Human cyclo-oxygenase-1 and an alternative splice variant: contrasts in expression of mRNA, protein and catalytic activities.

Schneider C, Boeglin WE, Brash AR
Biochem J. 2005 385 (Pt 1): 57-64

PMID: 15361066 · PMCID: PMC1134673 · DOI:10.1042/BJ20041115

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.

MeSH Terms (23)

Alternative Splicing Amino Acid Sequence Animals Blotting, Western Brain Catalysis Cell Line Chromatography, High Pressure Liquid Cloning, Molecular Colon Cyclooxygenase 1 Humans Membrane Proteins Models, Molecular Molecular Sequence Data Palatine Tonsil Prostaglandin-Endoperoxide Synthases Protein Biosynthesis Protein Conformation Reverse Transcriptase Polymerase Chain Reaction RNA, Messenger Sheep Transcription, Genetic

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