(Received for publication, October 4, 1994)
From the
The topology and post-translational modifications of microsomal
11-hydroxysteroid dehydrogenase (11
DH) 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 11
-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 11
-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 11
-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 11
-DH concluded an
absence of a flexible intradomain segment between the membranous and
the lumenal domains. The lumenal localization of the 11
-DH
requires a mechanism by which cortisol is transported to the
endoplasmic reticulum of the lumen.
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
11-hydroxysteroid dehydrogenase (11
-DH). (
)The
corticosteroid 11
-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 11
-DH
is associated with polycystic ovary syndrome (Rodin et al.,
1994). The 11
-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 11
-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 11
-DH have been documented (Lakshmi and
Monder, 1988). The 11
-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
11
-DH have also been reported (Tannin et al., 1991). In
spite of rapid progress in the study of 11
-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 11
-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 11
-DH in the membrane.
A second form of 11-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. 11
-DH was eluted upon developing the column with
buffer C containing 300 mM potassium phosphate. Fractions
containing 11
-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 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.
11-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.
Specific labeling of the
cysteine residues was as follows: 11-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 11
-DH in 3 ml of dialysis
buffer, 5 ml of neutralized 8 M guanidine chloride solution
were added, followed by 50 µCi of
[
C]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%
-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
C 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 11
-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
11
-DH activity determination, and the remaining portion was
analyzed by SDS-PAGE. Limited proteolysis of the 11
-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
or its reductase was used
as a positive control.
The purification procedure for 11-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
11
-DH as shown in Fig. 1. Affinity chromatography of the
CM-Sepharose column fractions containing 11
-DH on
2`,5`-ADP-agarose column afforded substantial purification of the
enzyme. Repetition of the affinity chromatography step gave a
homogeneous 11
-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 11
-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 11
-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 11-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 11
-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 11-DH.
A second form of
11-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 11
-DH. The
SDS-PAGE analysis and automated sequence analysis of some 25 N-terminal
residues of the two 11
-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 11-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
11-DH. Solvent A was 0.1% trifluoroacetic acid, solvent B was 0.1%
trifluoroacetic acid in 75% acetonitrile. The HPLC column was C4 (4
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 11-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,
11-DH was denatured with guanidine hydrochloride and treated with
iodoacetamide or [
C]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 [C]carboxyamidomethylated
11
-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 11-DH forms with endo H
resulted in identical decrease of their apparent molecular mass.
Limited proteolysis of 11
-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 11
-DH could not be
found.
Figure 8:
SDS-PAGE analysis of denatured 11-DH
after (+) and before(-) endo H
digestion.
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 11-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 11
-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 11-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 11
-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 11
-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 11-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
11
-DH appears at the C-terminal residues (Fig. 10).
Figure 10:
Comparison of amino acid sequences of
microsomal 11-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 11-DH.
Of interest is the presence of
Tyr-X-X-X-Lys sequence in the primary structure of 11-DH
beginning at residue 182. This motif is found in the family of
so-called short chain dehydrogenases which includes the 11
-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 3
,20
-hydroxysteroid dehydrogenase
(20
-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 20
-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 20
-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 3
-DH was reported (Hoog et al., 1994). The
x-ray structure of 3
-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 3
-DH is
kinetically unaltered from the wild type, supporting the noninvolvement
of the Tyr-205 in the catalysis of 3
-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 11
-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 11-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
11
-DH by non-reducing SDS-PAGE showed no evidence of
intermolecular disulfide bonds. Incubation of native 11
-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 11-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 11
-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 11
-DH
structure, remains to be determined.
Figure 9:
A, effect of deglycosylation on
enzyme activity. 11-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
11-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 11
-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
and the cytochrome b
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 11
-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 11-DH in the
membrane. CHO denotes the carbohydrate binding
sites.
The
establishment of 11-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 11
-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 11
-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 11-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 11
-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.
The protein sequence reported in this study has been submitted to the Protein Identification Resource (PIR) National Biomedical Research Foundation with accession no. A44619.