Comparison of prostaglandin H synthase isoform structures using limited proteolytic digestion
| Autor: | Qiupeng Guo, Laura Diekman, Richard J. Kulmacz, Guishan Xiao, Shiliang Chang |
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| Rok vydání: | 1997 |
| Předmět: |
Models
Molecular Protein Denaturation Protein Folding Arginine Protein Conformation Immunoblotting Molecular Sequence Data Biophysics Peptide Biology Cleavage (embryo) Biochemistry Guanidines medicine Animals Humans Trypsin Amino Acid Sequence Molecular Biology Guanidine chemistry.chemical_classification Chymotrypsin Sheep Proteolytic enzymes Membrane Proteins Proteinase K Molecular biology Peptide Fragments Recombinant Proteins Amino acid Isoenzymes chemistry Cyclooxygenase 2 Prostaglandin-Endoperoxide Synthases biology.protein Cyclooxygenase 1 Electrophoresis Polyacrylamide Gel Endopeptidase K medicine.drug |
| Zdroj: | Archives of biochemistry and biophysics. 344(1) |
| ISSN: | 0003-9861 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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