From the
Biocenter Oulu and Department of Biochemistry, University of Oulu, P.O.
Box 3000, Oulu FIN-90014 Finland, the
Department of Biosciences, University of Kent,
Canterbury, Kent CT2 7NJ, United Kingdom, and the
Department of Biochemistry, University of Oulu,
P.O. Box 3000, Oulu FIN-90014, Finland
Received for publication, May 2, 2003
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ABSTRACT |
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INTRODUCTION |
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The process of native disulfide bond formation in the endoplasmic reticulum (ER)1 is known to be catalyzed by several families of enzymes (3). However, whereas many of the participants in the cellular process are known, their individual roles are still largely confused. Furthermore, it is likely that not all of the participants in the process have yet been identified. This situation not only inhibits our understanding of the biogenesis of a range of important proteins but also prevents the effective manipulation of the cellular environment by the biotechnology industry for the efficient production of therapeutic proteins.
Native disulfide bond formation usually occurs via multiple parallel pathways (see Ref. 4 as an example). Each step in these pathways can be considered to be an oxidation or isomerization reaction. Oxidative reactions result in the formation of a disulfide bond from two free thiols. Isomerization reactions do not alter the overall number of disulfides, but they result in a rearrangement in their position within the substrate protein. Isomerization reactions are required due to the formation of non-native disulfide bonds in the substrate protein and may either occur directly or via a linked reduction-oxidation cycle. It is unclear to what extent, if any, reduction reactions (i.e. the formation of two free thiols from a disulfide) play a role in the formation of native disulfide bonds under physiological conditions. In vitro the rate-limiting step for native disulfide bond formation in proteins that contain multiple disulfides is late stage isomerization reactions, where disulfide bond formation is linked to conformational changes in protein substrates with substantial regular secondary structure (46).
The first reported catalyst of protein folding, protein-disulfide isomerase (PDI), is involved in native disulfide bond formation in the ER (7). Whereas PDI is an excellent catalyst of isomerization reactions, it is a relatively poor catalyst of oxidative processes in vitro. Furthermore, the participation of PDI in disulfide bond formation requires the presence of a system for reoxidizing PDI. For many years, it was presumed that the primary reoxidant in the ER was oxidized glutathione. However, it has become clear that there are parallel pathways for providing the oxidizing equivalents required for native disulfide bond formation. ERo1 (8, 9) from yeast and higher eukaryotes has been shown to provide oxidizing equivalents to this process either via oxidation of glutathione (10) or via direct oxidation of protein thiols, including reoxidizing PDI in vivo (11) and in vitro (12). Recently, a parallel oxidizing pathway has been reported based on flavin-dependent sulfydryl oxidases (13, 14). Whereas the gene products of ERO1 and PDI1 are essential for viability in yeast (8, 15) and those of the other components including the glutathione biosynthetic pathway are not, it is still unclear to what extent the three possible oxidative pathways contribute to native disulfide bond formation under physiologically normal conditions.
A further complicating factor in understanding the physiological process of native disulfide bond formation is the presence of a family of proteins related to PDI. In S. cerevisiae, there are five proteins that are related to PDI (Pdi1p, Eug1p, Mpd1p, Mpd2p, and Eps1p) (see Refs. 16 and 17 for characterization). Whereas of these five proteins only Pdi1p can be considered to be an efficient catalyst of disulfide bond isomerization, it is clear that in higher eukaryotes this is not the case. Instead, a range of PDI homologues have been found, including ERp72, ERp57, P5, PDIp, and PDIr (see Refs. 18 and 19). In addition, there are two PDI-related proteins that are not isomerases, ERp44 (20) and ERp28/Erp29 (21, 22), the function of both of which is currently unresolved.
Here we report a new addition to the ER-located PDI-related family of
proteins, which we call ERp18. ERp18 contains a single catalytic domain with
an unusual CGAC active site motif and a probable insertion between
3 and
3 of the thioredoxin fold. Although
it is listed in several data bases as a thioredoxin-like protein, the reduced
form of the protein is more stable than the oxidized form, suggesting that it
is involved in disulfide bond formation and not reduction. Furthermore, in
vitro ERp18 possesses significant peptide thiol-disulfide oxidase
activity. A putative physiological role for Erp18 in native disulfide bond
formation is discussed.
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EXPERIMENTAL PROCEDURES |
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Protein Expression and PurificationProtein production was
carried out in E. coli strain BL21 (DE3) pLysS. Strains were grown in
LB medium at 37 °C and 200 rpm and induced at an A600
of 0.3 for 4 h with 1 mM isopropyl
-D-thiogalactoside. Cells were pelleted by centrifugation
(6,500 rpm for 10 min), and the pellet was resuspended in one-tenth volume of
buffer A (20 mM sodium phosphate, pH 7.3) and one-one thousandth
volume of 10 mg/ml DNase (Roche Applied Science). The cells were lysed by
freeze-thawing twice, and the cell debris were removed by centrifugation
(12,000 rpm for 20 min). The supernatant was filtered through a 0.45-µm
filter before being applied to an immobilized metal affinity chromatography
column (chelating Sepharose fast flow; Amersham Biosciences), precharged with
Ni2+ and equilibrated in buffer A. After loading, the
column was washed in 20 mM sodium phosphate, 50 mM
imidazole, 0.5 M NaCl, pH 7.3, and then in buffer A, before the
His-tagged proteins were eluted using 20 mM sodium phosphate, 50
mM EDTA, pH 7.3. The eluant was diluted 5x into buffer A and
then applied to a Resource Q anion exchanger, from which the proteins were
eluted with a linear gradient from buffer A to buffer A containing 0.5
M NaCl over 9 column volumes. Eluted fractions were checked for
purity by SDS-polyacrylamide gel electrophoresis, and fractions containing
pure protein were pooled and buffer-exchanged into 20 mM sodium
phosphate, pH 7.3, using an Amicon ultra 15 centrifugal filter device (10-kDa
nominal molecular weight limit membrane filter). The concentration of the
protein was determined spectrophotometrically using a calculated absorption
coefficient of 16,680 M1
cm1 at 280 nm. 15N-Labeled ERp18 was
produced by growing the expressing strain in M9 medium using
15N-labeled NH4Cl (Cambridge Isotopes) with protein
purification as described for unlabeled protein. To ensure full oxidation of
the active site CXXC motif prior to NMR studies,
15N-labeled ERp18 was incubated with 0.5 mM oxidized
glutathione for 15 min at room temperature, and then the glutathione was
removed by buffer exchanging using an Amicon ultra 15 centrifugal filter
device (10-kDa nominal molecular weight limit membrane filter) into 20
mM sodium phosphate, 150 mM sodium chloride, pH 6.5. The
a-domain of human PDI was purified as per human ERp18 (calculated
absorption coefficient of 19,720 M1
cm1 at 280 nm). E. coli thioredoxin was
purified as per human ERp18 except that elution from the chelating Sepharose
column was with 25 mM EDTA, and the anion exchange column was run
in 20 mM Tris buffer, pH 8.6, instead of buffer A.
Cell TransfectionsCOS-7 cells (ATCC, Manassas, VA) were grown on 30-mm diameter Petri dishes with or without glass coverslips in Dulbecco's modified Eagle's medium-high glucose medium supplemented with Glutamax (Invitrogen), 10% fetal calf serum, and penicillin/streptomycin. Cells seeded 1 day earlier were transfected with the ERp18-GFP plasmid using 0.51 µg/plate and the Fugene6TM transfection reagent (Roche Applied Science) as suggested by the manufacturer. After 24 h, cells were rinsed with phosphate-buffered saline, fixed with 4% p-formaldehyde for 20 min, and processed for indirect immunofluorescence as described earlier (24). Monoclonal antibodies against protein-disulfide isomerase (PDI, Dako A/S, Glostrup, Denmark) and the Golgi matrix protein, Gm130 (BD Biosciences, Lexington, KY) were used as the ER and Golgi markers, respectively, to allow localization of the expressed ERp18-GFP in transfected cells. Fixed and stained cells were examined using an epifluorescence microscope (Olympus BX61) and photographed with a CCD camera.
Biophysical AnalysisFar UV CD spectra were recorded on a Jasco J600 spectrophotometer. All scans were collected at 25 °C as an average of eight scans, using a cell with a path length of 0.1 cm, scan speed 50 nm/min, a spectral bandwidth of 1.0 nm, and a time constant of 0.5 s. The maximal HT voltage was 750 V.
Fluorescence spectra were collected on a PerkinElmer Life Sciences LS50
spectrophotometer using a 1-ml cuvette. All scans were collected at 25 °C
as an average of four scans, excitation at 280 nm, emission at 300400
nm, slit widths of 5 nm, and scan speed of 200 nm/min. Fully oxidized and
reduced proteins were generated immediately prior to use by preincubating the
protein stock in 10 mM oxidized glutathione or 20 mM
reduced glutathione for 15 min at room temperature. Protein stocks were
diluted at least 200-fold into 0.2 M phosphate buffer, pH 7.0,
containing 06 M guanidinium chloride and equilibrated for 5
min at 25 °C before fluorescence spectra were recorded. All spectra were
corrected for the blank spectra with no protein added. The fluorescence
parameter examined to investigate the effects of guanidinium chloride on
protein structure was the ratio of the average fluorescence intensity 2 nm to
either side of the max for native protein (337 nm for
oxidized ERp18; 336 nm for reduced ERp18; 340 nm for PDI a-domain) to
the average fluorescence intensity over the range 320400 nm. This
parameter was chosen because it is independent of concentration and less
dependent on the direct effects of guanidinium chloride on tryptophan
fluorescence.
NMR spectra were collected on a Varian Inova 600-MHz spectrometer from samples of uniformly 15N-labeled ERp18 (0.3 mM) in 20 mM sodium phosphate buffer (pH 6.5) containing 150 mM NaCl and 10% (v/v) D2O. Reduced ERp18 was prepared by preincubating the oxidized protein with 3 mM dithiothreitol for 30 min at room temperature. 1H/15N HSQC spectra were collected at 25 °C over 6 h with acquisition times of 148 ms in 1H and 71 ms in 15N and with water suppression using the WATERGATE sequence.
Oxidase AssayThe method of Ruddock et al. (25) using a fluorescent decapeptide was used to determine the oxidase activity of each of the purified human PDI family members. McIlvaine buffer (0.2 M disodium hydrogen phosphate, 0.1 M citric acid, pH 3.07.5) to give a final assay volume of 1 ml was placed in a fluorescence cuvette. Except where noted, to this was added 10 µl of oxidized glutathione (50 mM stock solution in buffer A), 20 µl of reduced glutathione (100 mM stock solution in buffer A), and 510 µl of enzyme. After mixing, the cuvette was placed in a PerkinElmer Life Sciences LS50 spectrophotometer for 5 min to allow thermal equilibration of the solution. 6.3 µl of substrate peptide (539 µM in 30% acetonitrile, 0.1% trifluoroacetic acid) was added and mixed, and the change in fluorescence intensity (excitation at 280 nm, emission at 350 nm, slit widths at 5 nm) was monitored over an appropriate time (usually 15 min) with 6001800 data points being collected. In the absence of substrate or in the absence of oxidized glutathione, no significant change in fluorescence occurred (e.g. for ERp18 alone, the change in fluorescence over 30 min was only 0.65%, which probably represents photobleaching).
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RESULTS |
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Serial analysis of gene expression (26) indicates that ERp18 is widely expressed in humans including in the kidney, brain, prostate, lung, liver, heart, spinal cord, mammary gland, ovary, colon, and vascular epithelium. Unigene (J. U. Pontius, L. Wagner, and G. D. Schuler; available on the World Wide Web at www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unigene) cDNA sources include liver, stomach, uterus, bone, placenta, brain, adrenal gland, ovary, prostate, lung, testis, pancreas, and kidney. The ERp18 coding region used in these studies was PCR-amplified from a liver cDNA library.
Homologues of ERp18 can be found in mouse, rat, Xenopus, and Caenorhabditis elegans. Multiple alignments of these four proteins were performed using ClustalW (28), T-COFFEE (29), Match-box (30), and Multialin (31). These alignments showed considerable consensus, varying only in the extreme N- and C-terminal regions. A consensus alignment that takes into account the probable structural alignment with the thioredoxin fold (see below) is shown in Fig. 1. Over the mature protein (see below), the human and mouse proteins show 94.6% identity, human and rat proteins show 94.0% identity, human and Xenopus proteins show 81.2% identity, and human and C. elegans proteins show 41.6% identity.
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Analysis of the Domain Structure of ERp18 Analysis of the sequence of ERp18 using PSORT (available on the World Wide Web at psort.ims.u-tokyo.ac.jp/) and TargetP (33) indicated that the protein would be targeted by a cleavable N-terminal signal sequence to the secretory pathway, with the mature protein starting at Ser24 (PSORT) or His27 (TargetP). The C-terminal amino acids are EDEL, which probably acts as an ER retrieval motif (usual consensus KDEL).
If the mature protein starts at Ser24, then it is 149 amino acids long, with a molecular mass of 16.7 kDa and a theoretical pI of 5.1 (ProtParam). The protein contains one CXXC thioredoxin superfamily active site motif, located at Cys66Cys69, and no other cysteine residues. The nature of the two residues between the active site cysteines plays a key role in determining the redox potential of the thioredoxin superfamily and hence their biological activity. In the thioredoxins, which act as reductants, the motif is CGPC, whereas in the PDI family, which act as oxidants and isomerases, it is usually CGHC, and in the DsbA family, which act as oxidants, it is usually CPHC. The sequence in ERp18 is CGAC, which does not directly fit into any of these families.
Multiple alignments of the catalytic domains of the human PDI family with
ERp18 were performed using ClustalW
(28), T-COFFEE
(29), Match-box
(30), and Multialin
(31), and alignments of pairs
of proteins were performed with a wide range of programs. These alignments
showed some degree of consensus at the N terminus of the proteins, indicating
that the probable residue starting a putative thioredoxin fold in human ERp18
was Ile38 (equivalent to Val26 in human PDI) but showed
very significant differences for the alignment for the C-terminal part of
ERp18. ERp18 is clearly longer than a single PDI family member catalytic
domain, and the different alignments indicated that the additional sequence
could either occur (i) at the C terminus (i.e. beyond the end of the
thioredoxin domain), (ii) as an insert between secondary structure elements
3 and
3 or
3 and
4, or (iii) as a combination of these. To help to distinguish
between these three possibilities, four C-terminal deletion constructs were
generated that truncated the protein at a point where an alignment with the
PDI catalytic domain ended. If the alignment was correct, then it would be
reasonable to expect that the truncated protein might retain the ability to
fold and form stable structures, since they would still encompass the entire
thioredoxin-like fold. The constructs terminating with Gln146 or
Gly150 were expressed insolubly in the cytoplasm of E.
coli under all conditions tested, whereas a construct terminating with
Arg157 generated a small percentage of soluble material, which was
clearly unstable. Only constructs expressing mature human ERp18
(Ser24Leu172) or with the putative ER retention
signal deleted (Ser24Leu168) were solubly and
stably expressed in the cytoplasm of E. coli (data not shown).
Localization of ERp18 ERp18 contains a putative secretory pathway signal sequence and a putative ER retention signal; hence, it is most probably an ER-resident protein. To investigate the subcellular localization of ERp18, two ERp18-GFP chimeras were constructed. The first had the whole ERp18 gene fused in frame N-terminally to GFP; the second had amino acids Met1Leu168 of ERp18 fused in frame to the N terminus of GFP and Leu168-Leu172 of ERp18 fused in frame C-terminal to GFP. Direct microscopic examination of the transfected cells showed that the first ERp18-GFP fusion protein could not be visualized intracellularly. The second chimera localized in a fine reticular network and the nuclear envelope (Fig. 2), suggesting that the fusion protein localizes mainly in the ER. This was confirmed by staining of the transfected cells with antibodies against the known ER and Golgi markers (PDI and Gm130). The chimeric protein, with ERp18 (including the signal sequence) N-terminal to GFP and the retrieval signal (EDEL) C-terminal to GFP, co-localized well with PDI but not with the Golgi marker Gm130. The chimeric protein appears also to be efficiently retained in the ER (via the EDEL), since no marked accumulation of the protein in the Golgi region was observed (Fig. 2, right, ERp18-GFP).
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Biophysical Analysis of ERp18 Mature ERp18 (Ser24Leu172), with an N-terminal hexa-His tag to aid purification, was expressed solubly in the cytoplasm of E. coli, and the construct was purified to apparent homogeneity by a combination of immobilized metal affinity chromatography and anion exchange chromatography (data not shown). Mass spectrometric analysis of the purified protein by MALDI mass spectrometry (mass accuracy 0.1%) gave a mass of 17,768.6, close to the calculated mass for oxidized ERp18 of 17,768.9. Purified ERp18 was then analyzed by a variety of techniques.
The far UV CD spectra of purified ERp18
(Fig. 3A) indicated
that the protein was well structured and contained both -helix and
-sheet. All members of the thioredoxin superfamily including thioredoxin
and the catalytic a-domain of human PDI share the same
/
fold (see Refs. 34 and
35 as examples). Since ERp18
showed considerable homology with the N-terminal region of the catalytic
domains of the PDI family, the far UV CD spectra of purified ERp18 was
compared with those of the a-domain of human PDI and E. coli
thioredoxin. The resulting spectra are significantly different for all three
proteins (see Fig.
3A).
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Fluorescence spectra of ERp18 under nondenaturing conditions gave a peak
with a max of 337 nm and a shoulder at around 351 nm,
indicating that one of the two tryptophans of ERp18 is in a hydrophobic
environment, whereas the other is in a hydrophilic environment (see
Fig. 3B). Similar
observations were made for the a-domain of human PDI, which also
contains 2 tryptophan residues (
max 340 nm; data not
shown). Upon the addition of 6 M guanidinium chloride, the
fluorescence spectra of ERp18 had a
max of 356.5 nm,
indicative of a denatured protein (see Fig.
3B).
The redox potential of members of the thioredoxin superfamily is dependent
on the relative stability of the oxidized and reduced forms of the active site
CXXC motif, and this determines whether individual proteins act as
oxidants, reductants, and/or isomerases. Guanidinium chloride denaturation
curves for oxidized and reduced ERp18 (Fig.
4A) and the a-domain of human PDI
(Fig. 4B) indicated
that for both proteins the reduced form of the protein is significantly more
stable than the oxidized. This is consistent with an oxidative function for
both proteins. Whereas the midpoint for denaturation for both proteins in the
oxidized or reduced states is approximately equal, denaturation of ERp18
occurs over a narrower concentration range of guanidinium chloride. This
indicates that the stability of ERp18 under nondenaturing conditions is higher
than that of the a-domain of human PDI. Using the six-component
equation for denaturant-dependent changes in G
(36), midpoints for
denaturation,
G0, and
G0 between the oxidized and reduced forms
were calculated (see Table II).
These results indicate that ERp18 is more stable than PDI a-domain in
both the oxidized and reduced forms and that it has a nearly equivalent redox
potential. However, it should be noted that
G0
values calculated from denaturation curves, especially curves with sharp
transitions, are prone to error, and hence these values should be treated with
some caution.
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Structural Measurements of ERp18 To further analyze the structure of ERp18, 15N-labeled material was generated. MALDI mass spectrometry indicated a 95% labeling efficiency. The resolution and dispersion of the 1H/15N HSQC spectra for both oxidized and reduced ERp18 (Fig. 5) suggested that in both states the protein existed as a fully folded and compact molecule. The cross-peaks represent N-H pairs in the protein and derive mainly from the backbone, but they also include some side chain NH2 groups. A second HSQC experiment where the side chain NH2 signals were selectively suppressed (data not shown) allowed the number of backbone amide signals to be counted. The number was estimated to be 141, which is in good agreement with the number of nonproline residues (n = 147) in the His-tagged ERp18 construct.
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A comparison of the oxidized (Fig. 5A) and reduced (Fig. 5B) spectra of ERp18 showed that the majority of the backbone amide resonances remained unchanged in position upon reduction of the Cys66Cys69 active site disulfide bond. This finding suggests that the overall structure of the protein is unperturbed by a change in oxidation state. However, significant changes in position were seen for 5 or 6 residues, and about a further 25 residues shifted position slightly. This degree of change is consistent with a local structural rearrangement, presumably centered on the CGAC redox active motif.
Activity Measurements of ERp18 To measure the relative peptide oxidase activity of ERp18, a simple fluorescence-quenching assay was used (25). In this assay, the formation of a disulfide bond in the substrate peptide can be monitored in real time, since this brings an arginine residue in the peptide into close proximity with the single tryptophan, resulting in quenching of the intrinsic fluorescence. Under standard assay conditions at pH 6.5, the ERp18 showed significant peptide oxidase activity; however, this was considerably lower than that of the a-domain of human PDI (see Table III and Fig. 6). The rate of reaction was directly proportional to enzyme concentration (data not shown). Varying the concentration of oxidized glutathione in the assay indicated that the rate-limiting step for ERp18 peptide oxidase activity was oxidation of the substrate and not reoxidation of reduced enzyme by oxidized glutathione as is the norm for other PDI family members (e.g. see Ref. 25). To further investigate this, the peptide substrate concentration was varied to estimate the Km and Vmax for ERp18. The results (see Table III) indicate that the Km of ERp18 for the substrate peptide is greater than can be determined by the dynamic range of the fluorescence-based assay used ([substrate] is up to 8 µM; an estimate can be made of Km of the order of 25 µM, which is 1 order of magnitude greater than the apparent Km of the other human PDI family members, excluding PDIr, under standard assay conditions).2 The pH dependence of the oxidase activity of human ERp18 is similar to that reported for human PDI (Fig. 6B) (25).
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All thioredoxin superfamily members characterized to date have a CXXC active site motif, with both cysteine residues being required for oxidative activity. Single point mutants were generated in both active site cysteines of ERp18, C66S and C69S. The corresponding proteins were purified, the correct mass was confirmed by MALDI mass spectrometry, and the proteins were tested for peptide oxidase activity. Both mutants gave no detectable activity in the peptide oxidase assay (see Table II).
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DISCUSSION |
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Three motifs can be used as a diagnostic of which family in the thioredoxin
superfamily a protein is likely to belong to: the CXXC active site
motif, the residues from the end of 2 to the end of
2 (which include this motif), and the residues around the
conserved cis-proline (Pro100 in the a-domain of
human PDI). In the thioredoxins, the conserved active site motif is CGPC, in
the PDI family it is usually CGHC, and in the DsbA family it is usually CPHC.
The CGAC sequence found in ERp18 does not belong to any of these families but
is closer to the motif found in PDI and thioredoxin than to the DsbA motif.
The residues around the active site motif in ERp18, WCGACKALK-PKF, are the
same as the conserved motif of the human PDIs (excluding the uncharacterized
protein PDIr), WCGHCKX(L/M/F)XPX(Y/W/F) and the
thioredoxin consensus sequence, WCGPC(K/R)X(I/F/L)XP.
However, since it contains an aromatic residue +2 from the fully conserved
proline residue in the PDI sequence, which forms an important
structure-defining kink in
2 of the thioredoxins and in
human PDI a-domain (34,
35), the motif in ERp18 is
marginally closer to that of the PDIs. The third diagnostic motif is centered
on the conserved proline residue (Pro100 in the a-domain of
human PDI), which in all known superfamily structures is in the cis
conformation. The sequence in human ERp18, GNPSYKY, does not directly fit into
the consensus sequence of any of the thioredoxin superfamily families.
However, it most closely resembles that of the human PDI family,
G(F/Y)PT(I/L)X(F/Y/I), with X often representing lysine,
since it has a Gly at the 2-position (a feature shared with the DsbAs
but not the thioredoxins or glutaredoxins).
Whereas the comparison of the consensus sequences suggests that, although not a member of the PDI family, ERp18 more closely resembles the PDIs than the thioredoxins, it does not provide strong or direct evidence of function. However, the results reported in this study on the ER localization of ERp18 and the greater stability of the reduced over oxidized form of the enzyme (a property shared with PDI and DsbA but not with thioredoxin) are indicative of a role for ERp18 in disulfide bond formation. This is further reinforced by the significant peptide oxidase activity of ERp18, for which both active site cysteine residues were found to be required. The oxidase assay was carried out in a glutathione redox buffer that mimics the redox potential of the ER, the compartment in which ERp18 is localized, and hence indicates a physiologically relevant function.
It is clear that the sequence of mature ERp18 (149 amino acids) is
significantly longer than the catalytic domain of thioredoxin or a catalytic
domain of PDI (around 110 amino acids). It is possible that ERp18 contains
either an N-terminal or C-terminal extension, as is seen in all human PDIs
(18,
23) or that it contains an
insertion in the thioredoxin fold as is seen in the DsbAs. All of the
alignment programs used indicated that ERp18 has a 14-amino acid N-terminal
extension, which is larger than that found in human PDI or ERp57 but shorter
than that found in human PDIp or ERp72
(23). One of the four
alignment programs used placed another long extension at the C terminus of the
protein, whereas the others placed an insertion between either
3-
3 or
3-
4. The later programs align
Pro135 with the conserved cis-proline found in the
thioredoxin superfamily, and the region around this proline (GNPSY) does align
better with the highly conserved sequence, G(F/Y)PT(I/L), than the region that
aligns by the insertion being at the C terminus (Pro113, YIPRI). An
insertion into the thioredoxin fold at this proposed position would not be
unique, since the DsbA family have an all
-helical domain inserted
between
3 and
3 of this fold
(37,
38). The nature of the
insertion and the C-terminal extension varied between alignment programs, and
C-terminal truncations were made to elucidate which alignment was most
probable. The inability for C-terminal truncations deleted from
Val147, Met151, and Leu158 to fold in
vivo, whereas a truncation at Leu168 could still fold,
suggests that the most probable alignment is that which inserts the sequence
EEEPKDEDFSPDGGYIPRILFLD into the loop between
3 and
3 (Fig. 7). This alignment also places the insertion in the thioredoxin fold at the same
location as the large insertion (
75 amino acids) found in the thioredoxin
fold in the DsbAs and puts the insertions/deletions found in the homologous
Xenopus and C. elegans sequences
(Fig. 1) outside the limits of
the thioredoxin fold or in the loops between the secondary structural
elements. The insertion in human ERp18 being both acidic and containing a
significant number of P/G residues is reminiscent of the interdomain linkers
found in PDIr (23). Whereas
the CD spectra of ERp18 is significantly different from that of the
a-domain of human PDI, so is the spectra of E. coli
thioredoxin, which shares the same fold; hence, nothing can be inferred from
this other than the fact that human ERp18 is well structured. It is clear from
the 15N/1H HSQC NMR spectra that there are no
significant unstructured regions in ERp18 and that the overall structure of
ERp18 is unperturbed by the redox state of the active site (as seen for all
other superfamily members studied to date) (see Refs.
3942
for examples).
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Although the multiple alignments of the catalytic domains of the PDI family with ERp18 differed significantly, the consensus regions for all indicated that ERp18 does not possess the conserved (except in PDIr) glutamic acid (Glu47 in the a-domain of human PDI), which is important for the catalytic redox cycle of the PDI family3 and in the thioredoxins (43). In addition, the unusual active site motif, CGAC, does not contain a histidine residue found in both the DsbA (CPHC) and PDI (CGHC) families, which has been implicated in helping to generate an oxidizing redox potential for the active site (44, 45). However, ERp18 has significant oxidase activity (which is limited by substrate binding), and it shows nearly identical guanidinium chloride stability curves compared with the a-domain of human PDI for both the oxidized and reduced states. This indicates that ERp18 must stabilize the reduced state of the enzyme by a different method from that used by PDI. We have recently reported that modulations in His94 in the all-helical domain insert in Vibrio cholerae DsbA, which spatially is located close to the active site, modulates the activity of the enzyme (32), and it is possible that the putative insert in the thioredoxin fold in ERp18 plays a similar role.
Whereas the data presented here suggest a role for ERp18 in disulfide bond
formation in the ER, it does not define what that function might be. The
186-amino acid C. elegans homologue of human ERp18 (Y57A10A.23) shows
no observable RNA interference phenotype (WormBase Web site, available at
www.wormbase.org,
release WS99), implying that the function of this gene product is not
essential. However, C. elegans also contains a 257-amino acid protein
(F49H12.5) whose N-terminal region shows 44.3% identity with Y57A10A.23 and
whose C-terminal 88 amino acids are composed almost entirely (94.3%) of Lys,
Glu, and Asp. F49H12.5 also shows no observable RNA interference phenotype
(WormBase Web site, available at
www.wormbase.org,
release WS99). It is possible that there may be functional complementation
between these proteins. The results from the oxidase activity of ERp18 are
unusual in that the rate-limiting step in the process is not reoxidation of
the enzyme by oxidized glutathione, but instead oxidation is limited by
interactions of ERp18 with the substrate. Whereas it is possible that ERp18
has a different substrate specificity than PDI, it should be noted that with
this peptide substrate all human PDI family members (except PDIr) have nearly
equivalent molar active site activities, and all are rate-limited by
reoxidation by glutathione.2 Furthermore, to our knowledge, the
oxidation of all reported substrates by PDI is limited by reoxidation (see
Refs. 25 and
27 for examples). If ERp18 has
a thioredoxin fold with an insert between 3 and
4 (as seen in DsbA), then this insert will be spatially
close to the active site and may regulate access to it. The rapid reoxidation
of ERp18 in vitro by glutathione and the low affinity for peptide
substrates may suggest a role for ERp18 in shuffling oxidizing equivalents
from ERo1 to luminally located proteins, a role played in S.
cerevisiae by Mpd2p (8), a
protein with a single catalytic domain plus a region of unknown
structure/function with no known homologues in higher eukaryotes and with no
observable phenotype on deletion
(16).
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FOOTNOTES |
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¶ To whom correspondence should be addressed. Tel.: 358-8-553-1136; Fax: 358-8-553-1141; E-mail: lloyd.ruddock{at}oulu.fi.
1 The abbreviations used are: ER, endoplasmic reticulum; PDI,
protein-disulfide isomerase; GFP, green fluorescent protein; MALDI,
matrix-assisted laser desorption ionization.
2 H. I. Alanen and L. W. Ruddock, unpublished observations.
3 A. K. Lappi and L. W. Ruddock, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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