title: N-glycosylation site occupancy in human prostaglandin H synthases expressed in Pichia pastoris.
abstract: Prostaglandin H synthases (PGHSs) are N-glycosylated membrane proteins that catalyse the committed step in prostaglandin synthesis. Unlike PGHS-2, the production of recombinant PGHS-1 in non-mammalian expression systems is complicated. The majority of the heterologous enzyme is inactive due to misfolding. Correct N-glycosylation is proposed to be obligatory for proper folding of mammalian PGHSs. In this study, human PGHS-1 and -2 (hPGHS-1 and -2) were expressed in the yeast Pichia pastoris. Recombinant hPGHS-2 was catalytically active, whereas hPGHS-1 was inactive. Accumulation of non-glycosylated hPGHSs was not observed in the crude lysate of the yeast cells. The N-glycosylation patterns of the purified recombinant proteins were characterised using nano-LC/MS/MS. The isoforms exhibited similar N-glycosylation site occupancy. The results indicate that there are more complex grounds for the inactivity of the recombinant hPGHS-1 produced in yeast.
authors.affiliation: Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia.
authors.forename: Kaia
authors.initials: K
authors.lastname: Kukk
authors.affiliation: Proteomics Core Facility, Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia.
authors.forename: Sergo
authors.initials: S
authors.lastname: Kasvandik
authors.affiliation: Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia.
authors.forename: Nigulas
authors.initials: N
authors.lastname: Samel
keywords: COX; Cyclooxygenase; Mass spectrometry; N-glycosylation; PGHS; Pichia pastoris; Recombinant prostaglandin H synthase