N-GLYCOSYLATION OF PROSTAGLANDIN ENDOPEROXIDE SYNTHASES-1 AND SYNTHASES-2 AND THEIR ORIENTATIONS IN THE ENDOPLASMIC-RETICULUM

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 cycloox ygenase and the peroxidase activities of the enzyme. Inactive PGH synt hase-1 glycosylation site mutant proteins do not appear to achieve the ir native conformations. However, the native enzyme, once in an active conformation, does not appear to require attached carbohydrate for cy clooxygenase or peroxidase activities. N-Glycosylation consensus seque nces corresponding to the three glycosylation sites of ovine PGH synth ase-1 are conserved in the deduced amino acid sequences of PGH synthas es-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 mo lecules, resulting in two peptide bands on SDS-polyacrylamide gel elec trophoresis (72 and 74 kDa). Glycosylation of PGH synthase-2 is necess ary for expression of enzyme activity, but glycosylation of PGH syntha se-2 at Asn580 per se does not affect activity. Assuming that the N-gl ycosylation sites of PGH synthase-1 are on the luminal side of the end oplasmic reticulum (ER), and that the site of tryptic cleavage of ovin e PGH synthase-1 (Arg277) is on the cytoplasmic side of the ER, we pro pose 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 lo cated between Asn144 and Arg277 and between Arg277 and Asn410, respect ively. A similar orientation is predicted for PGH synthase-2.