©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Lumenal Orientation and Post-translational Modifications of the Liver Microsomal 11-Hydroxysteroid Dehydrogenase (*)

(Received for publication, October 4, 1994)

Juris Ozols

From the Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06030

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The topology and post-translational modifications of microsomal 11beta-hydroxysteroid dehydrogenase (11betaDH) was investigated using the approaches of protein structure analysis. Sequence analysis of peptides generated by chemical and enzymatic cleavages revealed that carbohydrate is attached at Asn-122, -161, and -206. Enzymatic deglycosylation reactions of the protein identified the attached glycans as high mannose carbohydrates, implying that the bulk of the protein molecule is oriented on the lumenal side of the endoplasmic membrane. The carbohydrate moiety of native dehydrogenase was cleaved by endo-N-acetylglucosaminidase H without significantly affecting the 11beta-DH activity. Chemical modification of cysteinyl residues, followed by amino acid sequence analysis, identified one disulfide bond linking Cys-77 and Cys-212. This disulfide bond was inaccessible to thiol reagents, unless the protein was denatured. Contrary to the partially purified 11beta-DH preparations, the purified enzymatically active protein failed to bind to a 2,5`-ADP affinity column, suggesting that a conformational change has occurred in the enzyme during purification. The proposed model of the 11beta-DH has a single trans-membrane segment at the N terminus, with the bulk of the polypeptide chain projecting into the lumen of endoplasmic reticulum. Limited proteolysis studies of 11beta-DH concluded an absence of a flexible intradomain segment between the membranous and the lumenal domains. The lumenal localization of the 11beta-DH requires a mechanism by which cortisol is transported to the endoplasmic reticulum of the lumen.


INTRODUCTION

In a previous study, I reported the isolation and covalent structure of the lumenal NADP-glucose-6-phosphate dehydrogenase (Ozols, 1993). During these studies a 35-kDa protein with affinity for 2`,5`-ADP-agarose was identified. Initial structural studies identified this protein as a glycoprotein related to the microsomal 11beta-hydroxysteroid dehydrogenase (11beta-DH). (^1)The corticosteroid 11beta-DH catalyzes NADP-dependent conversion of cortisol to the inactive metabolite cortisone (Tannin et al., 1991). Absence of cortisol/cortisone conversion activity leads to a potentially fatal form of childhood hypertension termed apparent mineralocorticoid excess (Monder, l991). Overactivity of the 11beta-DH is associated with polycystic ovary syndrome (Rodin et al., 1994). The 11beta-DH plays an essential role in corticosteroid action by regulating glucocorticoid access to both glucocorticoid and mineralocorticoid receptors (White, 1994). The type I mineralocorticoid receptor has equal affinities in vitro for cortisol and for aldosterone, and the concentration of cortisol in the blood is a thousandfold greater than that of aldosterone (Arriza et al., 1987). Thus, cortisol has the potential to overwhelm the mineralocorticoid receptor. However, cortisol does not act as a mineralocorticoid in vivo. The 11beta-DH is the key enzyme in conversion of cortisol to the inactive metabolite cortisone, thus eliminating cortisol competition with aldosterone for the mineralocorticoid receptor (Funder, et al., 1988). A number of enzymatic properties of 11beta-DH have been documented (Lakshmi and Monder, 1988). The 11beta-DH has been cloned from a rat liver expression library (Agarwal et al., 1989). The structure of cDNA clone, tissue distribution, and chromosomal localization of human 11beta-DH have also been reported (Tannin et al., 1991). In spite of rapid progress in the study of 11beta-DH, little is as yet known of its post-translational modifications and its orientation in the ER. In continuation of our interests in the glycosylated microsomal reductases and dehydrogenases, the structure and topology of this protein was investigated. The objectives of this study were severalfold: (a) to identify and to determine the chromatographic behavior of 11beta-DH during the solubilization and resolution of microsomal membranes; (b) identify its post-translational modifications; and (c) propose a model for the orientation of the 11beta-DH in the membrane.


EXPERIMENTAL PROCEDURES

Materials and Methods

Detergents, enzyme substrates, cofactors, chromatographic media, and chemicals, unless stated otherwise were obtained from Sigma. Hydroxyapatite (HA-Ultragel) was product of IBF Biotech, Villeneuve-la-Garenne, France. Adenosine 2`,5`-diphosphate, immobilized on cross-linked 4% beaded agarose, with seven atom spacer, lot no. 31H7830 was from Sigma. Trypsin and chymotrypsin were obtained from Worthington. Endopeptidases Asp-N, Glu-C, and endo-N-acetylglucosaminidase H (endo H) were from Boehringer Mannheim. Achromobacter peptidase Lys-C was obtained from Biochemical Diagnostics, Edgewood, NY. Cyanogen bromide was obtained from Pierce. Solvents for HPLC and gel filtration steps were from Burdick & Jackson.

Isolation of 11beta-Hydroxysteroid Dehydrogenase

Liver microsomes were isolated from New Zealand male rabbits and solubilized as described previously (Ozols, 1990a). Microsomes were suspended in 10 mM potassium phosphate, pH 7.4, containing 20% glycerol, 0.1 mM DTT, and 1 mM EDTA to a concentration of 15 mg/ml of protein. Solubilization of microsomes was achieved by the addition of sodium cholate, 1.2 mg/mg of protein. The solubilized material was fractioned with polyethylene glycol as described in detail in Ozols (1990a). The 6-12% polyethylene glycol precipitate was solubilized with Tergitol Nonidet P-10, 1.3 mg/mg of protein. The solubilized material was applied to a DEAE-cellulose column (250 ml) equilibrated with 5 mM potassium phosphate, pH 7.4, containing 20% glycerol, 0.5% Nonidet P-10, 1 mM EDTA, and 0.1 mM DTT. All buffers used in the subsequent purification steps contained 20% glycerol, 1 mM EDTA, and 0.1 mM DTT, abbreviated as GED. The unbound material eluting from DEAE-cellulose column was applied to a column containing 100 ml of CM-Sepharose equilibrated with 10 mM potassium phosphate, containing 0.2% Nonidet P-40 and GED (buffer A). The column was washed with 200 ml of buffer A, followed by a linear gradient of increasing concentration of potassium phosphate (10-300 mM). Each container of the gradient apparatus contained 220 ml of the appropriate buffer. Fractions containing 11beta-DH were dialyzed against 100 volumes of 20 mM Tris acetate, pH 8.1, containing 0.1% Nonidet P-40 and GED (buffer B), and applied to a 7-ml column of 2`,5`-ADP-agarose equilibrated with buffer B. The column was washed with 50 ml of buffer B, 50 ml of 10 mM potassium phosphate, pH 7.4, containing 0.1% of Nonidet P-40 and GED (buffer C). The column was further washed with buffer C containing 50 mM NaCl until the absorbance at 413 nm reached the base line. The 11beta-DH was eluted upon developing the column with buffer C containing 50 mM NaCl and 1 mM NADP. Fractions containing 11beta-DH were dialyzed twice against 1 liter of buffer B, and reapplied to the 2`,5`-ADP-agarose column equilibrated and developed as described above.

A second form of 11beta-DH was present in the void volume of the CM-Sepharose column. The unbound material from the CM-Sepharose was applied to a column (100 ml) of hydroxyapatite, equilibrated with buffer C. The column was washed with 300 ml of buffer C, followed by 250 ml of buffer C containing 125 mM potassium phosphate, pH 7.4. Fraction collection was initiated at the beginning of 125 mM potassium phosphate elution step. 11beta-DH was eluted upon developing the column with buffer C containing 300 mM potassium phosphate. Fractions containing 11beta-DH were dialyzed against 150 volumes of buffer B and further purified on the ADP-agarose column as described above.

Column fractions were monitored by SDS-PAGE using 4% acrylamide stacking and 8-, 10-, or 12% acrylamide resolving gels (8 times 7 cm, 1.0 mm). Electrophoresis was performed in the Bio-Rad minicell at room temperature for 40 min at 180 V. Gels were stained with 0.25% (w/v) Coomassie Blue in 4% (v/v) methanol, 10% (v/v) acetic acid, and destained in 10% (v/v) acetic acid, 40% (v/v) methanol.

11beta-DH activity was measured by monitoring corticosterone-dependent NADP reduction at 340 nm. The reaction mixture contained 5-50 µl of purified protein in 1 ml of 10 mM Tris acetate, pH 8.5, containing 0.2 mM NADP. The reaction was initiated by the addition of corticosterone (0.02 mM). The reduction of NADP was followed for 5 min at room temperature. Activity was expressed as at 340 nm/µg of protein/5 min.

Sequence Analysis

Protein/peptide hydrolysis for amino acid analysis were performed with 6 M HCl in the gas-phase, at 150 °C for 1 h (Ozols, 1990b). Reduction, carboxymethylation, succinylation, enzymatic, and chemical cleavages were performed as described previously (Ozols, 1990c). Purification peptide mixtures on a large scale were first separated using a 1.5 times 100-cm column of LH60-Sephadex equilibrated with formic acid/ethanol, 3:7 (v/v) as the solvent. Peptide mixtures from the gel-filtration column were resolved by reverse-phase HPLC. The latter methodology has been described in detail (Ozols et al., 1980; Ozols, 1990c). Reverse-phase columns employed for HPLC separations include C4, Vydac (Hesperia, CA) (4 times 0.46 cm or 15 times 0.46 cm), and Waters C18 Bondapac (30 times 0.39 cm). Peptide mixtures were dissolved in 88% formic acid prior to injection on the column. Solvent conditions for the reverse-phase columns were as follows: solvent A was 0.1% trifluoroacetic acid and solvent B was 0.1% trifluoroacetic acid in 75% (v/v) acetonitrile. A linear gradient from 0 to 100% of solvent B was applied at a flow rate of 1.0 ml/min. Sequence analysis was carried out on an Applied Biosystems model 470A sequencer equipped with model 120A PTH-analyzer according to the manufacturer's instructions. Solid-phase sequencing was carried out on a 6600 ProSequencer system (MilliGen/Biosearch, Novato, CA) as described previously (Ozols, 1990c).

Specific labeling of the cysteine residues was as follows: 11beta-DH preparation was dialyzed against 100 volumes of buffer (degassed with nitrogen) containing 20 mM Tris acetate, pH 8.1, 20% glycerol, 0.1% Nonidet P-40, and 1 mM EDTA. To 15 nmol of 11beta-DH in 3 ml of dialysis buffer, 5 ml of neutralized 8 M guanidine chloride solution were added, followed by 50 µCi of [^14C]iodoacetamide (specific radioactivity, 55 mCi/mmol). After 10 min, 100 µl of 1 M iodoacetamide were added, and the reaction mixture was incubated for 1 h at 37 °C. The reaction mixture was then reduced by the addition of 250 µl of 1 M DTT. After 1 h at 37 °C, 700 µl of 1 M iodoacetic acid were added, and alkylation was continued for 1 h at 37 °C. Following extensive dialysis against 5 mM ammonium bicarbonate containing 0.1% beta-mercaptoethanol, the preparation was lyophilized and subjected to trypsin or Lys-C protease hydrolysis. The tryptic digest was first resolved on a column of LH60-Sephadex. The Lys-C protease digest was resolved by HPLC directly. Fractions containing peptide material and radioactivity were pooled and resolved by HPLC. Aliquots of peptide-containing material were withdrawn to measure the ^14C content using a Packard scintillation counter. The remaining peptide fraction was used for the sequence analysis. Carboxymethyl- and carboxyamidomethyl-PTH-cysteine derivatives were distinguished by their characteristic elution positions on the PTH analyzer. Enzymatic deglycosylation of HCl/acetone-precipitated protein with endo H was performed as described by the manufacturer's instructions. Acid-acetone precipitation of protein solutions was performed by the addition of 20 volumes of cold (-20 °C) acetone containing 0.2% (v/v) HCl. Endoglycolytic cleavage of native 11beta-DH was conducted in 20 mM sodium phosphate buffer, pH 6.7, containing 15 milliunits of endo H for 1-3 h, at room temperature. Aliquots of the digest were used for 11beta-DH activity determination, and the remaining portion was analyzed by SDS-PAGE. Limited proteolysis of the 11beta-DH (50 µg) of protein were incubated with 5-25 µg of the appropriate protease for 1-15 h at 4 °C. The reaction was terminated by the addition of phenylmethylsulfonyl fluoride in dimethyl sulfoxide to a final concentration of 1 mM. After a 10-min interval, SDS-sample buffer was added, and the resulting digest was analyzed by SDS-PAGE. Cytochrome b(5) or its reductase was used as a positive control.


RESULTS

The purification procedure for 11beta-DH is outlined in Fig. 1-4. The enzyme eluted in the void volume of the DEAE-cellulose column effectively resolved from most of the acidic membrane proteins. Chromatography of the DEAE-cellulose column void volume on a CM-Sepharose column yielded the predominant form of 11beta-DH as shown in Fig. 1. Affinity chromatography of the CM-Sepharose column fractions containing 11beta-DH on 2`,5`-ADP-agarose column afforded substantial purification of the enzyme. Repetition of the affinity chromatography step gave a homogeneous 11beta-DH. The second affinity chromatography step, however, yielded a preparation which eluted in the void volume of the 2`,5`-ADP-Agarose column (Fig. 4). Extensive dialysis of the enzyme to remove NADP, even in the presence of the substrate, again gave the enzyme which did not bind to the affinity column. It is unclear why the purified 11beta-DH preparations failed to be retained upon further chromatography on ADP-affinity columns. Inclusion of NADP in the elution buffer was essential for elution of the enzyme. It appears that the affinity chromatography step or the inclusion of NADP in the elution buffer leads to a conformation that is not retained on an ADP-affinity column. It is also conceivable that the purified enzyme binds NADP refractory to extensive dialysis.


Figure 1: A, CM-Sepharose column chromatography profile. The unretained material from DEAE-cellulose column was applied to the column and developed with a linear gradient of potassium phosphate. Effluent column fractions were monitored at A, and 15-µl aliquot from each fraction was assayed for 11beta-DH activity. Relative activity was expressed as at 340 nm/15 µl/5 min. B, SDS-PAGE of the above column fractions. Lane B denotes position of the purified dehydrogenase. Lane S, molecular mass markers (97-kDa, phosphorylase B; 67-kDa, serum albumin; 45-kDa ovalbumin; 31-kDa, carbonic anhydrase; 21.5-kDa, trypsin inhibitor; and 14-kDa, lysozyme).




Figure 4: SDS-PAGE analysis of 11beta-DH fractions after 2nd 2`,5`-ADP-agarose column chromatography. The dialyzed sample (fractions 48-51, Fig. 3), was repurified on the same column. The equilibration and the elution of the column was performed as described in the legend of Fig. 3. Lane PC denotes an aliquot of the precolumn material. Position of the 11beta-DH is indicated by the arrow. The numbers above denote column fractions.




Figure 3: SDS-PAGE analysis of 2`,5`-ADP-Agarose column fractions. The numbers at the top denote column fractions. Samples were analyzed on 12% (Panels A and C) or 8% polyacrylamide gels (Panel B). Panel A, fractions from the void volume of the column. Panel B, fractions obtained upon developing the column with buffer C containing 50 mM NaCl. Panel C, fractions (47-51) obtained upon eluting the column with buffer C containing 50 mM NaCl and 1 mM NADP. PC indicates the aliquot of pre-column material. Lane S contains molecular mass markers: 97-kDa, phosphorylase B; 67-kDa, serum albumin; 45-kDa ovalbumin; and 31-kDa carbonic anhydrase. The arrow on the sides of the panel marks the position of 11beta-DH.



A second form of 11beta-DH was present in the void volume of the CM-Sepharose column. Chromatography of this fraction on hydroxyapatite column (Fig. 2), followed by afffinity chromatography on 2`,5`-ADP-agarose column yielded a homogeneous preparation with enzymatic activity identical to the major form of 11beta-DH. The SDS-PAGE analysis and automated sequence analysis of some 25 N-terminal residues of the two 11beta-DH forms were identical. The amino acid sequence determination was performed on the predominant form, the preparation which is initially retained on the CM-Sepharose column.


Figure 2: A, hydroxyapatite column chromatography profile. The unretained material from the CM-Sepharose column was applied to the column and developed with a stepwise gradient of potassium phosphate. At the fraction indicated by the arrow, the column was eluted with buffer containing 300 mM potassium phosphate. Relative activity was expressed as delta at 340 nm/50 µl/5 min. B, SDS-PAGE of the column fractions. Lanes S and B denote molecular mass markers and partially purified 11beta-DH, respectively.



The complete amino acid sequence analysis of the enzyme involved the reduction and carboxymethylation of the protein, followed by chemical and enzymatic cleavages. Resolution of the digests was completed by fractionation on an LH-60 Sephadex column followed by HPLC of individual fractions. Digestion of the protein with Achromobacter peptidase Lys-C gave a digest which could be resolved directly by HPLC (Fig. 5). Solid-phase and gas-phase Edman degradation of the purified peptides established the complete amino acid sequence of this protein (Fig. 6).


Figure 5: HPLC of Lys-C endoprotease digest of carboxyamidomethylated and then reduced and carboxymethylated 11beta-DH. Solvent A was 0.1% trifluoroacetic acid, solvent B was 0.1% trifluoroacetic acid in 75% acetonitrile. The HPLC column was C4 (4 times 0.46 cm). A linear gradient from 0 to 100% of solvent B was applied at a flow rate of 1.0 ml/min. Peptides K-7, -15, and -19 contain alkylated Cys-77, -212, and -240 respectively.




Figure 6: Complete amino acid sequence of rabbit liver microsomal 11beta-DH. Peptides obtained by CNBr, Achromobacter Lys-C endoprotease, and tryptic digest of succinylated protein are designated by CB-, K-, and R-, respectively. Subcleavage of peptides by endoprotease Glu-C or Asp-N are designated by E- and D-, respectively. Residues identified by automated Edman degradation are identified by a solid line. CHO denotes carbohydrate binding sites.



To determine the cysteine pairing, 11beta-DH was denatured with guanidine hydrochloride and treated with iodoacetamide or [^14C]iodoacetamide followed by reduction and alkylation with iodoacetic acid. The modified protein was fragmented with Lys-C peptidase or trypsin. To locate the carboxyamidomethyl- and carboxymethylated cysteinyl residues within the polypeptide chain, the resulting peptide mixture was subjected to HPLC analysis. Fig. 7shows the separation profile of the carboxyamidomethylated peptides and the amount of radioactivity associated with each fraction. The radioactivity was confined to peptide T-19 containing the Cys-240. No significant radioactivity was detected in peptides having residues Cys-77 or Cys-212. The cysteine containing peptides were subjected to sequence analysis, along with measurement of the radioactivity released following each cycle of degradation. In peptide T-19, radioactivity was confined to cycle three of the Edman degradation step, which corresponded to carboxyamidomethyl-PTH derivative of Cys-240. Lys-C protease digest was performed on protein preparations alkylated with unlabeled iodoacetamide followed by reduction and alkylation with iodoacetic acid (Fig. 5). Peptides K-7, K-15, and K-19 containined Cys-77, Cys-212, and Cys 240, respectively, and sequence analysis of K-7 and K-15 identified the expected carboxymethyl derivative at that particular degradation cycle.


Figure 7: HPLC of tryptic digest of [^14C]carboxyamidomethylated 11beta-DH. Top, elution of peptides was performed as described in the legend of Fig. 5. Peptide T-19 denotes the segment, residues 234-246, containing Cys-240. Peptide containing Cys-77 elutes at position corresponding to 33 min. Bottom, distribution of radioactivity in each fraction.



The carbohydrate status of the enzyme was examined by deglycosylation with endo H followed by SDS-PAGE ( Fig. 8and 9). Treatment of the two 11beta-DH forms with endo H resulted in identical decrease of their apparent molecular mass. Limited proteolysis of 11beta-DH was performed with endopeptidase Glu-C, chymotrypsin, and trypsin. The extent of proteolysis was followed by SDS-PAGE. Despite the use of a variety of enzyme/protease ratios, conditions for a partial cleavage of 11beta-DH could not be found.


Figure 8: SDS-PAGE analysis of denatured 11beta-DH after (+) and before(-) endo H digestion.




DISCUSSION

While great progress has been made in the understanding of the membrane-bound electron transport enzymes that have their catalytic domains oriented to the cytosolic side of the membrane, less is known about the structure and post-translational modifications of proteins, particularly the NAD/NADP dehydrogenases, that are present in the lumen or have their catalytic domains extruding into the lumen of ER. The enzymes involved in transport of reducing equivalents in the lumen remain undefined. One of the objectives of this study was to investigate the topological disposition of pyridine nucleotide-dependent enzymes present in the ER. Two forms of 11beta-DH differing in their chromatographic properties were identified in this study. However, upon SDS-PAGE and N terminus analysis, the two forms were identical (Fig. 2). All structure studies were performed on the predominant form of 11beta-DH.

The amino acid sequence of this form is summarized in Fig. 6. The protein consists of 291 residues. A PTH derivative was not identified at residues 122, 161 and 206 upon sequence analysis of the corresponding peptides. Since treatment of denatured 11beta-DH with endo H resulted in a decrease of its apparent molecular mass, as analyzed by SDS/PAGE (Fig. 8), the presence of oligosaccharide of the high mannose type rather than of the complex type in the 11beta-DH was indicated. Hence, the enzyme had not experienced oligosaccharide processing in the medial to trans-regions of the Golgi apparatus. The presence of high mannose oligosaccharide at three strategic sites of 11beta-DH implies that the bulk of the enzyme is extruding into the lumen of the ER.

Recently, sequences derived from the cDNA structure coding for rat and human liver 11beta-DH have appeared (Agarwal et al., 1989; Tannin et al., 1991). The comparison of the amino acid sequences of human, rat, and rabbit forms is shown in Fig. 10. The cDNA sequence of rat liver enzyme lacks two residues at the N terminus, and contains two deletions at residues 21 and 246, which are present in the rabbit and human proteins. The human and rabbit forms share 80% identical residues, while the identity between human and rat liver forms is 73%. A large stretch of identical residues is evident in the segment residues 17-49, and the most variant region of the 11beta-DH appears at the C-terminal residues (Fig. 10).


Figure 10: Comparison of amino acid sequences of microsomal 11beta-DH forms. The human (Tannin et al., 1991) and the rat (Agarwal, 1989) liver sequences were predicted from the cDNA structures. Identical residues are underlined.



Amino acid sequence studies of a number of pyridine nucleotide-dependent reductases and dehydrogenases have recognized the presence of a Gly-X-Gly-X-X-X-(Gly/Ala) motif in this group of enzymes. Crystal structure studies of several such dehydrogenases have identified this motif with the pyrophosphate binding domain of NADP/NADPH (Wierenga et al., 1985). This motif is present in the segment residues 45-51 in the amino acid sequence of 11beta-DH.

Of interest is the presence of Tyr-X-X-X-Lys sequence in the primary structure of 11beta-DH beginning at residue 182. This motif is found in the family of so-called short chain dehydrogenases which includes the 11beta-DH (Persson et al., 1991). The three-dimensional structure has been reported only for two members of this family: the NADP-dependent dihydropteridine dehydrogenase (Varughese et al., 1994), and the NAD-dependent 3alpha,20beta-hydroxysteroid dehydrogenase (20beta-DH) from Streptomyces hydrogenans (Ghosh et al., 1991). In the dihydropteridine reductase, the x-ray structure implies that Tyr-X-X-X-Lys residues may be part of the enzyme active site. In the 20beta-DH, a tyrosyl residue of the aforementioned motif is positioned in close proximity to a lysyl residue, 4 residues downstream in the sequence. Ghosh et al.(1991) propose that such spatial configuration of the Tyr-X-X-X-Lys segment facilitates tyrosine hydroxyl group participation in a proton-transfer reaction. Moreover, the x-ray structure of dihydropyridine reductase revealed that superimposition of the Tyr-X-X-X-Lys region conformation to the Tyr-X-X-X-Lys segment in the 20beta-DH leads to a remarkable similarity between the two three-dimensional structures, suggesting a common catalytic mechanism for the two enzymes. Very recently, the three-dimensional structure of rat liver NADP-dependent 3alpha-DH was reported (Hoog et al., 1994). The x-ray structure of 3alpha-DH implicates Tyr-55 and Lys-84 as part of the catalytic site. This enzyme contains the Tyr-X-X-X-Lys consensus sequence (residues 205-209). The x-ray structure, however, shows that these residues are on the periphery of the molecule facing the solvent, and are unlikely to participate in the catalysis of the enzyme. Furthermore, the Tyr-205-Phe mutant of 3alpha-DH is kinetically unaltered from the wild type, supporting the noninvolvement of the Tyr-205 in the catalysis of 3alpha-DH (Paulowski and Penning, 1994). These findings imply that the Tyr-X-X-X-Lys sequence may not always be involved in the active site of steroid dehydrogenases, and hence, the possible participation of this sequence segment in the active site of 11beta-DH remains to be determined.

Current experimental evidence suggests that, in eukaryotic cells, translocation of proteins across the ER into the lumen involves molecular chaperone assisted folding, disulfide bond formation, N-glycosylation and deglucosylation of the formed N-glycans. Subsequent discoveries in each of these steps have complicated the overall picture. Too many nascent polypeptide-chaperone protein, enzyme complexes appear to be formed at the same time. Therefore, a coherent model of the events occurring when polypeptides emerge from ribosomes and are translocated in the lumen of ER as correctly folded, catalytically active proteins remains to be established. It is generally accepted that disulfide bond formation in nascent polypeptides is assisted by the protein disulfide isomerase present in the lumen of ER (Lyles and Gilbert, 1991). Indirect evidence implies that the lumen of ER is more oxidative than cytosol (Hwang et al., 1992). Oxidative conditions would be thermodynamically and kinetically favorable to the disulfide formation as compared to the reducing environment normally found in the cytosol. How the lumen of ER maintains the proper oxidative environment is not known. Contrary to the secretory proteins, the extent of disulfide bond formation and N-glycosylation of peptide segments extruding into the lumen remains undefined. Which proteins are involved in the proper folding, glycosylation, and disulfide bond formation of the lumenally oriented polypeptide segments? Do the chaperone proteins bind to the peptide regions during the N-glycosylation, removal of glucose moieties, and the disulfide bond formation steps? Do these events occur in lumenal proteins that are anchored to the membrane, or are such proteins reinserted in the membrane after these events? Chemical modification studies on the disulfide pairing in 11beta-DH identified a single disulfide bond between Cys-77 and Cys-212, indicating that disulfide bond formation, similar to N-glycosylation, also occurs in peptide segments exposed to the lumen of ER. Analysis of the 11beta-DH by non-reducing SDS-PAGE showed no evidence of intermolecular disulfide bonds. Incubation of native 11beta-DH with iodoacetic acid or its amide failed to modify its enzymatic activity. Hence, the single unpaired Cys-240 is not involved in intermolecular disulfide bonding or in the catalytic function of the enzyme.

As oligosaccharide chains play different roles in the physiological activity of different proteins, it was of interest to learn how deglycosylation affected the 11beta-DH activity. Deglycosylation of the native enzyme could be achieved with endo H within 2 h of incubation. The catalytic activity of the deglycosylated enzyme was comparable to that of the native enzyme (Fig. 9). Presumably, the function of the glycosylation of 11beta-DH is to dictate the proper folding of the enzyme during biosynthesis and to prevent aggregation with other lumenal proteins. Whether the Asn-linked carbohydrates form hydrogen bonds or are involved in hydrophobic interactions with the polypeptide backbone, stabilizing the 11beta-DH structure, remains to be determined.


Figure 9: A, effect of deglycosylation on enzyme activity. 11beta-DH was incubated with endo H at room temperature. At the indicated time interval, samples were withdrawn and activity determined. Controls were run under identical conditions except that endo H was omitted from the reaction mixture. B, SDS-PAGE analysis of the above fractions.



The proposed topology for 11beta-DH in the microsomal membrane is depicted in Fig. 11. This orientation is based on the presence of three high mannose structures at residues 122, 161, 206, and a disulfide bond between Cys-77 and Cys-212. A putative membrane spanning segment between residues 5 and 24 is evident by the presence of stretch of hydrophobic residues. A search for potential membrane spanning regions predicts three other segments, residues 141-157, 164-180, and 201-217. These regions, however, are unlikely to span the membrane, since the presence of the single disulfide bond and the three carbohydrate moieties would preclude positioning of these peptide regions in the membrane. The two domain structure of 11beta-DH prompted the investigation to determine whether the enzyme contains a flexible and exposed interdomain region susceptible to limited proteolysis. Attempts to fragment the intact protein into domains by proteolysis were unsuccessful. Proteolysis conditions yielding partial fragmentation of the intact protein could not be found with trypsin, chymotrypsin, or endopeptidases Glu-C or Lys-C, despite the presence of amino acid residues susceptible to these proteases between residues 25 and 77. This is in contrast to several microsomal proteins with catalytic moieties on the cytosolic side of the ER membrane, such as cytochrome b(5) and the cytochrome b(5) reductase which have intradomain segments that are very susceptible to limited proteolysis, and yield functionally active proteins following limited proteolysis (Ozols, 1989a; Ozols et al., 1985). The observed resistance of the 11beta-DH to proteolysis indicates the absence of a flexible intradomain segment between the membrane binding polypeptide and the luminal domain.


Figure 11: Proposed orientation of 11beta-DH in the membrane. CHO denotes the carbohydrate binding sites.



The establishment of 11beta-DH in the lumen of ER raises a number of questions concerning the availability of the cofactors and the substrates for this enzyme. Cortisol occurs in the plasma in two forms: free cortisol and protein-bound cortisol. The exact pathway of cortisol from the plasma to ER membrane remains to be determined. A current model of steroid hormone action envisions passive diffusion of free steroids across the plasma membrane to the cell's interior. Thus, a simplified cortisol translocation pathway to the 11beta-DH would involve passive diffusion of unbound cortisol from plasma across the plasma membrane, into cytosol, and to the ER membrane. Several lines of evidence suggest that corticosteroids in the cytosol are complexed with specific proteins, and that the resulting hormone-receptor complex proceeds to its site of action, the nucleus (Pratt, 1987; Yamamoto et al., 1988). It is conceivable that cortisol in cytoplasm associates with receptor proteins which may also interact with the ER membrane resulting in the delivery of cortisol into the lumen. Transport of cortisol to the ER lumen by way of specific proteins would lend an additional degree of specificity and control to cortisol action and metabolism. Consequently, additions and modifications of the cortisol transport to the 11beta-DH in the lumen may be forthcoming.

Two carboxylesterases and a unique form of glucose-6-phosphate dehydrogenase have been identified as resident proteins of the ER lumen (Ozols, 1989b, 1993). Moreover, the catalytic moiety of oligosaccharyl transferase, the enzyme complex catalyzing the transfer of a high mannose oligosaccharide to asparaginyl residues with an Asn-X-(Ser/Thr) consensus sequence in nascent polypeptides, appears lumenally oriented (Kumar et al., 1994). Evidence that hepatic microsomal UDP-glucuronyltransferase is a transmembrane protein with its catalytic domain oriented in the lumen of ER has also been reported (Yokota et al., 1992). The latter enzyme catalyzes the glucurodination of many physiological compounds such as bilirubin and steroid hormones. Hence, it may be inferred that the lumenal surface of ER is a catalytically active membrane structure and not a relatively empty space through which newly synthesized proteins pass en route to the cell surface, but an important subcellular compartment with its own set of enzymes and cofactors.

Another unresolved problem that arises concerning the proposed orientation of 11beta-DH is the origin and fate of reduced pyridine nucleotides in the lumen. As mentioned earlier, we reported the existence of a structurally unique form of NAD/NADP-glucose-6-phosphate dehydrogenase in the lumen of the ER. Hence, there are at least two dehydrogenases on the lumenal side of ER generating reduced pyridine nucleotides. It is unlikely that pyridine nucleotides can passively diffuse across the membrane. Clearly, the activity of 11beta-DH will depend on the presence and activity of the enzymes utilizing the pyridine nucleotides in the lumenal compartment of the ER. Reactions that utilize the lumenal reducing equivalents are at present unknown. Further studies are necessary to identify the reductases that are present on the lumenal side of the ER.


FOOTNOTES

*
This work was supported by National Institutes of Health, National Institute of General Medical Sciences Grant RO1 GM-26351. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The protein sequence reported in this study has been submitted to the Protein Identification Resource (PIR) National Biomedical Research Foundation with accession no. A44619.

(^1)
The abbreviations used are: DH, hydroxysteroid dehydrogenase; DTT, dithiothreitol; HPLC, high performance liquid chromatography; ER, endoplasmic reticulum; PTH, phenylthiohydantoin; PAGE, polyacrylamide gel electrophoresis; endo H, endo-N-acetylglucosaminidase H.


ACKNOWLEDGEMENTS

I am grateful to George Korza for his outstanding technical assistance.


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