Title : N-glycosylation of
prostaglandin endoperoxide synthases-1 and -2 and their orientations in the endoplasmic reticulum
Abstract :
- Using site-directed mutagenesis, we have determined that Asn68, Asn144, and Asn410 of ovine prostaglandin endoperoxide ( PGH) synthase-1 are N-glycosylated
- A fourth consensus N-glycosylation sequence at Asn104 is not glycosylated
- Glycosylation of PGH synthase-1 at Asn410 and at either Asn68 or Asn144 was required for expression of both the cyclooxygenase and the peroxidase activities of the enzyme
- Inactive PGH synthase-1 glycosylation site mutant proteins do not appear to achieve their native conformations
- However, the native enzyme , once in an active conformation, does not appear to require attached carbohydrate for cyclooxygenase or peroxidase activities
- N-Glycosylation consensus sequences corresponding to the three glycosylation sites of ovine PGH synthase-1 are conserved in the deduced amino acid sequences of PGH synthases-2
- Using site-directed mutagenesis, we determined that there is an additional site of N-glycosylation in murine PGH synthase-2 located at Asn580
- This site is N-glycosylated in about 50% of PGH synthase-2 molecules, resulting in two peptide bands on SDS-polyacrylamide gel electrophoresis (72 and 74 kDa)
- Glycosylation of PGH synthase-2 is necessary for expression of enzyme activity, but glycosylation of PGH synthase-2 at Asn580 per se does not affect activity
- Assuming that the N-glycosylation sites of PGH synthase-1 are on the luminal side of the endoplasmic reticulum (ER), and that the site of tryptic cleavage of ovine PGH synthase-1 ( Arg277 ) is on the cytoplasmic side of the ER, we propose that both the NH2 and COOH termini of PGH synthase-1 are located in the lumen of the ER and that there are two transmembrane domains located between Asn144 and Arg277 and between Arg277 and Asn410 , respectively
- A similar orientation is predicted for PGH synthase-2