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Titolo:
INVESTIGATION OF HUMAN CYCLOOXYGENASE-2 GLYCOSYLATION HETEROGENEITY AND PROTEIN EXPRESSION IN INSECT AND MAMMALIAN-CELL EXPRESSION SYSTEMS
Autore:
PERCIVAL MD; BASTIEN L; GRIFFIN PR; KARGMAN S; OUELLET M; ONEILL GP;
Indirizzi:
MERCK FROSST CTR THERAPEUT RES,DEPT BIOCHEM & MOL BIOL,POB 1005 POINTE CLAIRE PQ H9R 4P8 CANADA MERCK RES LABS,DEPT ANALYT BIOCHEM RAHWAY NJ 07065
Titolo Testata:
Protein expression and purification
fascicolo: 3, volume: 9, anno: 1997,
pagine: 388 - 398
SICI:
1046-5928(1997)9:3<388:IOHCGH>2.0.ZU;2-I
Fonte:
ISI
Lingua:
ENG
Soggetto:
PROSTAGLANDIN-G/H SYNTHASE-1; NONSTEROIDAL ANTIINFLAMMATORY DRUGS; INDUCIBLE CYCLOOXYGENASE; ENDOPEROXIDE SYNTHASE; SELECTIVE-INHIBITION; BACULOVIRUS SYSTEM; VACCINIA VIRUS; PURIFICATION; BIOSYNTHESIS; ACID;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
33
Recensione:
Indirizzi per estratti:
Citazione:
M.D. Percival et al., "INVESTIGATION OF HUMAN CYCLOOXYGENASE-2 GLYCOSYLATION HETEROGENEITY AND PROTEIN EXPRESSION IN INSECT AND MAMMALIAN-CELL EXPRESSION SYSTEMS", Protein expression and purification, 9(3), 1997, pp. 388-398

Abstract

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 consensussequence at Asn(580) (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 mu mol O-2/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 thissite, 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 Asn(580) and Asn(592) N-linked oligosaccharide profiling of purified VV and BV WT andS582A 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-2by liquid chromatography-electrospray ionization-mass spectrometry showed an envelope of peaks separated by approximately 160 Ha, corresponding to differences of a single monosaccharide. The difference betweenthe highest mass peaks of the two envelopes, of approximately 1500 Da, is consistent with the wild-type enzyme containing an additional high mannose oligosaccharide. (C) 1997 Academic Press.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 11/07/20 alle ore 14:30:04