title: Human cyclo-oxygenase-1 and an alternative splice variant: contrasts in expression of mRNA, protein and catalytic activities.
abstract: The two COX (cyclo-oxygenase) isoenzymes COX-1 and -2 catalyse the initial step in the conversion of arachidonic acid into PG (prostaglandin) hormones. The identification of an mRNA transcript encoding a splice variant of human COX-1 was reported more than a decade ago [Diaz, Reginato and Jimenez (1992) J. Biol. Chem. 267, 10816-10822], yet catalytic activity and tissue expression of the corresponding spliced protein remained uncharacterized. The splice variant lacks amino acids 396-432, corresponding to the last 37 amino acids of exon 9 of the gene encoding COX-1. These amino acids form a loop at one side of the peroxidase active site of the protein. We expressed the full-length and spliced COX-1 cDNAs in COS-7 and Sf9 insect cells, and determined the PG-forming activity using incubations with radiolabelled arachidonic acid and HPLC analyses. When expressed in either system, abundant PG formation was observed with the full-length COX-1, whereas the spliced protein did not form any detectable product. Peroxidase activity was readily detected in microsomes prepared from COS-7 cells transfected with COX-1 but not with the splice variant. In reverse transcriptase-PCR experiments, we detected the mRNA for the alternatively spliced and full-length COX-1 in human brain, tonsil and colon tissue, yet we were unable to detect expression of the spliced protein in the same tissues using immunoprecipitation and Western-blot analyses. We conclude that, whereas the mRNA transcript for the spliced COX-1 is present in various human tissues, the corresponding protein is either not formed or subject to rapid proteolytic degradation.
authors.affiliation: Department of Pharmacology, Vanderbilt University School of Medicine, 23rd Ave. at Pierce, Nashville, TN 37232-6602, USA. claus.schneider@vanderbilt.edu
authors.forename: Claus
authors.initials: C
authors.lastname: Schneider
authors.forename: William E
authors.initials: WE
authors.lastname: Boeglin
authors.forename: Alan R
authors.initials: AR
authors.lastname: Brash
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology / Q000378:metabolism; D002384:Catalysis; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003106:Colon / Q000201:enzymology / Q000378:metabolism; D051545:Cyclooxygenase 1; D006801:Humans; D008565:Membrane Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D014066:Palatine Tonsil / Q000201:enzymology / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012756:Sheep; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D008565:Membrane Proteins; D012333:RNA, Messenger; D051545:Cyclooxygenase 1; C496511:PTGS1 protein, human; D011451:Prostaglandin-Endoperoxide Synthases