From the Collagen Research Unit, Biocenter Oulu and
Department of Medical Biochemistry, University of Oulu, P. O. Box
5000, FIN-90014 Oulu, Finland and the ¶ Department of
Biosciences, University of Kent,
Canterbury CT2 7NJ, United Kingdom
Received for publication, November 26, 2000, and in revised form, December 29, 2000
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ABSTRACT |
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Protein disulfide isomerase (PDI) is a
modular polypeptide consisting of four domains, a,
b, b', and a', plus an acidic
C-terminal extension, c. PDI carries out multiple
functions, acting as the Protein disulfide isomerase
(PDI)1 (EC 5.3.4.1), a major
protein within the lumen of the eukaryotic endoplasmic reticulum, is a
catalyst of disulfide bond formation and rearrangement in protein
folding (for reviews, see Refs. 1 and 2). PDI is a modular protein
consisting of four domains, a, b, b',
and a', plus an acidic C-terminal extension, c
(3, 4). The a and a' domains show sequence
similarity to thioredoxin, contain the catalytic site motif CGHC (1),
and have the thioredoxin fold (3, 5). The b and
b' domains show no amino acid sequence similarity to
thioredoxin and have no catalytic site sequence, but recent NMR studies
have indicated that the b domain (and by homology the
b' domain) also has the thioredoxin fold (2, 6).
PDI is a multifunctional polypeptide. In addition to its role in
protein folding within the endoplasmic reticulum (12-20), PDI serves
as the Previous cross-linking studies with various peptides and polypeptides
have indicated that the principal peptide binding site of PDI is
located in the b' domain and that this domain alone is
sufficient for binding peptides of 10-15 residues (26). However, binding of longer peptides requires the additional presence of either
the ab domains or the a' domain (26). In
agreement with these data, it has been found that the isolated
a and a' domains function effectively as simple
thiol:disulfide oxidoreductases but that the remaining domains are
required for full catalytic activity in assisting protein folding
associated with the formation of native disulfide bonds (27). It thus
seems likely that the nonnative protein binding region extends beyond
the principal peptide binding region present in domain b',
through all four domains (26, 28). The C-terminal extension
c plays no reported role in any of the functions of PDI
(29).
ERp57 is a PDI-related polypeptide (1, 9) that forms complexes with
both calnexin and calreticulin, these complexes being specifically
involved in the modulation of glycoprotein folding within the lumen of
the endoplasmic reticulum (30, 31). ERp57 resembles PDI in size, has
thioredoxin-like domains with CGHC catalytic site motifs in positions
corresponding to the a and a' domains of PDI, and
also shows significant sequence similarity to PDI in regions
corresponding to the b and b' domains (32). However, it does not substitute for PDI as the The present work sets out to study which domains of the PDI polypeptide
are required for the assembly of a prolyl 4-hydroxylase tetramer and
whether some of these domains can be substituted by the corresponding
domains of ERp57. The vertebrate prolyl 4-hydroxylase Construction of Baculovirus Expression Vectors and Generation of
Recombinant Baculoviruses--
PDI domain constructs were synthesized
by polymerase chain reaction using a human PDI cDNA (7) as a
template. The PDI signal sequence was added in front of the constructs
that did not begin at the a domain. Similarly, ER-retention
signals were added at the end of constructs that did not include region
c. Thus, construct PDIabb' codes for amino acids
1-350 of the mature PDI polypeptide, PDIbb'a'c for
119-491, PDIb'a'c for 217-491, PDIa'c for
348-491, PDIbb'a for 119-351 and 1-120, and
PDIabb'a for 1-352 and 1-120. cDNAs for
PDIabb' and PDIabb'a were ligated into the
EcoRI-BamHI site of pVL1392 (Invitrogen),
PDIb'a'c into the XbaI-BamHI site, and
PDIbb'a into the EcoRI-SmaI site of
this plasmid, whereas those for PDIbb'a'c and
PDIa'c were ligated into the EcoRI site of
pVL1393. Expression constructs for PDI/ERp57 hybrids were synthesized by polymerase chain reaction using the cDNAs for human PDI (7) and
ERp57 (32) as templates. Two polymerase chain reaction products, one
coding for the domains of PDI and the other for those of ERp57, were
ligated blunt-ended together and into cohesive sites of pVL1392. Thus,
PDIabb'ERp57a' codes for amino acids 1-350 of
PDI, 353-465 of ERp57, and an-AVKDEL retention signal;
ERp57aPDIbb'a'c codes for amino acids 1-107 of
ERp57, an R, and amino acids 117-491 of PDI;
ERp57abPDIb'a'c codes for amino acids 1-218 of
ERp57 and 219-491 of PDI; ERp57abb'PDIa'c codes
for amino acids 1-352 of ERp57 and 351-491 of PDI; and
PDIaERp57bb'a'c codes for amino acids 1-117 of
PDI and 110-481 of ERp57. The constructs for
PDIabb'ERp57a', ERp57aPDIbb'a'c, and
PDIaERp57bb'a'c were ligated into the
EcoRI-BamHI site, and those for
ERp57abPDIb'a'c and
ERp57abb'PDIa'c were ligated into the
EagI-BamHI site of pVL1392.
Spodoptera frugiperda Sf9 insect cells (Invitrogen)
were cultured as monolayers in TNM-FH medium (Sigma) supplemented with 10% fetal bovine serum (Bioclear) at 27 °C. The recombinant
baculovirus transfer vectors were cotransfected into Sf9 insect
cells with a modified Autographa californica nuclear
polyhedrosis virus DNA (BaculoGold, PharMingen) by calcium phosphate
transfection (35). The resultant viral pools were collected 4 days
later, amplified twice, and used for recombinant protein production.
Other recombinant baculoviruses used in this work were human PDI, human
ERp57, and human Expression and Analysis of Recombinant Proteins--
For the
expression of recombinant proteins, Sf9 insect cells were
infected with the recombinant baculoviruses at a multiplicity of 5. In
coexpression experiments, viruses were used at a ratio of 1:1. The
cells were harvested 3 days after infection; washed with a solution of
0.15 M NaCl and 0.02 M phosphate, pH 7.4;
homogenized in a solution of 0.1 M glycine, 0.1 M NaCl, 10 µM dithiothreitol, 0.1% Triton
X-100, and 0.01 M Tris, pH 7.8; and centrifuged at 10,000 × g for 20 min at 4 °C. The resulting
supernatants were analyzed by SDS-PAGE or by nondenaturing PAGE,
followed by Coomassie staining or Western blotting, and assayed for
prolyl 4-hydroxylase activity. In Western blotting, polyclonal
antibodies against human PDI, human Prolyl 4-Hydroxylase Activity--
Prolyl 4-hydroxylase activity
was assayed by a method based on the hydroxylation-coupled
decarboxylation of 2-oxo[1-14C]glutarate as described
previously (36). Total cellular protein concentrations were determined
using a Bio-Rad protein assay kit.
Synthesis and Labeling of Binding and Cross-linking of Expression of PDI Domain Constructs and PDI/ERp57 Hybrids in Insect
Cells--
Recombinant baculoviruses coding for the polypeptides
described in Fig. 1 were generated and
used to infect Sf9 insect cells. The cells were harvested
72 h after infection, homogenized in a buffer containing Triton
X-100, and centrifuged. The 0.1% Triton X-100-soluble proteins were
analyzed by 10% SDS-PAGE under reducing conditions, followed by
Coomassie staining. Bands corresponding to polypeptides of the expected
size were found in the Triton X-100-soluble fractions of the samples
(Fig. 2).
Coexpressions of PDI Domain Constructs with Prolyl 4-Hydroxylase
When PDIabb' was coexpressed with either the
PDI domains a and a' both contain the catalytic
site motif CGHC, show a high degree of amino acid sequence similarity
(7), and have very similar folded structures (3, 5). We therefore investigated whether the a' domain could be substituted by
the a domain. A baculovirus coding for PDIbb'a
was generated and used to coinfect insect cells with a virus coding for
either the Coexpression of PDI/ERp57 Hybrid Polypeptides with Prolyl
4-Hydroxylase Binding of Radiolabeled Interaction of Scrambled RNase with PDI Domain Constructs and
PDI/ERp57 Hybrids--
When crude cell extracts expressing the PDI
domain constructs were incubated in the presence of scrambled RNase and
subsequently cross-linked, cross-linking products were seen for PDI,
PDIabb', PDIbb'a'c, PDIbb'a, and
PDIabb'a (Fig. 6A, lanes
2, 4, 6, 10, and 12) but not for PDIb'a'c or
PDIa'c (Fig. 6A, lanes 8 and 14). The
cross-linking product of PDIb'a'c only became visible after a longer exposure time (Fig. 6B, lane 4). When the
corresponding polypeptide produced in Escherichia coli was
incubated in the presence of scrambled RNase and subsequently
cross-linked, a cross-linking product was also obtained (Fig. 6B,
lane 2). The result obtained with PDIabb' differs from
that previously obtained with the corresponding construct in crude cell
extracts of E. coli, in which no binding of biotinylated
scrambled RNase was detected using a streptavidin-horseradish peroxidase conjugate (26). This conjugate could not be used in insect
cell extracts because the biotinylated scrambled RNase was bound by
some polypeptide(s) present in the lysate. When PDIabb' produced in E. coli was analyzed by the method used here, a
cross-linking product was also obtained (data not shown).
Studies on the binding of scrambled RNase to the PDI/ERp57 hybrids were
limited to those constructs that could be recognized by the anti-PDI
antibody used. Cross-linking products were observed in the presence of
scrambled RNase for the PDIabb'ERp57a',
ERp57aPDIbb'a'c, and
ERp57abPDIb'a'c constructs (Fig. 6C, lanes
2, 4, and 6). ERp57aPDIbb'a'c exhibited a doublet band on cross-linking, probably resulting from an
intramolecular cross-linking event. No cross-linking to the
ERp57abb'PDIa'c construct was observed.
PDI has been shown to be a multifunctional protein. Because PDI is
a modular polypeptide, one important issue in its molecular analysis is
to understand the roles played by each of the individual PDI domains in
its multiple functions. The data presented here allow us to analyze the
roles of the individual domains of PDI in prolyl 4-hydroxylase assembly
and the degree to which ERp57 domains can substitute for PDI in
assembly and in the binding of peptides and nonnative proteins.
By coexpressing PDI domain constructs with the The two types of human prolyl 4-hydroxylase PDI and ERp57 have homologous domain structures, the highest degree of
identity and similarity being found in the a and
a' domains. It is therefore perhaps not surprising that the
role of the a domain of PDI in prolyl 4-hydroxylase assembly
could, in part, be substituted by the a domain of ERp57,
this effect being more significant with the The primary peptide binding site of PDI is located in the b'
domain, and this domain alone is sufficient for the binding of peptides
of 10-15 residues (26). In agreement with this requirement, Whereas the primary peptide binding site of PDI is located in the
b' domain, binding of longer substrates (for example, a 28-amino acid fragment derived from bovine pancreatic trypsin inhibitor) has been shown to minimally require either abb'
or b'a'c (26). The binding of nonbiotinylated scrambled
RNase was likewise found here to require either PDI abb' or
b'a'c.
A remarkable finding was that the a' domain of PDI could not
be replaced in prolyl 4-hydroxylase assembly by either a' of ERp572 or a of
PDI, even though these three domains are highly similar in their amino
acid sequences (7, 32) and folded structures (3, 5). Previously, it has
been demonstrated that substitution in PDI of the 78 most C-terminal
residues of domain a' and the C-terminal extension
c by the corresponding residues of ERp57 totally abolishes
the assembly of a prolyl 4-hydroxylase tetramer (32). Similarly,
several mutations introduced into the C-terminal region of the PDI
domain a' prevent prolyl 4-hydroxylase assembly (29). It
thus seems that the a' domain contains a region that is
highly critical for the assembly of the prolyl 4-hydroxylase tetramer
(Table II), as is the primary peptide binding region present in the
b' domain. As no assembly was seen with
PDIabb' and either the The results presented here indicate that in PDI there is a broad
binding region for folding substrates and functional partners to which
domains contribute to different extents. For small peptides, the
b' domain is essential and sufficient. For large peptides, nonnative proteins such as scrambled RNase, and for the subunit in the animal prolyl
4-hydroxylases and in the microsomal triglyceride transfer protein and
independently acting as a protein folding catalyst. We report here that
the minimum sequence requirement for the assembly of an active prolyl
4-hydroxylase
2
2 tetramer in insect cell
coexpression experiments is fulfilled by the PDI domain construct
b'a' but that the sequential addition of the b
and a domains greatly increases the level of enzyme
activity obtained. In the assembly of active prolyl 4-hydroxylase
tetramers, the a and b domains of PDI, but not
b' and a', can in part be substituted by the
corresponding domains of ERp57, a PDI isoform that functions naturally
in association with the lectins calnexin and calreticulin. The
a' domain of PDI could not be substituted by the PDI
a domain, suggesting that both b' and
a' domains contain regions critical for prolyl
4-hydroxylase assembly. All PDI domain constructs and PDI/ERp57 hybrids
that contain the b' domain can bind the 14-amino acid
peptide
-somatostatin, as measured by cross-linking; however,
binding of the misfolded protein "scrambled" RNase required the
addition of domains ab or a' of PDI. The human prolyl 4-hydroxylase
subunit has at least two isoforms,
(I) and
(II), which form with the PDI polypeptide the
(
(I))2
2 and (
(II))2
2 tetramers. We report here
that all the PDI domain constructs and PDI/ERp57 hybrid polypeptides
tested were more effectively associated with the
(II) subunit than
the
(I) subunit.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit in the animal prolyl 4-hydroxylase
2
2 tetramers and
dimers (7-9) and
in the microsomal triglyceride transfer protein
dimer (10, 11).
Prolyl 4-hydroxylase plays a central role in the synthesis of all
collagens (8, 9, 21), whereas the microsomal triglyceride transfer
protein is essential for the assembly of apoB-containing lipoproteins
(10, 11). The main function of PDI in both of these proteins appears to
be to keep their highly insoluble
subunits in a catalytically active, nonaggregated conformation (8, 9, 11, 22-24). This function is
likely to be related to the peptide binding and chaperone functions of
PDI, and it does not require the catalytic site cysteine residues (24,
25).
subunit of prolyl 4-hydroxylase (32).
subunit has
at least two isoforms,
(I) and
(II), which form with the PDI
polypeptide the (
(I))2
2, type I, and
(
(II))2
2, type II, enzyme tetramers (33,
34). We therefore also studied whether any differences exist in the
assembly of the PDI domains and PDI/ERp57 hybrid polypeptides between
the
(I) and
(II) subunits. To correlate the prolyl 4-hydroxylase
assembly data with the less specific binding capabilities of PDI for
folding substrates, we also analyzed the ability of the various PDI
domains and PDI/ERp57 hybrid constructs to bind the peptide
-somatostatin and the misfolded protein "scrambled" RNase.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(I) and
(II), coding for the corresponding
prolyl 4-hydroxylase
subunits (22, 32, 34).
(I) subunit and mouse
(II)
subunit were used.
-Somatostatin and Scrambled
RNase--
Scrambled RNase, the homobifunctional cross-linking reagent
disuccinimidyl glutarate (DSG), and all other chemicals were obtained from Sigma. 125I-Labeled Bolton-Hunter labeling
reagent, ECL reagent, and x-ray films were purchased from Amersham
Pharmacia Biotech. The somatostatin derivative without cysteine
residues (
-somatostatin, AGSKNFFWKTFTSS) was synthesized as
described previously for other peptides (37). The polyclonal antibody
raised against PDI was from Stressgene. 125I-Labeled
Bolton-Hunter labeling of
-somatostatin was performed as recommended
by the manufacturer.
-Somatostatin and Scrambled
RNase--
After precipitation with trichloroacetic acid, the
radiolabeled
-somatostatin was dissolved in distilled water. Labeled
-somatostatin (approximately 3 µM) or scrambled RNase
(approximately 50 µM) was added to Buffer A (100 mM NaCl, 25 mM KCl, 25 mM phosphate buffer, pH 7.5) containing crude cell extracts (approximately 20 mg/ml). The samples (10 µl) were incubated for 10 min on ice before
cross-linking (26). Cross-linking was performed using DSG (26). The
samples were supplied with
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the PDI
domain constructs and PDI/ERp57 hybrids prepared.
Domains are marked at the top by a, b, b', and
a', and the C-terminal extension is marked by c.
Domains coding for PDI are indicated by dark gray and those
coding for ERp57 by light gray.
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Fig. 2.
Analysis of expression of PDI, ERp57, PDI
domain constructs, and PDI/ERp57 hybrids in insect cells by SDS-PAGE
under reducing conditions. Triton X-100-soluble samples from
insect cells infected with baculoviruses coding for human PDI
(lane 1), PDIabb' (lane 2),
PDIbb'a'c (lane 3), PDIb'a'c
(lane 4), PDIa'c (lane 5),
PDIbb'a (lane 6), PDIabb'a (lane
7), ERp57 (lane 8),
PDIabb'ERp57a' (lane 9),
ERp57aPDIbb'a'c (lane 10),
ERp57abPDIb'a'c (lane 11),
ERp57abb'PDIa'c (lane 12), and
PDIaERp57bb'a'c (lane 13). The samples
were analyzed by 10% SDS-PAGE and Coomassie staining.
Asterisks indicate migration of the polypeptides.
Subunits--
PDI domains required for the assembly of a prolyl
4-hydroxylase tetramer were studied by coexpression of the various
domain constructs (Fig. 1) with either the
(I) or
(II) subunit of
human prolyl 4-hydroxylase in insect cells. The cells were harvested and homogenized as described above. Then the Triton X-100-soluble proteins were analyzed by nondenaturing PAGE followed by Western blotting with a polyclonal antibody against human PDI (Fig.
3) and assayed for prolyl 4-hydroxylase
activity by a procedure based on the hydroxylation-coupled
decarboxylation of 2-oxo[1-14C]glutarate (Table
I).
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Fig. 3.
Analysis of coexpression of the human prolyl
4-hydroxylase (I) or
(II) subunit with PDI domain constructs in insect
cells by PAGE under nondenaturing conditions. Triton X-100-soluble
samples from insect cells coinfected with baculoviruses coding for
human
(I) subunit and PDI (lane 1), PDIabb'
(lane 3), PDIbb'a'c (lane 5),
PDIb'a'c (lane 7), PDIa'c (lane
9), PDIbb'a (lane 11), and
PDIabb'a (lane 13). Triton X-100-soluble samples
from coinfections with baculoviruses coding for human
(II) subunit
and PDI (lane 2), PDIabb' (lane 4),
PDIbb'a'c (lane 6), PDIb'a'c
(lane 8), PDIa'c (lane 10),
PDIbb'a (lane 12), and PDIabb'a
(lane 14). The samples were analyzed by 8% PAGE under
nondenaturing conditions. Western blotting was carried out by using an
anti-PDI antibody. Asterisks indicate migration of complexes
formed with the
subunits and the PDI domain constructs. Migration
of the free monomers is indicated by M.
Prolyl 4-hydroxylase activity of Triton X-100-soluble proteins from
insect cells expressing the (I) or
(II) subunit of human prolyl
4-hydroxylase together with either the wild-type PDI, ERp57, PDI domain
constructs, or PDI/ERp57 hybrids
(I) (typically about 600 dpm) and
(II) (typically about 800 dpm)
subunit alone were subtracted from all values.
(I) or
(II) subunit, no enzyme tetramer was detected by Western blotting
(Fig. 3, lanes 3 and 4) and no
significant level of enzyme activity was generated (Table I).
Coexpression of PDIbb'a'c with the
(I) subunit led to
assembly of a product with slow mobility in Western blotting (Fig. 3,
lane 5), but very little enzyme activity, if any, was
generated (Table I). However, when this same construct was coexpressed
with the
(II) subunit, an enzyme tetramer with a mobility that
corresponded to the reduced size of the PDI polypeptide was produced
(Fig. 3, lane 6), and this tetramer was an active prolyl
4-hydroxylase (Table I). Nevertheless, the amount of enzyme activity
generated in insect cells using this construct was only about 40% of
that obtained with wild-type PDI (Table I), due to either a less
efficient assembly level obtained with the mutant or a lower specific
activity of the mutant enzyme tetramer. When the PDI construct was
shortened further to contain just b'a'c, no assembly was
detected with the
(I) subunit (Fig. 3, lane 7). However,
assembly was still detected with the
(II) subunit (Fig. 3,
lane 8), although the amount of enzyme activity generated
was only about 9% of that obtained with the wild-type control (Table I). PDIa'c gave no assembly (Fig. 3, lanes 9 and
10) or activity (Table I) with either the
(I) or
(II) subunit.
(I) or
(II) subunit. No assembly was obtained with
this mutant polypeptide with either
subunit (Fig. 3, lanes
11 and 12), and correspondingly, no enzyme activity was
generated (Table I). To ensure that the result was not influenced by
the missing N-terminal a domain, a baculovirus coding for
PDI abb'a was generated and used for additional coexpression
experiments. However, no assembly or activity was seen with this
construct either (Fig. 3, lanes 13 and
14; Table I). The data thus indicate that the a
domain of PDI cannot functionally substitute for the a'
domain in prolyl 4-hydroxylase assembly.
Subunits--
To study whether the PDI domains can
be functionally replaced by the corresponding domains of ERp57,
baculoviruses coding for several hybrid PDI/ERp57 polypeptides (Fig. 1)
were generated. Coexpression of PDIabb'ERp57a'
with either the
(I) or
(II) subunit led to no assembly (Fig.
4A, lanes 5 and 6)
and generated no enzyme activity (Table I). In contrast, coexpression
of ERp57aPDIbb'a'c with either the
(I) or
(II) subunit resulted in tetramer assembly (Fig. 4A,
lanes 7 and 8) and in the generation of prolyl
4-hydroxylase activity (Table I). In the case of the
(I) subunit,
the amount of enzyme activity obtained with
ERp57aPDIbb'a'c was about 23% of that obtained
with the wild-type PDI. In contrast, the construct PDIbb'a'c
without the ERp57 a domain gave less than 5% of the wild-type activity (Table I). In the case of the
(II) subunit, no
difference could be observed between the
ERp57aPDIbb'a'c and PDIbb'a'c
constructs, both of which gave about 40% of the activity level seen
with the wild-type PDI (Table I). A positive effect of the addition of
ERp57 domains was seen even more distinctly with the construct
ERp57abPDIb'a'c. Cells expressing this
polypeptide formed an active enzyme with both the
(I) and
(II)
subunits even though detection of the complex with the
(I) subunit
failed by nondenaturing PAGE, most probably due to lability of this
tetramer (Fig. 4A, lanes 9 and 10; Table I),
whereas the PDIb'a'c construct showed no tetramer formation
with the
(I) subunit and gave less enzyme activity with the
(II)
subunit (8.7% versus 17.4%; Table I). The addition of
ERp57abb' in front of PDI a'c did not rescue prolyl 4-hydroxylase assembly (Fig. 4A, lanes 11 and 12; Table I). Similarly,
PDIaERp57bb'a'c resulted in no tetramer assembly (Fig. 4A, lanes 13 and 14; Table I), as confirmed
by Western blotting with anti-
(I) and anti-
(II) subunit
antibodies (Fig. 4B, lanes 3 and 6).
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Fig. 4.
Analysis of coexpression of the human prolyl
4-hydroxylase (I) or
(II) subunit with PDI/ERp57 hybrid polypeptides in
insect cells by PAGE under nondenaturing conditions. A,
Triton X-100-soluble samples from insect cells coinfected with
baculoviruses coding for human
(I) subunit and PDI (lane
1), ERp57 (lane 3),
PDIabb'ERp57a' (lane 5),
ERp57aPDIbb'a'c (lane 7),
ERp57abPDIb'a'c (lane 9),
ERp57abb'PDIa'c (lane 11), and
PDIaERp57bb'a'c (lane 13). Triton
X-100-soluble samples from coinfections with baculoviruses coding for
human
(II) subunit and PDI (lane 2), ERp57 (lane
4), PDIabb'ERp57a' (lane 6),
ERp57aPDIbb'a'c (lane 8),
ERp57abPDIb'a'c (10),
ERp57abb'PDIa'c (lane 12), and
PDIaERp57bb'a'c (lane 14). The samples
were analyzed by 8% PAGE under nondenaturing conditions, and Western
blotting was carried out by using an anti-PDI antibody.
Asterisks indicate migration of the tetramers; D
and M indicate migration of dimers and monomers,
respectively. B, Triton X-100-soluble samples from insect
cells coinfected with baculoviruses coding for human
(I) subunit and
PDI (lane 1), ERp57 (lane 2), and
PDIaERp57bb'a'c (lane 3); coinfections
with baculoviruses coding for human
(II) subunit and PDI (lane
4), ERp57 (lane 5), and
PDIaERp57bb'a'c (lane 6). The samples
were analyzed as in A, except that Western blotting was
carried out using an anti-
(I) subunit antibody in lanes
1-3 and an anti-
(II) subunit antibody in lanes
4-6. Asterisks indicate migration of the
tetramers.
-Somatostatin to PDI Domain Constructs
and PDI/ERp57 Hybrids--
125I-Labeled Bolton-Hunter
labeled
-somatostatin was added to the insect cell extracts of PDI
domain constructs and PDI/ERp57 hybrids to investigate their
interactions. After cross-linking with DSG, single cross-linking
products could be detected in cell extracts expressing PDI,
PDIabb', PDIbb'a'c, PDIb'a'c,
PDIbb'a, PDIabb'a,
PDIabb'ERp57a',
ERp57aPDIbb'a'c, and
ERp57abPDIb'a'c (Fig.
5A, lanes 1-4, 6, and
7; Fig. 5B, lanes 2-4). No
cross-linking products were detected with
-somatostatin when cell
extracts expressing PDIa'c (Fig. 5A, lane 5),
ERp57, ERp57abb'PDIa'c, or PDIaERp57bb'a'c (Fig. 5B, lanes 1, 5, and 6) were tested. These results (Table
II) are consistent with previous data
(26) indicating that the minimum sequence requirement for binding of
-somatostatin to PDI is fulfilled by the b' domain, and
they extend the data by demonstrating that the b' domain of
ERp57 is not capable of replacing the corresponding PDI domain in this
assay.
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Fig. 5.
Interaction of radiolabeled
-somatostatin with PDI domain constructs and
PDI/ERp57 hybrid polypeptides expressed in insect cells.
Radiolabeled
-somatostatin was cross-linked to insect cells
expressing the PDI domain constructs and PDI/ERp57 hybrids. The samples
were analyzed by 12.5% SDS-PAGE, and detection was performed by
autoradiography. A, human PDI (lane 1),
PDIabb' (lane 2), PDIbb'a'c
(lane 3), PDIb'a'c (lane 4),
PDIa'c (lane 5), PDIbb'a (lane
6), and PDIabb'a (lane 7). B,
ERp57 (lane 1), PDIabb'ERp57a'
(lane 2), ERp57aPDIbb'a'c (lane
3), ERp57abPDIb'a'c (lane 4),
ERp57abb'PDIa'c (lane 5), and
PDIaERp57bb'a'c (lane 6).
Correlation between binding of -somatostatin and scrambled RNase and
the assembly of a functional prolyl 4-hydroxylase tetramer
View larger version (12K):
[in a new window]
Fig. 6.
Interaction of scrambled RNase with PDI
domain constructs and PDI/ERp57 hybrid polypeptides expressed in insect
cells. Equal amounts of total cell extracts from insect cells were
incubated with scrambled RNase with subsequent cross-linking. The
samples were analyzed by 12.5% SDS-PAGE, and detection was done by
Western blotting using a polyclonal antibody against PDI. Cross-linking
products are marked by asterisks. A, cell
extracts expressing PDI (lanes 1 and 2),
PDIabb' (lanes 3 and 4),
PDIbb'a'c (lanes 5 and 6),
PDIb'a'c (lanes 7 and 8),
PDIbb'a (lanes 9 and 10), and
PDIabb'a (lanes 11 and 12)
polypeptides. Lanes 2, 4, 6, 8, 10, 12, and 14, polypeptides were cross-linked with DSG after incubation with scrambled
RNase. B, E. coli extracts expressing PDIb'a'c
(lanes 1 and 2) and insect cell extracts
expressing PDIb'a'c (lanes 3 and 4).
Lanes 2 and 4 show polypeptides cross-linked with
DSG after incubation with scrambled RNase. A longer exposure time was
used than in A. C, cell extracts of
PDIabb'ERp57a' (lanes 1 and
2), ERp57aPDIbb'a'c (lanes
3 and 4), ERp57abPDIb'a'c
(lanes 5 and 6), and
ERp57abb'PDIa'c (lanes 7 and
8). Lanes 2, 4, 6, and 8 show
polypeptides incubated with scrambled RNase after subsequent
cross-linking.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits of prolyl
4-hydroxylase, we have determined that the minimum sequence requirement
for the assembly of an active tetramer is fulfilled by the PDI
construct b'a'c (Table I). As a previous study (29) has
demonstrated that the presence or absence of the C-terminal extension
c has no observable effect on any of the main functions of
PDI (i.e. the oxidoreductase, disulfide isomerase,
chaperone, or protein subunit functions), the minimum requirement for
prolyl 4-hydroxylase assembly is in fact fulfilled by the PDI domains b'a'. However, the addition of the b domain gave
a higher level of prolyl 4-hydroxylase activity, and all four domains
of PDI were required for the highest level of activity measured. It is not clear whether this represents an increase in the absolute level of
active prolyl 4-hydroxylase or in the specific activity of the enzyme
tetramer formed using the PDI constructs. In either case, these data
indicate that the primary sites of interaction between PDI and the
subunit lie in the b' and a' domains of PDI but
that both the b and a domains contribute to the
structure or stability of the tetramer. Such a contribution may either
come from direct interactions between these domains and the
subunit or may come from a contribution due to interactions between the
subunits (i.e. a dimerization event in PDI). Further
evidence for the absolute requirement for the b' and
a' domains and also the involvement of the b and
a domains in tetramerization comes from an analysis of the
differences between
(I) and
(II) subunit tetramerization and the
use of PDI/ERp57 hybrids.
subunit, the
(I) and
(II) subunits, readily form the
(
(I))2
2 and
(
(II))2
2 enzyme tetramers in insect cell
coexpression experiments with PDI. The catalytic properties of the two
types of enzyme tetramer are highly similar, but there are some
distinct differences in the binding properties and binding sites for
proline-rich peptide substrates and peptide inhibitors between the two
isoenzymes (34, 38). To date, no differences have been reported in the
tetramer assembly between these subunits (22, 34). However, the data presented here indicate a major difference between the two types of
subunit, in that the
(II) subunit became more effectively associated
with the shortened PDI constructs, as seen by both the appearance of
tetramers on native gels and by prolyl 4-hydroxylase activity. Detailed
structural data are needed to fully understand the differences found
here in the tetramer assembly between the
(I) and
(II) subunits.
(I) subunit (Table I).
The degree of amino acid sequence identity between the b
domains of human PDI and ERp57 is only 23% (based on the domain
boundaries used here), but the ab fragment of PDI could
likewise be, in part, substituted by the ab fragment of
ERp57 (Tables I and II). Neither PDIabb'ERp57a'
nor the ERp57abb'PDIa'c constructs formed prolyl 4-hydroxylase tetramers, further indicating the importance of the
b' and a' domains of PDI in the assembly process.
-somatostatin could be cross-linked here to all PDI domain and hybrid constructs that contain the PDI b' domain. In
contrast, the b' domain of ERp57 does not appear to contain
a binding site for small peptides such as
-somatostatin, as this
peptide could not be cross-linked to ERp57 or to any hybrid lacking PDI
domain b' (Table II). Because the b' domain of
PDI could not be substituted by b'of ERp57 in the assembly
of a prolyl 4-hydroxylase tetramer, it seems reasonable to speculate
that the primary peptide binding domain of PDI, which is also required
for nonnative protein binding, is involved in interacting with the
subunit during the assembly process.
(I) or
(II) subunit, the
implication is that the requirements for the assembly of a prolyl
4-hydroxylase tetramer are more stringent than those for the binding of
nonnative proteins such as scrambled RNase (Table II). Detailed
structural comparison between PDI domains a and
a' and ERp57 domain a' and a determination of the
relative spatial positions of the domains of PDI in the native
structure are needed to understand these functional differences.
subunit of
prolyl 4-hydroxylase, the a' domain contributes
significantly to the binding and the b and a
domains enhance binding. The inability of the b' and
a' domains of ERp57 to substitute for the analogous PDI
domains suggests that these domains have very specific binding properties, e.g. to their permanent partners, the lectins
calnexin and calreticulin, which are therefore analogous to the
subunit in prolyl 4-hydroxylase.
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ACKNOWLEDGEMENTS |
---|
We thank Eeva Lehtimäki, Merja Nissilä, and Jaana Träskelin for expert technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Health Sciences Council of the Academy of Finland, the Finnish Center of Excellence Program 2000-2005 Grant 44843, and European Union Contract BIO 4 CT 960436.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Biocenter Oulu and Department of Biochemistry, University of Oulu, P. O. Box 3000, FIN-90014 Oulu, Finland.
To whom correspondence should be addressed. Tel.:
358-8-537-5831; Fax: 358-8-537-5811; E-mail:
peppi.koivunen@oulu.fi.
Published, JBC Papers in Press, December 29, 2000, DOI 10.1074/jbc.M010656200
2 L. Silvennoinen, P. Karvonen, P. Koivunen, J. Myllyharju, K. I. Kivirikko, and I. Kilpeläinen, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PDI, protein disulfide isomerase; PAGE, polyacrylamide gel electrophoresis; DSG, disuccinimidyl glutarate.
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