Comparison of prostaglandin H synthase isoform structures using limited proteolytic digestion.
Prostaglandin H synthase (PGHS) catalyzes a key step in the biosynthesis of a variety of bioactive lipid mediators. The two known isoforms (PGHS-1 and -2) share about 60% amino acid identity, but exhibit distinct interactions with substrates, activators, and inhibitors. Ovine PGHS-1 has previously been shown to have a distinctive protease-sensitive site near Arg277; cleavage by trypsin, chymotrypsin, or proteinase K produces fragments of 33 and 38 kDa and loss of activity. The ovine PGHS-1 crystal structure shows Arg277 located in an exposed loop structure; homology modeling predicts similar loop structures for both human isoforms (hPGHS-1 and -2). We have used limited proteolytic digestion of recombinant hPGHS-1 and hPGHS-2 to probe their structures. Incubation of hPGHS-1 with either trypsin or proteinase K produced 33- and 38-kDa fragments and loss of activity. In contrast, incubation of hPGHS-2 with the same proteases led to cleavage of only a 2- to 3-kDa fragment, with no decrease in activity. Immunoblotting with site-specific antibodies demonstrated that the cleaved fragment originated from the hPGHS-2 C-terminus. Similar immunoblotting experiments indicated that trypsin did not attack the ovine PGHS-1 C-terminus. Mutagenesis was used to replace Pro263 of hPGHS-2 (corresponds to Arg277 of ovine PGHS-1) with arginine, inserting a potential trypsin site. Incubation of this P263R hPGHS-2 mutant with either trypsin or proteinase K resulted in cleavage near the C-terminus and retention of activity, just as with wild-type hPGHS-2. A peptide containing residues 259-268 of the P263R mutant was cleaved by trypsin at the same rate as a peptide corresponding to hPGHS-1 residues 272-281, demonstrating that the sequence differences were not responsible for the lack of tryptic cleavage at residue 263 in the hPGHS-2 mutant. Preincubation of hPGHS-2 with graded levels of guanidinium HCl before incubation with proteinase K did not produce large proteolytic fragments, indicating that the hPGHS-2 loop region was not selectively unfolding. The results point to two regions of significant structural difference between PGHS-1 and -2: the Arg277 loop, which is protease-sensitive in PGHS-1 but protease-resistant in PGHS-2, and the C-terminus, which is protease-sensitive in PGHS-2 but not in PGHS-1.
Guo
Q
Q
Department of Internal Medicine, University of Texas Health Science Center at Houston, 77030, USA.
Chang
S
S
Diekman
L
L
Xiao
G
G
Kulmacz
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012756:Sheep; D014357:Trypsin / Q000378:metabolism
D006146:Guanidines; D007527:Isoenzymes; D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D014357:Trypsin; D019286:Endopeptidase K; D019791:Guanidine