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J. Biol. Chem., Vol. 279, Issue 30, 31131-31138, July 23, 2004

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Appropriate Function of 11-Hydroxysteroid Dehydrogenase Type 1 in the Endoplasmic Reticulum Lumen Is Dependent on Its N-terminal Region Sharing Similar Topological Determinants with 50-kDa Esterase^*

Christoph Frick, Atanas G. Atanasov, Peter Arnold, Juris Ozols, and Alex Odermatt¶

From the Division of Nephrology and Hypertension, Department of Clinical Research, University of Berne, 3010 Berne, Switzerland and the Biochemistry Department, University of Connecticut Health Center, Farmington, Connecticut 06030

Received for publication, December 15, 2003 , and in revised form, April 28, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
By interconverting glucocorticoids, 11-hydroxysteroid dehydrogenase type 1 (11-HSD1) exerts an important pre-receptor function and is currently considered a promising therapeutic target. In addition, 11-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 11-HSD1 orientation into the endoplasmic reticulum (ER) lumen. Previous studies revealed that the luminal orientation of 11-HSD1 and 50-kDa esterase/arylacetamide deacetylase (E3) is determined by their highly similar N-terminal transmembrane domains. Substitution of Lys⁵ by Ser in 11-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 11-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 11-HSD1. Moreover, activity measurements in intact cells expressing wild-type 11-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 11-HSD1, but not mutant K5S/K6S with cytoplasmic orientation, catalyzes the oxoreduction of 7-ketocholesterol. 11-HSD1 and E3 constructs with cytosolic orientation of their catalytic moiety should prove useful in future studies addressing the physiological function of these proteins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In humans, 11-HSD1¹ 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 11-HSD1. Mice deficient of 11-HSD1 were resistant to hyperglycemia induced by obesity or stress (1), whereas transgenic mice overexpressing 11-HSD1 developed visceral obesity with insulin resistance and dyslipidemia. In addition, overexpression of 11-HSD1 in adipose tissue caused salt-sensitive hypertension mediated by an activated renin-angiotensin system (2, 3). Experiments in obese and diabetic mice treated with a specific 11-HSD1 inhibitor showed reduced blood glucose levels and increased insulin sensitivity (4, 5). Therefore, 11-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 (6, 7). However, whether 11-HSD1 is indeed a suitable target for therapeutic treatment of excessive glucocorticoid actions remains to be tested.

Recently, we provided evidence that 11-HSD1 plays a role in the rapid hepatic metabolism of 7-ketocholesterol (7KC) (8), the major oxysterol in processed cholesterol-rich food and, after 27-hydroxycholesterol, in advanced atherosclerotic plaques (9–11). In rats treated with 7KC and the 11-HSD inhibitor carbenoxolone, 7KC tended to accumulate in liver and plasma (8). In addition, 11-HSD1 seems to catalyze the interconversion of 7-hydroxylated dehydroepiandrosterone metabolites (12) 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 (13–16).

Despite its importance, relatively little is known about the molecular mechanisms by which 11-HSD1 exerts its physiological function. 11-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 (17, 18). Several studies using 11-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 (19–22). The residues involved, however, were not identified.

We have shown previously (19, 23) that the single N-terminal transmembrane helix is responsible for the luminal orientation of the catalytic moiety of 11-HSD1. However, in contrast to 11-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 (24), the physiological role of the luminal orientation of 11-HSD1 remained unclear. In our previous study (19), we demonstrated that substitution of Lys⁵ by Ser in the short cytoplasmic N terminus of 11-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 11-HSD1 upon expression in HEK-293 cells.

The N-terminal transmembrane span of 11-HSD1 is highly similar to that of the 50-kDa esterase/arylacetamide deacetylase (E3) (23), 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 (25), acts as an N-deacetylase catalyzing hydrolytic reactions and plays a potential role in the prevention of arylamine-induced carcinogenesis (26). E3 might be involved, like the putative triacylglycerol hydrolase E1, in the assembly of hepatic very low density lipoprotein in the ER lumen (25). 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 11-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 (19, 23).

View larger version (36K): [in this window] [in a new window]   FIG. 1. A, schematic representation of the N-terminal membrane anchoring region of 11-HSD1 and E3. The positively charged residues on the cytoplasmic side and the negatively charged residues on the luminal side that are important for determining the orientation of 11-HSD1 and E3 are indicated in boldface italic. B, wild-type and mutant constructs of rabbit E3; C, human 11-HSD1. The name of the mutant protein is given on the left. Mutated residues are in boldface italic, and the orientation of the corresponding construct in the ER membrane, as determined by immunohistochemistry and protease protection assay, is indicated on the right.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 7-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 11-HSD1 was constructed as described (19). Mutant 11-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 (23). 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 (24). 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 11-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 11-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 (19). 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 x 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 x g for 10 min at 4 °C, followed by centrifugation at 11,000 x g for 10 min at 4 °C and 100,000 x 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 11-HSD—11-HSD1-dependent oxidation and oxoreduction were measured as described previously (19, 27). 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 x 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 (8). 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Sequence Comparison of the N Termini of 11-HSD1 and E3—An alignment of the sequences of human 11-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 11-HSD1 in the ER membrane (19), whereas substitution of the analogous Lys⁴ by Ile in E3 had no effect on topology (23), 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 (23). 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).

View larger version (84K): [in this window] [in a new window]   FIG. 2. Orientation in the ER membrane of constructs containing wild-type or mutant membrane anchoring regions of rabbit E3. Wild-type or mutant proteins indicated at the top of each lane were coexpressed with EGFP in HEK-293 cells. Plasma membranes of cells were selectively permeabilized with 25 µM digitonin (1st two rows) or membranes completely permeabilized with 1% saponin (3rd and 4th rows), followed by detection of the C-terminally attached FLAG epitope by mouse monoclonal anti-FLAG antibody M2 and secondary red fluorescent ALEXA-594 anti-mouse antibody using confocal microscopy. Representative experiments of cells expressing key mutant constructs are shown (results summarized in Fig. 1).

View larger version (49K): [in this window] [in a new window]   FIG. 3. Orientation of wild-type (wt) or mutant human 11-HSD1 in the ER membrane. HEK-293 cells were cotransfected with C-terminally FLAG-tagged wild-type or mutant 11-HSD1 (indicated at the top of each lane) and EGFP. Plasma membranes of cells were selectively permeabilized with 25 µM digitonin (1st two rows) or membranes completely permeabilized with 0.5% Triton X-100 (3rd and 4th rows), followed by immunofluorescence detection. Representative samples from cells expressing key mutant constructs are shown (results are summarized in Fig. 1).

To confirm the effect of these mutations on 11-HSD1 topology, microsomal vesicles expressing FLAG-tagged wild-type or mutant 11-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 11-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 11-HSD1 in the ER membrane.

View larger version (12K): [in this window] [in a new window]   FIG. 4. Proteinase K sensitivity of wild-type and mutant 11-HSD1 in ER microsomes. HEK-293 cells were transfected with FLAG epitope-tagged wild-type 11-HSD1 (A), mutant E25K/E26K (B), K6S/E25K/E26Q (C), E25K/E26Q (D), or K6S/E25K/E26K (E), and microsomal vesicles were isolated as described under "Experimental Procedures." Microsomal preparations were incubated for 30 min at 4 °C in the presence (+) or absence (–) of 0.25 µg/µl proteinase K and 0.5% Triton X-100. Proteins were separated on 12.5% SDS-PAGE and transferred to nitrocellulose, followed by detection with mouse monoclonal anti-FLAG antibody M2 and secondary anti-mouse horseradish peroxidase antibody.

View this table: [in this window] [in a new window]   TABLE I Reduction of cortisone by wild-type and mutant 11-HSD1 in lysates Reduction of cortisone by lysates of HEK-293 cells transfected with various 11-HSD1 constructs was determined as described under "Experimental Procedures." Activities are expressed in terms of apparent Km and apparent Vmax. Data were obtained from 4 to 6 independent experiments and are presented as mean ± S.D.

Effect of the Orientation of 11-HSD1 in the ER Membrane on the Interconversion of Glucocorticoids in Intact Cells—To elucidate the potential role of the luminal orientation of 11-HSD1 on its activity, we compared the interconversion of cortisone and cortisol in lysates and intact HEK-293 cells expressing either luminally oriented wild-type 11-HSD1 or cytoplasmically oriented mutant K5S/K6S. Measurements in lysates yielded similar kinetic parameters for the oxidation of cortisol and the oxoreduction of cortisone with both enzymes (Tables II and III). In intact cells, the oxoreduction of cortisone was not different between wild-type 11-HSD1 and mutant K5S/K6S; however, mutant K5S/K6S showed 50% lower V_max (p < 0.01) than wild-type for the oxidation of cortisol without a change in K_m. Approximately 50% lower V_max but similar K_m was also observed for the oxidation of corticosterone by mutant K5S/K6S (not shown).

View this table: [in this window] [in a new window]   TABLE II A comparison of the interconversion of cortisone and cortisol between wild-type 11-HSD1 and mutant K5S/K6S HEK-293 cells were transfected with cDNA for wild-type 11-HSD1 or mutant K5S/K6S and cultured in steroid-free medium 16 h prior to the determination of activities in lysates or intact cells as described under "Experimental Procedures." Activities (mean ± S.D.) are expressed in terms of apparent Km and apparent Vmax values and were obtained from at least four independent experiments.

View this table: [in this window] [in a new window]   TABLE III Effect of cyclosporin A on oxoreduction of cortisone by wild-type 11-HSD1 and mutant K5S/K6S in intact cells HEK-293 cells transiently expressing wild-type 11-HSD1 or mutant K5S/K6S were cultured in steroid-free medium for 16 h, preincubated with 50 µ m cyclosporin A, followed by determination of activities as described under "Experimental Procedures." Activities (mean ± S.D.) are expressed in terms of apparent Km, Vmax, and kcat (Vmax/Km) values and were obtained from four independent experiments.

The Luminal Orientation of 11-HSD1 Is Required for the Oxoreduction of 7KC in Intact Cells—Recently, we have shown (8) that 11-HSD1 plays an essential role in the metabolism of 7KC in intact cells. Here we compared the ability of luminally oriented wild-type enzyme and cytoplasmic mutant K5S/K6S to catalyze the oxoreduction of 7KC in lysates and intact HEK-293 cells. Both enzymes readily converted 7KC to 7-hydroxycholesterol in lysates, but only wild-type 11-HSD1 catalyzed the oxoreduction of 7KC in intact cells (Fig. 5). Mutant K5S/K6S showed background activity comparable with that of untransfected cells. These results demonstrate that the luminal orientation of 11-HSD1 is essential for the oxoreduction of 7KC.

View larger version (25K): [in this window] [in a new window]   FIG. 5. The luminal orientation of 11-HSD1 is required for the reduction of 7KC. HEK-293 cells were transfected either with a control plasmid, wild-type 11-HSD1, or mutant K5S/K6S and cultured in steroid-free medium. The reduction of 7KC to 7-hydroxycholesterol was determined by incubation of lysates in the presence of NADPH and 400 nM 7KC for 15 (A) or 30 min (B) at 37 °C. Intact cells were incubated in steroid-free medium containing 400 nM 7KC without cofactor for 30 (C) or 60 min (D). 7-Oxycholesterol metabolites were analyzed by separation on TLC and scintillation counting as described under "Experimental Procedures." White bars, 7KC; black bars, 7-hydroxycholesterol. Results are expressed as a percentage of initially supplied 7KC (mean ± S.D. from four independent experiments).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

According to the positive-inside rule, net charges on the polypeptide sequences flanking the transmembrane span determine the orientation, with the more positive of the two remaining on the cytoplasmic side of the membrane (31, 32). The positive-inside rule correctly predicts both the topology of wild-type E3, with N-terminal charge +1 and C-terminal charge –3(+1/–3), when considering six residues downstream of the membrane span, and 11-HSD1 (+2/–2). However, the present analysis of several mutant proteins revealed that specific amino acid residues rather than net charge distribution determine the topology of these two enzymes.

We have shown previously that E3 mutant K4I (0/–3) still adopts wild-type topology (N_cyt/C_lum) (23). Therefore, we now analyzed the role of luminal, negatively charged amino acid residues. Substitution by Lys of the three negatively charged residues that are closest to the membrane span (mutant D25K/E27K/E28K (+1/+3)) did not alter its orientation in the ER membrane despite the excess of positive charges introduced at the luminal side (Fig. 2). Thus, the topology of this mutant clearly does not follow the positive-inside rule. However, substitution of Asp²⁵ by Lys (mutant K4I/D25K (0/–1)) or of Asp²⁵ by Asn and Glu²⁵/Glu²⁶ by Gln (mutant K4I/D25N/E27Q/E28Q (0/0)) led to an inverted orientation in the ER membrane. This indicates that Lys⁴ is the major determinant of E3 topology, whereby the introduction of several positively charged residues on the C-terminal end of the membrane span cannot overcome the dominant N-terminal signal. In mutants lacking the N-terminal signal, Asp²⁵, the negatively charged residue closest to the membrane span, dictates the topology.

That the topology of 11-HSD1 is determined by the appropriate charge at a specific position rather than by the net charge distribution is demonstrated by the fact that mutant K5S (+1/–2) adopts N_lum/C_cyt topology, whereas K6S shows wild-type N_cyt/C_lum orientation (19). The importance of Lys⁵ is also reflected by its conservation in all known species, whereas Lys⁶ is replaced by Thr in squirrel monkey and by Asn in mouse. Substitution of both Glu²⁵ and Glu²⁶ by Lys (Fig. 1C) led to an inverted N_lum/C_cyt orientation (Figs. 3 and 4), indicating that in 11-HSD1 the introduction of two positive charges at the C-terminal side of the membrane span is dominant over the N-terminal Lys⁵ signal. This stands in contrast to E3, where the effect of Lys⁴ is not affected by the substitution of three negative by positive charges at the C-terminal side of the membrane span. In mutant K6S, like in wild-type 11-HSD1, both Glu²⁵ and Glu²⁶ had to be replaced by Lys to invert its topology, further demonstrating that Lys⁶ is not important for topology.

In addition to determining the luminal orientation in the ER membrane, the N-terminal regions of 11-HSD1 and E3 are sufficient to mediate retention in the ER membrane. We show that removal by mutagenesis of the cytoplasmic Lys signal, the luminal negatively charged residues immediately downstream of the membrane helix or of Lys³⁵ and Lys³⁶ in 11-HSD1, does not affect retention in the ER membrane. Similarly, all of the constructed mutants of the fusion between the N-terminal 34 residues of E3 and green fluorescent protein showed restricted localization to the ER membrane, suggesting that intrinsic properties of the transmembrane sequence may be responsible. Furthermore, the truncated splice variant 11-HSD1B, lacking the transmembrane span, was found to be attached to the ER membrane, suggesting that the hydrophobic domain located at amino acid residues 136–158 also sufficiently mediates retention to the ER membrane (21).

Previous studies provided evidence that the N-terminal region of 11-HSD1 has an important stabilizing effect on enzymatic activity (19–22); however, the residues involved were not identified. A fusion protein containing the transmembrane anchor of 11-HSD2 followed by residues 40–292 of 11-HSD1 as well as the truncated 11-HSD1 protein alone (residues 40–292, starting with Met-Thr-Gly) were catalytically inactive (19). This may be explained by the present finding that Lys³⁵ and Lys³⁶ are essential for enzymatic activity. The positive charge at positions 35/36 is highly conserved throughout different species with only cattle and sheep having Arg instead of Lys³⁶. Recently, Blum et al. (21) reported that the splice variant 11-HSD1B, lacking the first 30 amino acids but containing Lys³⁵ and Lys³⁶, could be partially reactivated upon solubilization from the microsomal fraction. Substitution of Val¹⁴⁹ by Arg in this truncated splice variant rendered the protein more soluble; however, this protein was like 11-HSD1B and not stable, indicating that the more N-terminal residues are required for the stability, e.g. for full activity of the enzyme. In line with this assumption, Walker et al. (20) purified a truncated enzyme lacking the first 23 amino acids, which retained its activity. The present finding that mutagenesis of residues Glu²⁵ and Glu²⁶ led to a significantly reduced V_max, independent of the orientation of 11-HSD1 in the ER membrane and not affecting expression level, suggests that these residues stabilize a conformationally active enzyme.

In a previous study, we demonstrated that the N-terminal region determines the topology of 11-HSD1 to the ER membrane (19), but no difference in oxoreduction of 11-dehydrocorticosterone was found between luminally oriented wild-type 11-HSD1 and a cytoplasmic mutant enzyme; thus the physiological consequences of the luminal orientation remained unclear. Here we addressed the question of the impact of luminal orientation on enzymatic function in more detail and compared the function of the luminally oriented wild-type 11-HSD1 with the cytoplasmic mutant K5S/K6S in intact HEK-293 cells not expressing endogenous 11-HSD activity. In line with the previous study, no difference was found for the oxoreduction of cortisone between wild-type and mutant enzyme. Similar results were also obtained when Chinese hamster ovary cells were used, a cell line not expressing endogenous 11-HSD activity (not shown). This is rather surprising because the cytoplasm is considered to have a more reducing environment than the ER lumen, and cofactor concentrations may be different between the two compartments. This finding also implies that the substrate has similar access to both compartments. However, regeneration systems exist for cofactors in the cytoplasm and in the ER lumen, and 11-HSD1 accepts both cofactors with a slight preference for NADP(H), and neither glycosylation nor disulfide bridges are required for the catalytic activity of the enzyme (19, 33, 34).

It was shown previously (28, 29) that P-glycoprotein acts as an energy-dependent efflux pump that exports a wide range of substrates including corticosteroids. P-glycoprotein is endogenously expressed in HEK-293 cells, although at a relatively low level, and can be efficiently inhibited by cyclosporin A (30).² The more pronounced increase in catalytic efficiency of the oxoreduction of cortisone observed for the cytoplasmic mutant K5S/K6S compared with wild-type 11-HSD1 indicates that the inhibition of P-glycoprotein by cyclosporin A predominantly enhanced cortisone concentrations in the cytoplasm. The tendency of an increase of catalytic efficiency of wild-type 11-HSD1 upon cyclosporin treatment indicates that the substrate is exchanged between the cytoplasm and the ER lumen.

In contrast to the oxoreduction of cortisone, a 50% decrease in the oxidation of cortisol was observed for mutant K5S/K6S in the cytoplasm compared with wild-type 11-HSD1, thus demonstrating that the luminal orientation is essential for appropriate function of 11-HSD1. The oxidative activity of 11-HSD1 has been suggested to protect mature Leydig cells from the inhibitory effect of glucocorticoids on testosterone production (35). In addition, dehydrogenase activity of 11-HSD1 is predominant in adipose stromal cells, where it may represent an autocrine protective mechanism that facilitates preadipocyte proliferation, but upon differentiation to adipocytes 11-HSD1 switches to oxoreductase activity (36). Recent studies (37) provided evidence that this switch from oxidation to oxoreduction is dependent on the activity of hexose-6-phosphate dehydrogenase, which may act as a regulator of the redox potential in the ER. Thus, the luminal orientation may not only be essential for efficient dehydrogenase activity as demonstrated in the present work but also for adequate functional interaction with hexose-6-phosphate dehydrogenase.

Moreover, we demonstrate that the luminal orientation of 11-HSD1 is required for oxoreduction of 7KC, because no conversion of 7KC to 7-hydroxycholesterol was detected for mutant K5S/K6S with cytoplasmic orientation. The molecular mechanism underlying the required luminal orientation of 11-HSD1 for oxoreduction of 7KC is unclear but may be dependent on other enzymes localized in the ER lumen. 7-KC has been suggested to play a role in atherosclerosis, for example, by inhibiting free cholesterol efflux from macrophages (38) and disturbing cholesterol trafficking between the ER membrane and the plasma membrane (39). In macrophages, the subcellular distribution of 7KC mirrors that of cholesterol, which is localized in the plasma membrane and intracellular membranes but also cycles constitutively between the plasma membrane and subcellular organelles (reviewed in Refs. 10 and 40). The cholesterol content of mammalian cells is controlled by the sterol regulatory element-binding protein pathway. This pathway is regulated by sterol regulatory element-binding protein cleavage-activating protein (SCAP) (41). Upon sterol accumulation, SCAP no longer moves from the ER to the Golgi, thus restricting the cleavage of sterol regulatory element-binding proteins and preventing the transcription of the cholesterol-synthesizing enzymes (42, 43). Recently, Brown et al. (44) showed that among other sterols, 7-hydroxycholesterol but not 7KC reduces the activity of SCAP in the ER membrane presumably by causing cholesterol to translocate from the plasma membrane to the ER, where it interacts with the sterol sensing domain of SCAP and produces conformational change of the protein. In line with this hypothesis, conversion of 7KC to 7-hydroxycholesterol in the ER lumen by 11-HSD1 may have a beneficial effect by protecting cells from lipid accumulation and may play a role in macrophage pathophysiology in atherosclerosis (45). The molecular mechanism of the oxoreduction of 7KC by 11-HSD1 require further studies; nevertheless, this novel role of 11-HSD1 should be kept in mind for the design of specific inhibitor molecules for therapeutic purposes.

In conclusion, we identified the residues determining the orientation of 11-HSD1 and E3 toward the ER lumen. Analysis of the charged residues in the N-terminal region of 11-HSD1 revealed that the di-lysine motif at position 35/36 and the di-glutamate motif at position 25/26 are essential for enzymatic activity. Most important, we demonstrate that the luminal orientation of 11-HSD1 is essential for efficient oxidation of cortisol and for the oxoreduction of 7KC. In future studies, the characterization of the expression and activities of E3 and 11-HSD1 constructs with cytosolic orientations of their catalytic moiety should contribute further to the understanding of the physiological function of these proteins.

	FOOTNOTES

 
^* This work was supported by grants from the Cloëtta Research Foundation and Swiss National Science Foundation Grant 3100AO-100060. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Division of Nephrology and Hypertension, Dept. of Clinical Research, University of Berne, Freiburgstrasse 15, 3010 Berne, Switzerland. Tel.: 41-31-632-9438; Fax: 41-31-632-9444; E-mail: alex.odermatt{at}dkf1.unibe.ch.

¹ The abbreviations used are: 11-HSD, 11-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.

² David Clarke, University of Toronto, personal communication.

	ACKNOWLEDGMENTS

 
We thank Mario Ziegler, Heidi Jamin, and Regula Suter for excellent technical support and Dr. Zoltan Balazs, Roberto Schweizer, and Dr. Bernhard Dick for helpful discussion and advice.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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