Examination of prostaglandin h synthase (PGHS) isoform structures with limited proteolytic digestion

The two PGHS isoforms catalyze key stops in the biosynthesis of bioactive lipid mediators. The isoforms share about 60% sequence identity, but have distinct interactions with substrates, activators and inhibitors. Ovine PGHS 1 (oPGHS-1) has a distinctive protease-sensitivr site near Arg277; cleavage produces fragments of 33 and 38 kDa and loss of activity. The oPGHS-1 cryhtal structure places Arg277 in an exposed loop; homology modelling predicts analogous loop structures in both human isoforms (hPGHS-1 and -2). We used limited proteolytic digestion of hPGHS-1 and -2 to probe their structures. Incubation of hPGHS-I with trypsin or proteinase K produced 33 and 38 kl)a fragments and loss of activity. Incubation of hPGHS-2 wit h the proteases led to cleavage of only a 2-3 kDa fragment, with no loss of activity; immunoblotting showed the cleavage to be near the C-terminus. Similar experiments indicated that trypsin did not attack the oPGHS-1 C-terminus. Mutation of hPGHS 2 Pro263 (corresponds to Arg277 of oPGHS-1) to Arg did not change the trypsin susceptibility. A peptide comprising residues 259-268 of the P263R mutant was cleaved by trypsin as quickly as the corresponding hPGHS-1 peptide, demonstrating that sequence differences did not account for the protea.se resistance of the hPGHS-2 mutant. The results highlight two regions of significant structural difference between the isoforms: the Arg277 loop, which is protease sensitive in PGHS-1 but resistant in PGHS-2, and the C-terminus. which is protease-sensitive in PGHS-2 but not in PGHS-1. Support: NIH GM 52170.