Investigation of human cyclooxygenase-2 glycosylation heterogeneity and protein expression in insect and mammalian cell expression systems.
Human cyclooxygenase-2 (hCox-2) is a key enzyme in the biosynthesis of prostaglandins and the target of nonsteroidal anti-inflammatory drugs. Recombinant hCox-2 overexpressed in a vaccinia virus (VV)-COS-7 system comprises two glycoforms. Removal of the N-glycosylation consensus sequence at Asn580 (N580Q and S582A mutants) resulted in the expression of protein comprising a single glycoform, consistent with the partial N-glycosylation at this site in the wild-type (WT) enzyme. The specific cyclooxygenase activities of the purified WT and N580Q mutant were equivalent (40 +/- 3 mumol O2/min/mg) and titrations with diclofenac showed no difference in inhibitor sensitivities of WT and both mutants. Results of the expression of WT and N580Q hCox-2 in a Drosophila S2 cell system were also consistent with the N-glycosylation at this site, but low levels of activity were obtained. High levels of N-glycosylation heterogeneity are observed in hCox-2 expressed using recombinant baculovirus (BV) in Sf9 cells. Expression of a double N-glycosylation site mutant in Sf9 cells, N580Q/N592Q, resulted in a decrease in glycosylation but no clear decrease in heterogeneity, indicating that the high degree of N-glycosylation heterogeneity observed with the BV-Sf9 system is not due to partial glycosylation of both Asn580 and Asn592. N-linked oligosaccharide profiling of purified VV and BV WT and S582A mutant hCox-2 showed the presence of high mannose structures, (Man)n (GlcNAc)2, n = 9, 8, 7, 6. The S582A mutant was the most homogeneous with (Man)9(GlcNAc)2 comprising greater than 50% of oligosaccharides present. Analysis of purified VV WT and S582A mutant hCox-2 by liquid chromatography-electrospray ionization-mass spectrometry showed an envelope of peaks separated by approximately 160 Da, corresponding to differences of a single monosaccharide. The difference between the highest mass peaks of the two envelopes, of approximately 1500 Da, is consistent with the wild-type enzyme containing an additional high mannose oligosaccharide.
Percival
M D
MD
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Quebec, Canada. david_percival@merck.com
Bastien
L
L
Griffin
P R
PR
Kargman
S
S
Ouellet
M
M
O'Neill
G P
GP
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D019556:COS Cells; D002240:Carbohydrate Sequence; D002460:Cell Line; D016384:Consensus Sequence; D051546:Cyclooxygenase 2; D004330:Drosophila; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics*; D013058:Mass Spectrometry; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D009844:Oligosaccharides / Q000737:chemistry / Q000235:genetics; D017354:Point Mutation; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D018411:Spodoptera; D014616:Vaccinia virus / Q000235:genetics
D007527:Isoenzymes; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases