%), K185T (59+/-6%), G230S (57+/-4%), and L237M (51+/-3%) variant proteins had significantly lower metabolic activity relative to wild-type (100+/-7%), while no significant differences were observed with the R8W (104+/-10%), P17L (113+/-7%), and V481I (121+/-10%) variants. Inhibition studies with indomethacin demonstrated that the P17L and G230S variants had significantly lower IC50 values compared to wild-type, suggesting these variants significantly increase COX-1 sensitivity to indomethacin inhibition. Consistent with the metabolic activity data, protein modeling suggested the G230S variant may disrupt the active conformation of COX-1.
  
  
  CONCLUSIONS
  Our findings demonstrate that several genetic variants in human COX-1 significantly alter basal COX-1-mediated arachidonic acid metabolism and indomethacin-mediated inhibition of COX-1 activity in vitro. Future studies characterizing the functional impact of these variants in vivo are warranted.
authors.affiliation: Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, Chapel Hill, North Carolina, USA.
authors.forename: Craig R
authors.initials: CR
authors.lastname: Lee
authors.forename: Frank G
authors.initials: FG
authors.lastname: Bottone
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Krahn
authors.forename: Leping
authors.initials: L
authors.lastname: Li
authors.forename: Harvey W
authors.initials: HW
authors.lastname: Mohrenweiser
authors.forename: Molly E
authors.initials: ME
authors.lastname: Cook
authors.forename: Robert M
authors.initials: RM
authors.lastname: Petrovich
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Bell
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Eling
authors.forename: Darryl C
authors.initials: DC
authors.lastname: Zeldin
mesh_terms: D000595:Amino Acid Sequence; D016718:Arachidonic Acid / Q000378:metabolism; D051545:Cyclooxygenase 1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007213:Indomethacin; D020128:Inhibitory Concentration 50; D015810:Linkage Disequilibrium; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D017433:Protein Structure, Secondary
chemical_list: D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D050505:Mutant Proteins; D016718:Arachidonic Acid; D051545:Cyclooxygenase 1; C496511:PTGS1 protein, human; D007213:Indomethacin