Topology of catalytic portion of prostaglandin I(2) synthase: identification by molecular modeling-guided site-specific antibodies.
Prostaglandin I(2) synthase (PGIS) is an eicosanoid-synthesizing cytochrome P450, located in the endoplasmic reticulum (ER) membrane. The membrane topology of the catalytic portion of PGIS is still unknown. General models of the membrane topology of microsomal P450s have been proposed in two forms: (a) large part of the polypeptide exposed on the cytoplasmic side with an NH(2)-terminal membrane anchor to the ER membrane and (b) deep immersion of the polypeptide in the membrane, as described by J. P. Miller et al. (1996, Biochemistry 35, 1466-1474). We have characterized the membrane topology of catalytic portion of PGIS using molecular modeling-guided site-specific antibodies. A 3D working model of PGIS was constructed by homology modeling using P450(BM-3) crystal structure as a template (S. K. Shyue et al., 1997, J. Biol. Chem. 272, 3657-3662). Three hydrophilic peptides corresponding to different regions of the surface portion of PGIS with residues 109-127 (P109-127), 353-368 (P353-368), and 411-431 (P411-431) predicted from the model and an NH(2)-terminal hydrophobic peptide (residues 1-28, P1-28) were synthesized and used to prepare site-specific antibodies. All three of the hydrophilic peptide antibodies have high titer and are specifically recognized human PGIS, as shown by binding assays and Western blot analysis. In contrast, the hydrophobic NH(2)-terminal peptide has a much lower titer binding to the PGIS protein. The overall arrangement of the PGIS polypeptide with respect to the endoplasmic reticulum (ER) membrane was examined by immunocytochemistry techniques in transiently transfected COS-1 cells with recombinant human PGIS cDNA and in ECV cells expressing endogenous PGIS. The immunofluorescence staining for the cells with selective permeabilization of the plasma membrane using streptolysin O indicated that all three of the hydrophilic peptide antibodies bound to the cytoplasmic surface of the ER membrane. These results provide direct experimental evidence supporting the predicted 3D protein topological model in which the segments are located on the protein surface and the membrane topological model in which PGIS is largely exposed on the cytoplasmic side of the ER membrane. It also led us to conclude that the PGIS substrate, prostaglandin H(2) (PGH(2)), produced by prostaglandin H(2) synthase (PGHS) in the ER lumenal side must pass through the ER membrane barrier to the catalytic site of the PGIS in the cytoplasmic side of the ER membrane.
Lin
Y Z
YZ
Vascular Biology Research Center, The University of Texas Health Science Center, Houston, Texas 77030, USA.
Deng
H
H
Ruan
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KH
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D001665:Binding Sites; D020134:Catalytic Domain; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D004721:Endoplasmic Reticulum / Q000201:enzymology; D000939:Epitopes / Q000276:immunology; D005455:Fluorescent Antibody Technique; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D044262:Prostaglandin H2; D011463:Prostaglandins H / Q000378:metabolism; D011487:Protein Conformation; D014162:Transfection
D000906:Antibodies; D000939:Epitopes; D008565:Membrane Proteins; D010446:Peptide Fragments; D011463:Prostaglandins H; D044262:Prostaglandin H2; D003577:Cytochrome P-450 Enzyme System; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase