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• W1970036469 abstract "By interconverting glucocorticoids, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) exerts an important pre-receptor function and is currently considered a promising therapeutic target. In addition, 11β-HSD1 plays a potential role in 7-ketocholesterol metabolism. Here we investigated the role of the N-terminal region on enzymatic activity and addressed the relevance of 11β-HSD1 orientation into the endoplasmic reticulum (ER) lumen. Previous studies revealed that the luminal orientation of 11β-HSD1 and 50-kDa esterase/arylacetamide deacetylase (E3) is determined by their highly similar N-terminal transmembrane domains. Substitution of Lys5 by Ser in 11β-HSD1, but not of the analogous Lys4 by Ile in E3, led to an inverted topology in the ER membrane, indicating the existence of a second topological determinant. Here we identified Glu25/Glu26 in 11β-HSD1 and Asp25 in E3 as the second determinant for luminal orientation. Our results suggest that the exact location of specific residues rather than net charge distribution on either side of the helix is critical for membrane topology. Analysis of charged residues in the N-terminal domain revealed an essential role of Lys35/Lys36 and Glu25/Glu26 on enzymatic activity, suggesting that these residues are responsible for the observed stabilizing effect of the N-terminal membrane anchor on the catalytic domain of 11β-HSD1. Moreover, activity measurements in intact cells expressing wild-type 11β-HSD1, facing the ER lumen, or mutant K5S/K6S, facing the cytoplasm, revealed that the luminal orientation is essential for efficient oxidation of cortisol. Furthermore, we demonstrate that 11β-HSD1, but not mutant K5S/K6S with cytoplasmic orientation, catalyzes the oxoreduction of 7-ketocholesterol. 11β-HSD1 and E3 constructs with cytosolic orientation of their catalytic moiety should prove useful in future studies addressing the physiological function of these proteins. By interconverting glucocorticoids, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) exerts an important pre-receptor function and is currently considered a promising therapeutic target. In addition, 11β-HSD1 plays a potential role in 7-ketocholesterol metabolism. Here we investigated the role of the N-terminal region on enzymatic activity and addressed the relevance of 11β-HSD1 orientation into the endoplasmic reticulum (ER) lumen. Previous studies revealed that the luminal orientation of 11β-HSD1 and 50-kDa esterase/arylacetamide deacetylase (E3) is determined by their highly similar N-terminal transmembrane domains. Substitution of Lys5 by Ser in 11β-HSD1, but not of the analogous Lys4 by Ile in E3, led to an inverted topology in the ER membrane, indicating the existence of a second topological determinant. Here we identified Glu25/Glu26 in 11β-HSD1 and Asp25 in E3 as the second determinant for luminal orientation. Our results suggest that the exact location of specific residues rather than net charge distribution on either side of the helix is critical for membrane topology. Analysis of charged residues in the N-terminal domain revealed an essential role of Lys35/Lys36 and Glu25/Glu26 on enzymatic activity, suggesting that these residues are responsible for the observed stabilizing effect of the N-terminal membrane anchor on the catalytic domain of 11β-HSD1. Moreover, activity measurements in intact cells expressing wild-type 11β-HSD1, facing the ER lumen, or mutant K5S/K6S, facing the cytoplasm, revealed that the luminal orientation is essential for efficient oxidation of cortisol. Furthermore, we demonstrate that 11β-HSD1, but not mutant K5S/K6S with cytoplasmic orientation, catalyzes the oxoreduction of 7-ketocholesterol. 11β-HSD1 and E3 constructs with cytosolic orientation of their catalytic moiety should prove useful in future studies addressing the physiological function of these proteins. In humans, 11β-HSD1 1The abbreviations used are: 11β-HSD, 11β-hydroxysteroid dehydrogenase; 7KC, 7-ketocholesterol; E3, 50-kDa esterase/arylacetamide deacetylase; EGFP, enhanced green-fluorescent protein; ER, endoplasmic reticulum; HEK, human embryonic kidney; SCAP, sterol regulatory element-binding protein cleavage-activating protein. catalyzes the reduction of biologically inactive cortisone to active cortisol, thereby playing an essential role in the local activation of the glucocorticoid receptor. Recent animal experiments provided insight into the pathophysiological role of 11β-HSD1. Mice deficient of 11β-HSD1 were resistant to hyperglycemia induced by obesity or stress (1Kotelevtsev Y. Holmes M.C. Burchell A. Houston P.M. Schmoll D. Jamieson P. Best R. Brown R. Edwards C.R. Seckl J.R. Mullins J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14924-14929Google Scholar), whereas transgenic mice overexpressing 11β-HSD1 developed visceral obesity with insulin resistance and dyslipidemia. In addition, overexpression of 11β-HSD1 in adipose tissue caused salt-sensitive hypertension mediated by an activated renin-angiotensin system (2Masuzaki H. Paterson J. Shinyama H. Morton N.M. Mullins J.J. Seckl J.R. Flier J.S. Science. 2001; 294: 2166-2170Google Scholar, 3Masuzaki H. Yamamoto H. Kenyon C.J. Elmquist J.K. Morton N.M. Paterson J.M. Shinyama H. Sharp M.G. Fleming S. Mullins J.J. Seckl J.R. Flier J.S. J. Clin. Investig. 2003; 112: 83-90Google Scholar). Experiments in obese and diabetic mice treated with a specific 11β-HSD1 inhibitor showed reduced blood glucose levels and increased insulin sensitivity (4Barf T. Vallgarda J. Emond R. Haggstrom C. Kurz G. Nygren A. Larwood V. Mosialou E. Axelsson K. Olsson R. Engblom L. Edling N. Ronquist-Nii Y. Ohman B. Alberts P. Abrahmsen L. J. Med. Chem. 2002; 45: 3813-3815Google Scholar, 5Alberts P. Nilsson C. Selen G. Engblom L.O. Edling N.H. Norling S. Klingstrom G. Larsson C. Forsgren M. Ashkzari M. Nilsson C.E. Fiedler M. Bergqvist E. Ohman B. Bjorkstrand E. Abrahmsen L.B. Endocrinology. 2003; 144: 4755-4762Google Scholar). Therefore, 11β-HSD1 is currently considered a promising drug target for the treatment of cognitive dysfunction in elderly men and patients with obesity and type 2 diabetes mellitus (6Chrousos G.P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6329-6330Google Scholar, 7Sandeep T.C. Yau J.L. MacLullich A.M. Noble J. Deary I.J. Walker B.R. Seckl J.R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 6734-6739Google Scholar). However, whether 11β-HSD1 is indeed a suitable target for therapeutic treatment of excessive glucocorticoid actions remains to be tested. Recently, we provided evidence that 11β-HSD1 plays a role in the rapid hepatic metabolism of 7-ketocholesterol (7KC) (8Schweizer R.A. Zurcher M. Balazs Z. Dick B. Odermatt A. J. Biol. Chem. 2004; 279: 18415-18424Google Scholar), the major oxysterol in processed cholesterol-rich food and, after 27-hydroxycholesterol, in advanced atherosclerotic plaques (9Bjorkhem I. Andersson O. Diczfalusy U. Sevastik B. Xiu R.J. Duan C. Lund E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8592-8596Google Scholar, 10Brown A.J. Jessup W. Atherosclerosis. 1999; 142: 1-28Google Scholar, 11Schroepfer Jr., G.J. Physiol. Rev. 2000; 80: 361-554Google Scholar). In rats treated with 7KC and the 11β-HSD inhibitor carbenoxolone, 7KC tended to accumulate in liver and plasma (8Schweizer R.A. Zurcher M. Balazs Z. Dick B. Odermatt A. J. Biol. Chem. 2004; 279: 18415-18424Google Scholar). In addition, 11β-HSD1 seems to catalyze the interconversion of 7-hydroxylated dehydroepiandrosterone metabolites (12Robinzon B. Michael K.K. Ripp S.L. Winters S.J. Prough R.A. Arch. Biochem. Biophys. 2003; 412: 251-258Google Scholar) and has a potential role in biotransformation by reducing reactive ketones such as the potent tobacco carcinogen nicotine-derived nitrosamine ketone, the anti-cancer drug oracin, as well as metyrapone and its derivatives (13Sampath-Kumar R. Yu M. Khalil M.W. Yang K. J. Steroid. Biochem. Mol. Biol. 1997; 62: 195-199Google Scholar, 14Bannenberg G. Martin H.J. Belai I. Maser E. Chem. Biol. Interact. 2003; 143: 449-457Google Scholar, 15Maser E. Friebertshauser J. Volker B. Chem. Biol. Interact. 2003; 143: 435-448Google Scholar, 16Wsol V. Szotakova B. Skalova L. Maser E. Chem. Biol. Interact. 2003; 143: 459-468Google Scholar). Despite its importance, relatively little is known about the molecular mechanisms by which 11β-HSD1 exerts its physiological function. 11β-HSD1 belongs to the family of short-chain dehydrogenases-reductases, characterized by a core domain with conserved regions, including the Rossmann fold for binding of the cofactor NADP(H) and the Tyr-(Xaa)3-Lys motif in the catalytic site, and less conserved N- and C-terminal sequences (17White P.C. Mune T. Agarwal A.K. Endocr. Rev. 1997; 18: 135-156Google Scholar, 18Stewart P.M. Krozowski Z.S. Vitam. Horm. 1999; 57: 249-324Google Scholar). Several studies using 11β-HSD1 constructs with N-terminal deletions demonstrated that this part of the enzyme is not only essential to anchor the enzyme to the ER membrane but also for stability and catalytic activity (19Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Google Scholar, 20Walker E.A. Clark A.M. Hewison M. Ride J.P. Stewart P.M. J. Biol. Chem. 2001; 276: 21343-21350Google Scholar, 21Blum A. Raum A. Maser E. Biochemistry. 2003; 42: 4108-4117Google Scholar, 22Blum A. Maser E. Chem. Biol. Interact. 2003; 143: 469-480Google Scholar). The residues involved, however, were not identified. We have shown previously (19Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Google Scholar, 23Mziaut H. Korza G. Hand A.R. Gerard C. Ozols J. J. Biol. Chem. 1999; 274: 14122-14129Google Scholar) that the single N-terminal transmembrane helix is responsible for the luminal orientation of the catalytic moiety of 11β-HSD1. However, in contrast to 11β-HSD2, whose cytoplasmic orientation is required to tether the mineralocorticoid receptor to the ER membrane in the absence of steroid and that prevents its occupation by cortisol through conversion of cortisol to cortisone (24Odermatt A. Arnold P. Frey F.J. J. Biol. Chem. 2001; 276: 28484-28492Google Scholar), the physiological role of the luminal orientation of 11β-HSD1 remained unclear. In our previous study (19Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Google Scholar), we demonstrated that substitution of Lys5 by Ser in the short cytoplasmic N terminus of 11β-HSD1 inverted its topology in the ER membrane. Surprisingly, no difference was found for the oxoreduction of 11-dehydrocorticosterone between the mutant enzyme with cytoplasmic orientation and wild-type 11β-HSD1 upon expression in HEK-293 cells. The N-terminal transmembrane span of 11β-HSD1 is highly similar to that of the 50-kDa esterase/arylacetamide deacetylase (E3) (23Mziaut H. Korza G. Hand A.R. Gerard C. Ozols J. J. Biol. Chem. 1999; 274: 14122-14129Google Scholar), two proteins that are otherwise unrelated (Fig. 1A). E3, which is highly expressed in liver and adrenal glands and to a lesser extent in small intestine, stomach, kidney, and pancreas (25Trickett J.I. Patel D.D. Knight B.L. Saggerson E.D. Gibbons G.F. Pease R.J. J. Biol. Chem. 2001; 276: 39522-39532Google Scholar), acts as an N-deacetylase catalyzing hydrolytic reactions and plays a potential role in the prevention of arylamine-induced carcinogenesis (26Probst M.R. Beer M. Beer D. Jeno P. Meyer U.A. Gasser R. J. Biol. Chem. 1994; 269: 21650-21656Google Scholar). E3 might be involved, like the putative triacylglycerol hydrolase E1, in the assembly of hepatic very low density lipoprotein in the ER lumen (25Trickett J.I. Patel D.D. Knight B.L. Saggerson E.D. Gibbons G.F. Pease R.J. J. Biol. Chem. 2001; 276: 39522-39532Google Scholar). A significantly reduced expression of E3 was observed in insulin-deficient diabetes, which is characterized by a severe decrease in the secretion of hepatic very low density lipoprotein triacylglycerol. Unlike Lys5 in 11β-HSD1, substitution of the analogous Lys4 in E3 had no effect on topology, suggesting the existence of a second determinant for the orientation of these enzymes in the ER membrane (19Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Google Scholar, 23Mziaut H. Korza G. Hand A.R. Gerard C. Ozols J. J. Biol. Chem. 1999; 274: 14122-14129Google Scholar). Here we tested the hypothesis whether the negatively charged residues on the luminal side of the transmembrane span might act as a second determinant for the orientation of these two enzymes in the ER membrane, and we studied the effect of charged amino acid residues on 11β-HSD1 activity. In addition, we investigated the role of the luminal orientation of 11β-HSD1 on the oxidation of cortisol and the oxoreduction of cortisone and 7KC. Materials—Cell culture reagents were purchased from Invitrogen; [1,2,6,7-3H]cortisone and [1,2,6-3H]7KC were from American Radiolabeled Chemicals, St. Louis, MO; [1,2,6,7-3H]cortisol was from Amersham Biosciences; 7KC and 7β-hydroxycholesterol were from Steraloids (Wilton, NH); and fluorescence-labeled antibodies were from Molecular Probes. Proteinase K was from Roche Diagnostics, and all other chemicals were from Fluka AG, Buchs, Switzerland, and were of the highest grade available. Construction of Plasmids—The plasmid for expression of FLAG epitope-tagged human 11β-HSD1 was constructed as described (19Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Google Scholar). Mutant 11β-HSD1 constructs were generated by site-directed mutagenesis according to the QuickChange mutagenesis kit (Stratagene, La Jolla, CA). The rabbit E3-enhanced green fluorescent protein (EGFP) chimera, containing the N-terminal membrane anchor sequence of E3 with a C-terminal EGFP, was constructed as described (23Mziaut H. Korza G. Hand A.R. Gerard C. Ozols J. J. Biol. Chem. 1999; 274: 14122-14129Google Scholar). For immunofluorescence experiments, a construct containing the N-terminal 34 amino acids of rabbit E3 followed by a FLAG epitope tag was generated by three-piece ligation of an EcoRI-BamHI fragment containing the N-terminal 34 amino acids of E3, a BamHI-XbaI fragment encoding the FLAG epitope (MDYKDDDD), and an EcoRI-XbaI pcDN3 expression vector fragment. Mutant E3-FLAG and-EGFP constructs were made by site-directed mutagenesis. All constructs were verified by sequencing. Cell Culture and Transient Transfection—HEK-293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 4.5 g/liter glucose, 50 units/ml penicillin, 50 mg/ml streptomycin, and 2 mm glutamine. For microscopy experiments, cells were seeded at ∼10% confluence onto glass coverslips placed in 6-well plates. After growth for 24 h at 37 °C under 5% CO2, cells were transfected by Ca2+-phosphate precipitation with 2 μg/well of the corresponding expression plasmid and 1 μg/well EGFP control plasmid. After incubation for 8 h, the medium was replaced to remove the Ca2+-phosphate precipitate. For isolation of microsomes and activity analysis, cells were seeded to 50% confluence in 10-cm dishes and transfected, and medium was replaced or cells were washed three times with steroid-free Dulbecco's modified Eagle's medium (doubly charcoal-treated). Selective Permeabilization and Immunofluorescence Analysis—Immunofluorescence analysis was performed 48 h post-transfection as described previously (24Odermatt A. Arnold P. Frey F.J. J. Biol. Chem. 2001; 276: 28484-28492Google Scholar). Briefly, paraformaldehyde (4%) fixed cells coexpressing the corresponding FLAG-tagged construct and EGFP control were washed four times with the buffer NAPS (150 mm sodium phosphate, pH 7.4, 120 mm sucrose). For complete permeabilization of membranes, cells were blocked in NAPS buffer containing 1% milk powder and either 0.5% Triton X-100, for 11β-HSD1 constructs, or 1% saponin, for E3 constructs. The fluorescence signal obtained for E3 constructs was much stronger when saponin was used instead of Triton X-100, an observation made previously with N-terminally FLAG-tagged 11β-HSD1. The reason is unknown, but it seems that Triton X-100 interferes with antibody-epitope interactions in close proximity to the membrane. For semipermeabilization of the plasma membrane, 25 μm digitonin was used (19Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Google Scholar). FLAG-tagged constructs were detected using anti-FLAG antibody M2 as primary antibody and ALEXA-594 goat anti-mouse secondary antibody. EGFP served as a transfection efficiency control. Following incubation with antibodies in NAPS containing 0.1% milk, samples were washed four times with NAPS and treated with Slow Fade Antifade kit (Molecular Probes). Samples were analyzed on a Carl Zeiss confocal microscope LSM410 (Carl Zeiss, Goettingen, Germany). Isolation of Microsomes from HEK-293 Cells—HEK-293 cells were transfected with 8 μg of the corresponding expression plasmid per 10-cm dish. Cells from four dishes were collected 48 h post-transfection, washed with phosphate-buffered saline, and centrifuged at 150 × g for 3 min, and the pellet was resuspended in 1.2 ml of buffer containing 10 mm Tris-HCl, pH 7.5, 0.5 m MgCl2, and protease inhibitor (Complete, Roche Diagnostics). Cells were sonicated; 1.2 ml of isotonic buffer (1 m sucrose, 10 mm Tris-HCl, pH 7.5, 2.5 m NaCl, 1 mm dithiothreitol) was added, and lysates were centrifuged at 1,000 × g for 10 min at 4 °C, followed by centrifugation at 11,000 × g for 10 min at 4 °C and 100,000 × g for 1 h at 4 °C. The pellet was resuspended in 300 μl of buffer containing 10 mm Tris-HCl, pH 7.5, 0.5 m sucrose, 1.25 m NaCl, and 0.5 mm dithiothreitol. The protein concentration was adjusted to 1 mg/ml, and microsomal preparations were shock-frozen in liquid nitrogen and stored at –70 °C until analysis. Protease Protection Assay and Immunoblotting—Microsomes (20 μg of proteins) were incubated in a total volume of 25 μl with 0.25 μg/μl proteinase K (Roche Diagnostics) for 30 min on ice in the presence or absence of 0.5% Triton X-100. Proteinase K was inactivated by adding 2.5 μl of 10 mm phenylmethylsulfonyl fluoride in isopropyl alcohol for 5 min, followed by solubilization of proteins with SDS sample buffer and boiling for 5 min. Proteins were subjected to SDS-PAGE and Western blot analysis using anti-FLAG antibody M2 or anti-EGFP antibody as primary antibodies and secondary horseradish peroxidase-conjugated antibody (Roche Diagnostics). Antibody binding was visualized using the enhanced chemiluminescence Western detection system (Pierce). Assay for 11β-HSD—11β-HSD1-dependent oxidation and oxoreduction were measured as described previously (19Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Google Scholar, 27Schweizer R.A. Atanasov A.G. Frey B.M. Odermatt A. Mol. Cell. Endocrinol. 2003; 212: 41-49Google Scholar). Briefly, the rate of conversion of cortisol to cortisone or the reverse reaction was determined in a final volume of 20 μl in a TG1 buffer (20 mm Tris-HCl, pH 7.4, 1 mm EGTA, 1 mm EDTA, 1 mm MgCl2, 100 mm NaCl, 20% glycerol) supplemented with 400 μm NADP+ or NADPH, 30 nCi of 3H-labeled substrate, and unlabeled substrate at different concentrations ranging from 12.5 nm to 2 μm. Cell lysates were prepared by washing transfected cells with phosphate-buffered saline, centrifugation for 3 min at 150 × g, removal of the supernatant, and quick-freezing the cell pellet in a dry ice ethanol bath. Cell pellets were resuspended in buffer TG1, sonicated, and used immediately for activity assays. The reactions were started by mixing 10 μl of cell extract corresponding to 2–5 μg of total proteins with 10 μl of reaction mixture and incubated for 10–20 min at 37 °C. When measuring activities in intact cells, steroid-free medium was used, and cofactor was omitted. To assess the effect of cyclosporin A, cells were preincubated for 15 min with 50 μm cyclosporin A prior to addition of cortisone. The reactions were stopped by adding an excess of unlabeled steroids in methanol, followed by separation of steroids by TLC. Enzyme kinetics were analyzed by nonlinear regression using Data Analysis Toolbox (MDL Information Systems Inc.) assuming first-order rate kinetics. Hill coefficients for measurements in lysates were ranging between 0.94 and 1.17. Data represent mean ± S.D. of at least four independent transfections. Oxoreduction of 7KC was determined as described (8Schweizer R.A. Zurcher M. Balazs Z. Dick B. Odermatt A. J. Biol. Chem. 2004; 279: 18415-18424Google Scholar). Briefly, freshly prepared lysates were suspended in buffer TG1 and 400 μm NADP+, 400 nm 7KC, and 30 nCi of 3H-labeled 7KC added, followed by incubation at 37 °C for 15 or 30 min. Intact cells were incubated in steroid-free medium in the absence of cofactor for 30 or 60 min. The reactions were stopped by adding an excess of unlabeled oxycholesterols in methanol, followed by separation by TLC and scintillation counting. Sequence Comparison of the N Termini of 11β-HSD1 and E3—An alignment of the sequences of human 11β-HSD1 and rabbit E3 revealed that both proteins share a similar N-terminal region (Fig. 1A) with a short and positively charged N-terminal cytoplasmic part, a single transmembrane span followed by negatively charged residues immediately downstream of the membrane helix, and a large C-terminal luminal domain. In previous studies, we have shown that substitution of Lys5 by Ser led to inverted orientation of 11β-HSD1 in the ER membrane (19Odermatt A. Arnold P. Stauffer A. Frey B.M. Frey F.J. J. Biol. Chem. 1999; 274: 28762-28770Google Scholar), whereas substitution of the analogous Lys4 by Ile in E3 had no effect on topology (23Mziaut H. Korza G. Hand A.R. Gerard C. Ozols J. J. Biol. Chem. 1999; 274: 14122-14129Google Scholar), indicating the existence of a second determinant for the topology of these two proteins. The Cytoplasmic Lys4 and the Luminal Asp25 Determine the Topology of E3—To test the hypothesis that the negatively charged residues immediately downstream of the membrane span represent this second topology determining motif, we generated a fusion protein encoding the N-terminal 34 amino acids of E3 followed by a FLAG epitope for facilitated immunodetection, and we subjected this construct to site-specific mutagenesis (Fig. 1B). Cells coexpressing the corresponding FLAG-tagged construct and EGFP control protein were either completely permeabilized with 1% saponin or the plasma membrane was selectively permeabilized with 25 μm digitonin, allowing restricted access of the anti-FLAG antibody to the cytosolic compartment. Cells were then incubated with anti-FLAG antibody and red fluorescent secondary antibody and analyzed by fluorescence microscopy. Cells expressing EGFP were analyzed for the presence of red fluorescence signal from the corresponding E3 construct, whereby 200–300 cells were counted in a typical experiment. By using saponin, over 95% of cells expressing wild-type E3 stained positive but less than 5% when digitonin was used, in line with the luminal orientation of E3 (Fig. 2). Mutant K4I showed wild-type orientation, confirming previous findings (23Mziaut H. Korza G. Hand A.R. Gerard C. Ozols J. J. Biol. Chem. 1999; 274: 14122-14129Google Scholar). To investigate the role of negatively charged residues at the luminal side of the transmembrane helix, we replaced Asp25, Glu28, and Glu29 to Lys in either wild-type E3 or mutant K4I (Fig. 1B). Substitution of the negatively charged residues by Lys led to inverted insertion into the ER membrane in mutant K4I/D25K/E28K/E29K but not in mutant D25K/E28K/E29K (Fig. 2). Additional mutagenesis revealed that substitution of Asp25/Glu28/Glu29 to Asn25/Gln28/Gln29 and a single substitution of Asp25 by Lys in mutant K4I both were sufficient to invert the orientation in the ER membrane. All of the mutant proteins analyzed showed exclusive localization to the ER membrane, indicating that they are not important for ER retention. These results demonstrate that both Lys4 and Asp25 are essential for determining the topology of E3. The effects on topology were confirmed by subjecting microsomal vesicles expressing wild-type or mutant constructs to proteinase K digestion in the presence or absence of detergent (data not shown). The Luminal Residues Glu25 and Glu26 Are Important Determinants for the Topology of 11β-HSD1—In analogy to Asp25 of E3, 11β-HSD1 contains two glutamate residues (Glu25 and Glu26) immediately downstream of the membrane span. To investigate whether the luminal di-glutamate motif plays a role in determining the topology of 11β-HSD1, we generated a series of constructs with a C-terminal FLAG epitope (Fig. 1C) and analyzed them in fully permeabilized (0.5% Triton X-100) or semipermeabilized (25 μm digitonin) cells by immunofluorescence detection. Substitution of both Glu25 and Glu26 to Lys led to an inverted orientation of 11β-HSD1 in the ER membrane (Fig. 3). Substitution of Glu25 by Lys and Glu26 by Gln (mutant E25K/E26Q) did not change the orientation of the mutant enzyme, indicating that the change from positive to negative charge at both positions is necessary to invert the topology of 11β-HSD1. We have shown previously that mutant K5S but not K6S showed inverted topology. To assess a potential role of Lys6 in determining topology, we mutated Glu25 and Glu26 in mutant K6S. Independent of the residue at position 6, the orientation of the enzyme was only inverted when both Glu25 and Glu26 were changed to Lys (Fig. 1C and Fig. 3). These results indicate that Lys6 is not involved in determining the topology of 11β-HSD1. In addition, all mutations analyzed in the present study did not alter the restricted expression of 11β-HSD1 in the ER membrane (not shown). To confirm the effect of these mutations on 11β-HSD1 topology, microsomal vesicles expressing FLAG-tagged wild-type or mutant 11β-HSD1 constructs were subjected to proteinase K digestion in the presence or absence of detergents, followed by separation on SDS-PAGE and detection with anti-FLAG antibody on Western blots. As seen in Fig. 4, in intact vesicles mutants E25K/E26K and K6S/E25K/E26K were completely digested by proteinase K, whereas wild-type 11β-HSD1 and all other mutants were protected from proteinase K-dependent digestion. Upon addition of Triton X-100, all constructs were digested. These results are consistent with the findings from fluorescence microscopic analyses of semipermeabilized cells, demonstrating an essential role of Glu25 and Glu26 in determining the orientation of 11β-HSD1 in the ER membrane. The Effect of Charged Residues in the N-terminal Region of 11β-HSD1 on Enzymatic Activity in Lysates—To assess the effect of charged residues in the N-terminal region of 11β-HSD1 on enzymatic activity, the reduction of cortisone to cortisol was measured in lysates of cells expressing wild-type or mutant 11β-HSD1. None of the mutations had a significant effect on apparent Km values (Table I); however, replacement of both Glu25 and Glu26 by Gln or Lys led to a significant decrease in activity (p < 0.01), with a tendency for a loss of Vmax when substituting the negatively charged Glu residues first by polar Gln and then by positively charged Lys. The fact that mutant K6S/E25K/E26Q with luminal orientation and mutant K6S/E25K/E26K with cytoplasmic orientation both had similarly reduced activities suggests that the di-glutamate motif at position 25/26 stabilizes enzymatic activity independent of the orientation of 11β-HSD1 in the ER membrane. In addition, mutant K5S/K6S showed kinetic values comparable with that of wild-type 11β-HSD1, indicating that the orientation in the ER membrane itself does not affect enzymatic activity. Similar effects of these mutations were observed for the oxidation of corticosterone (data not shown).Table IReduction of cortisone by wild-type and mutant 11β-HSD1 in lysatesConstructKmaApparent Km (nm) and apparent Vmax (nmol × h-1 × mg-1 of total protein) values were calculated by nonlinear regression using Data Analysis Toolbox (MDL Information Systems Inc.) assuming first-order rate kinetics (Hill coefficients ranging between 0.94 and 1.17).VmaxaApparent Km (nm) and apparent Vmax (nmol × h-1 × mg-1 of total protein) values were calculated by nonlinear regression using Data Analysis Toolbox (MDL Information Systems Inc.) assuming first-order rate kinetics (Hill coefficients ranging between 0.94 and 1.17).,bFor calculation of Vmax, the amount of 11β-HSD1 protein per mg of total proteins was determined, and values were normalized to the expression of FLAG-tagged wild-type 11β-HSD1 by semiquantitative densitometric analysis of Western blotsnmnmol × h-1 × mg-111β-HSD1 (wild type)336 ± 391.88 ± 0.23E25Q307 ± 351.55 ± 0.15E25K346 ± 201.66 ± 0.27E26K303 ± 371.23 ± 0.33cp < 0.05 compared with wild type.K6S/E25K314 ± 211.63 ± 0.30E25Q/E26Q308 ± 291.07 ± 0.13dp < 0.01 compared with wild type.K6S/E25Q/E26Q302 ± 220.80 ± 0.17dp < 0.01 compared with wild type.E25K/E26Q300 ± 280.84 ± 0.15dp < 0.01 compared with wild type.K6S/E25K/E26Q314 ± 340.57 ± 0.17dp < 0.01 compared with wild type.E25K/E26K299 ± 220.37 ± 0.08dp < 0.01 compared with wild type.K6S/E25K/E26K344 ± 490.59 ± 0.13dp < 0.01 compared with wild type.K5S/K6S/E25K/E26K340 ± 360.39 ± 0.02dp < 0.01 compared with wild type.K5S/K6S295 ± 251.75 ± 0.14K35S/K36SNDeActivity was not detectable despite normal expression levels.NDeActivity was not detectable despite normal expression levels.a Apparent Km (nm) and apparent Vmax (nmol × h-1 × mg-1 of total protein) values were calculated by nonlinear regression using Data Analysis Toolbox (MDL Information Systems Inc.) assuming first-order rate kinetics (Hill coefficients ranging between 0.94 and 1.17).b For calculation of Vmax, the amount of 11" @default.
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• W1970036469 date "2004-07-01" @default.
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• W1970036469 title "Appropriate Function of 11β-Hydroxysteroid Dehydrogenase Type 1 in the Endoplasmic Reticulum Lumen Is Dependent on Its N-terminal Region Sharing Similar Topological Determinants with 50-kDa Esterase" @default.
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