The membrane binding domains of prostaglandin endoperoxide H synthases 1 and 2. Peptide mapping and mutational analysis
| Autor: | A G, Spencer, E, Thuresson, J C, Otto, I, Song, T, Smith, D L, DeWitt, R M, Garavito, W L, Smith |
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| Rok vydání: | 1999 |
| Předmět: |
Models
Molecular Protein Folding Binding Sites Glycosylation Sheep Azirines Molecular Sequence Data Membrane Proteins Photoaffinity Labels Transfection Peptide Fragments Protein Structure Secondary Isoenzymes Peroxidases Solubility Cyclooxygenase 2 Prostaglandin-Endoperoxide Synthases COS Cells Cyclooxygenase 1 Animals Humans Amino Acid Sequence |
| Zdroj: | The Journal of biological chemistry. 274(46) |
| ISSN: | 0021-9258 |
| Popis: | Prostaglandin endoperoxide H synthases 1 and 2 (PGHS-1 and -2) are the major targets of nonsteroidal anti-inflammatory drugs. Both isozymes are integral membrane proteins but lack transmembrane domains. X-ray crystallographic studies have led to the hypothesis that PGHS-1 and -2 associate with only one face of the membrane bilayer through a novel, monotopic membrane binding domain (MBD) that is comprised of four short, consecutive, amphipathic alpha-helices (helices A-D) that include residues 74-122 in ovine PGHS-1 (oPGHS-1) and residues 59-108 in human PGHS-2 (hPGHS-2). Previous biochemical studies from our laboratory showed that the MBD of oPGHS-1 lies somewhere between amino acids 25 and 166. In studies reported here, membrane-associated forms of oPGHS-1 and hPGHS-2 were labeled using the hydrophobic, photoactivable reagent 3-trifluoro-3-(m-[(125)I]iodophenyl)diazirine, isolated, and cleaved with AspN and/or GluC, and the photolabeled peptides were sequenced. The results establish that the MBDs of oPGHS-1 and hPGHS-2 reside within residues 74-140 and 59-111, respectively, and thus provide direct provide biochemical support for the hypothesis that PGHS-1 and -2 do associate with membranes through a monotopic MBD. We also prepared HelA, HelB, and HelC mutants of oPGHS-1, in which, for each helix, three or four hydrophobic residues expected to protrude into the membrane were replaced with small, neutral residues. When expressed in COS-1 cells, HelA and HelC mutants exhibited little or no catalytic activity and were present, at least in part, as misfolded aggregates. The HelB mutant retained about 20% of the cyclooxygenase activity of native oPGHS-1 and partitioned in subcellular fractions like native oPGHS-1; however, the HelB mutant exhibited an extra site of N-glycosylation at Asn(104). When this glycosylation site was eliminated (HelB/N104Q mutation), the mutant lacked cyclooxygenase activity. Thus, our mutational analyses indicate that the amphipathic character of each helix is important for the assembly and folding of oPGHS-1 to a cyclooxygenase active form. |
| Databáze: | OpenAIRE |
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