N-glycosylation of prostaglandin endoperoxide synthases-1 and -2 and their orientations in the endoplasmic reticulum.
Department of Biochemistry, Michigan State University, East Lansing 48824.
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.
PMID: 8349699 [PubMed - indexed for MEDLINE]