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.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-436) contains supplementary material, which is available to authorized users.
Highlights
Prostaglandin H synthases (PGHSs, known as cyclooxygenases or prostaglandin-endoperoxide synthases) are dimeric membrane proteins that catalyse the bis-oxidation of arachidonic acid (AA) to prostaglandin G2 (PGG2) and the subsequent reduction of PGG2 to PGH2
The glycosylation sites of murine PGHS-2 expressed in insect cells have been characterised showing that N53 and N130 are close to 100% glycosylated and that N396 (N409 in human PGHSs (hPGHSs)-1) and N580 are partially glycosylated (Nemeth et al 2001). hPGHSs contain 5 disulphide bonds, three of which are located in the epidermal growth factor
Expression of the recombinant hPGHSs in P. pastoris GS115 So far, functional mammalian PGHSs have been expressed in the baculoviral system (Vecchio and Malkowski 2011; Zou et al 2012) and in mammalian cell lines (Mbonye et al 2006, 2008), and hPGHS-2 recently in the yeast P. pastoris (Kukk et al 2012)
Summary
Prostaglandin H synthases (PGHSs, known as cyclooxygenases or prostaglandin-endoperoxide synthases) are dimeric membrane proteins that catalyse the bis-oxidation of arachidonic acid (AA) to prostaglandin G2 (PGG2) and the subsequent reduction of PGG2 to PGH2. The amino acid sequences of PGHS-1 and PGHS-2 are about 60% identical, unlike PGHS-2, the production of recombinant PGHS-1 in insect cells is obstructed as most of the enzyme is inactive and likely misfolded due to deficient glycosylation (Kulmacz et al 2003; Shimokawa and Smith 1992). N67, N143 and N409 (hPGHS-1 numbering) have been shown to be occupied in both isoforms and an additional site, N580 (human PGHS-2 numbering), in hPGHS-2 (Otto et al 1993). HPGHSs contain 5 disulphide bonds, three of which are located in the epidermal growth factor (EGF)-like domain, whereas there is an 8 amino acid insertion in the N-terminal part of hPGHS-1 preceding the disulphide rich region (Kulmacz et al 2003; Simmons et al 2004). An ovine PGHS-1 mutant that had a disrupted disulphide bond (C59-C69) lacked activity, affirming the importance of an intact EGF-like domain for proper folding (Smith et al 2000b)
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