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.

DOI: 10.1186/2193-1801-3-436

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@inproceedings{Kukk2014NglycosylationSO, title={N-glycosylation site occupancy in human prostaglandin H synthases expressed in Pichia pastoris}, author={Kaia Kukk and Sergo Kasvandik and Nigulas Samel}, booktitle={SpringerPlus}, year={2014} }