Cloning, expression, and functional characterization of human cyclooxygenase-1 splicing variants: evidence for intron 1 retention.
Recently, a splicing variant of cyclooxygenase (COX)-1, arising via the retention of its intron 1, was identified in canine. It was called COX-3 and was reported to be differentially sensitive to inhibition by various nonsteroidal anti-inflammatory drugs (NSAIDs) as well as acetaminophen (Chandrasekharan et al., 2002). However, the existence of an orthologous splicing variant in human tissues has been questioned due to a reading frame shift and premature termination. In this study, we first confirmed the existence of intron 1-retained COX-1 in certain human tissues at both the mRNA and protein levels. Molecular biology studies revealed that three distinct COX-1 splicing variants exist in human tissues. The most prevalent of these variants, called COX-1b1, arises via retention of the entire 94 base pair (bp) of intron 1, leading to a shift in the reading frame and termination at bp 249. However, the other two variant types, called COX-1b2 and COX-1b3, retain entire intron 1, but they are missing a nucleotide in one of two different positions, thereby encoding predicted full-length and likely COX-active proteins. Functional studies revealed that the COX-1b2 is able to catalyze the synthesis of prostaglandin F2alpha from arachidonic acid with Km and Vmax values of 0.54 microM and 3.07 pmol/mg/min, respectively. However, no significant differential selectivity for inhibition by selected NSAIDs was observed. Accordingly, we conclude that intron 1-retained human COX-1 is not likely to be the therapeutic target of acetaminophen or a candidate of COX-3.
Qin
Ning
N
Analgesics, Drug Discovery, Johnson & Johnson Pharmaceutical Research and Development, P.O. Box 776, Welsh and McKean Roads, Spring House, PA 19477-0776, USA. nqin@prdus.jnj.com
Zhang
Sui-Po
SP
Reitz
Tasha L
TL
Mei
Jay M
JM
Flores
Christopher M
CM
D000082:Acetaminophen / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D051545:Cyclooxygenase 1 / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D014644:Genetic Variation; D006801:Humans; D007313:Insecta / Q000166:cytology; D007438:Introns; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics; D014018:Tissue Distribution
D000894:Anti-Inflammatory Agents, Non-Steroidal; D007527:Isoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000082:Acetaminophen; D051545:Cyclooxygenase 1