From the School of Biological Sciences, The University of Manchester, 2.205 Stopford Building, Manchester M13 9PT, United Kingdom
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ABSTRACT |
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Protein-disulfide isomerase (PDI) has been shown to be a multifunctional enzyme catalyzing the formation of disulfide bonds, as well as being a component of the enzymes prolyl 4-hydroxylase (P4-H) and microsomal triglyceride transfer protein. It has also been proposed to function as a molecular chaperone during the refolding of denatured proteins in vitro. To investigate the role of this multifunctional protein within a cellular context, we have established a semi-permeabilized cell system that reconstitutes the synthesis, folding, modification, and assembly of procollagen as they would occur in the cell. We demonstrate here that P4-H associates transiently with the triple helical domain during the assembly of procollagen. The release of P4-H from the triple helical domain coincides with assembly into a thermally stable triple helix. However, if triple helix formation is prevented, P4-H remains associated, suggesting a role for this enzyme in preventing aggregation of this domain. We also show that PDI associates independently with the C-propeptide of monomeric procollagen chains prior to trimer formation, indicating a role for this protein in coordinating the assembly of heterotrimeric molecules. This demonstrates that PDI has multiple functions in the folding of the same protein, that is, as a catalyst for disulfide bond formation, as a subunit of P4-H during proline hydroxylation, and independently as a molecular chaperone during chain assembly.
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INTRODUCTION |
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Protein-disulfide isomerase is now firmly established as a multifunctional protein that both catalyzes the formation of disulfide bonds and acts as a subunit of prolyl 4-hydroxylase and microsomal triglyceride transfer protein (1). The function of PDI1 as a component of these enzymes appears to be to maintain the catalytic subunits in a soluble form rather than directly participating in catalysis (2, 3). In this respect, its function is independent of disulfide isomerase activity (4). More recently, PDI has been proposed to act as a molecular chaperone by binding to unfolded proteins, thereby preventing aggregation (5-8). This proposal is based on the observation that PDI assists in the refolding of certain denatured proteins in vitro, but this activity appears to be substrate specific, with no activity or even negative (antichaperone) activity being observed with some protein substrates (9-11). PDI also has been shown to interact with newly synthesized proteins (12, 13) and with cysteine mutants of human lysozyme (14), but whether this interaction reflects chaperone activity or the binding of PDI to its substrate during disulfide bond formation still needs to be determined.
PDI is clearly a key cellular folding enzyme that is important for the maturation of several secreted and membrane associated proteins. This is particularly true for the folding and maturation of procollagen, where PDI is involved in a number of key stages. As the polypeptide chain is translocated across the membrane of the endoplasmic reticulum, intrachain disulfide bonds are formed within the N-propeptide and C-propeptide, and hydroxylation of proline and lysine residues occurs within the triple helical domain (15). Chains then associate via the C-propeptides to form homo- or heterotrimeric molecules. This allows the triple helical domain to form a nucleation point at its C-terminal end, ensuring correct alignment of the chains. The triple helix then folds in a C- to N-direction, with the N-propeptides finally associating and in some cases forming interchain disulfide bonds (16). PDI participates during proline hydroxylation as a subunit of prolyl 4-hydroxylase and also catalyzes the formation of both intra- (17) and interchain disulfide bonds (18).
Most of our understanding of how procollagen folds and assembles within the cell has come from studies of cells grown in culture, particularly either skin or tendon fibroblasts. Although this approach has provided us with a clear outline of the intracellular folding and assembly of procollagen, it does not lend itself to a more detailed analysis of the molecular recognition events occurring during assembly. To facilitate these studies, a semi-permeabilized cell system has been developed that reconstitutes the initial stages in the assembly and modification of procollagen as they would occur in an intact cell (19). Using this system, the translocation, disulfide bond formation, and assembly of procollagen into a correctly aligned triple helical molecule has been reconstituted, in a system that mimics the processes as they would occur within an intact cell (20). Here, we have extended these studies to investigate the role of PDI in the folding and assembly of procollagen. We have demonstrated that this protein not only participates in disulfide bond formation and proline hydroxylation but also acts as a molecular chaperone interacting specifically and independently with procollagen chains that remain monomeric, thereby preventing premature assembly or aggregation.
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MATERIALS AND METHODS |
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Construction of Recombinant Plasmids--
Recombinant
p1(III)
1 and p
2(I)
1 have been described previously (21).
Recombinant p
1(I)
1 was generated from COL1A1-CMV (22) by excision
of an internal 2.5-kb ApaI fragment and religation of the
parental plasmid. An additional nucleotide was inserted by
Pfu mutagenesis using the QuikChange mutagenesis kit
(Stratagene Ltd., Cambridge, UK) at the ApaI cleavage site
to preserve the correct reading frame. Recombinant plasmid constructs
were generated by polymerase chain reaction overlap extension using the
principles outlined by Horton (23). Polymerase chain reactions (100 µl) comprised template DNA (500 ng), oligonucleotide primers (100 pmol each), in 10 mM KCl, 20 mM Tris-HCl, pH
8.8, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% (v/v) Triton X-100, 300 µM each dNTP. Ten rounds of amplification were performed
in the presence of 1unit Vent DNA polymerase (New England Biolabs,
Beverly, MA). Recombinant C-propeptide-minus was generated using a
5'-oligonucleotide primer (5' GATTACGCCAAGCGCGCA 3') complementary to
the T3 promoter sequence upstream of the initiation codon in
p
1(III)
1 and a 3'-oligonucleotide primer (5'
TCGCTAGGTACCCTATTATCCATAATACGGGGCAAAAC 3') complementary to sequence in
p
1(III)
1 up to the C-proteinase cleavage site and incorporating a
KpnI site to facilitate subsequent sub-cloning. Polymerase
chain reaction yielded a 1200-bp fragment that was cut with
HindIII and KpnI and subcloned into
pBS-SK
(Stratagene Ltd., Cambridge, UK). Recombinant
p
1(III)
1:alt was generated using a 5'-oligonucleotide primer (5'
AATGGAGCTCCTGGACCCATG 3') complementary to a sequence 100 bp upstream
of an XhoI site in p
1(III)
1 and a 3' amplification
primer (5' TCGCAGGGTACCGTCGGTCACTTGCACTGGTT 3') complementary to a
region 100 bp downstream of the stop codon in p
1(III)
1. A
KpnI site was incorporated to facilitate subsequent subcloning. Pairs of internal oligonucleotides, of which one included a
19 nucleotide overlap, were designed to generate a molecule with
sequence coding for the B-G region from pro
2(I) as described previously (25). Overlap extension yielded a product of approximately 1000 bp, which was purified, digested with XhoI and
KpnI, and ligated into p
1(III)
1 from which a 1080 bp
XhoI-KpnI fragment had been excised.
Transcription in Vitro-- Transcription reactions were carried out as described by Gurevich et al. (26). Recombinant plasmids were linearized and transcribed using T3 RNA polymerase (Promega, Southampton, UK). Reactions (100 µl) were incubated at 37 °C for 4 h. Following purification over RNeasy columns (Qiagen, Dorking, UK), the RNA was resuspended in 100 µl of RNase-free water containing 1 mM DTT and 40 units of RNasin (Promega).
Translation in Vitro--
RNA was translated using a rabbit
reticulocyte lysate (FlexiLysate, Promega) for 60 min. at 30 °C. The
translation reaction (25 µl) contained 17.5 µl of reticulocyte
lysate, 0.5 µl of 1 mM amino acids (minus methionine),
0.5 µl of 100 mM KCl, 0.25 µl of ascorbic acid (5 mg/ml), 15 µCi of L-[35S] methionine, (NEN
Life Science Products), 1 µl of transcribed RNA and 1 µl
(approximately 2 × 105) of semipermeabilized cells
(SP cells) prepared as described previously (19). Translations were
incubated either in the presence or absence of 1 µl of 25 mM ,
'-dipyridyl (Sigma) to inhibit the activity of
endoplasmic reticulum (ER) hydroxylase enzymes.
Posttranslational Incubations-- After 60 min of translation, cycloheximide was added to 5 mM, and samples were incubated for a further time periods up to 60 min at 30 °C in the presence or absence of 5 mM Fe(II) sulfate to allow hydroxylation to occur posttranslationally (20). SP cells were isolated by centrifugation at 13,000 × g for 5 min. Pellets were resuspended in KHM buffer prior to subsequent analysis. Samples were prepared for electrophoresis and treated with proteases or the chemical cross-linkers BMH or DSP (Pierce and Warriner Ltd., Cheshire, UK).
Proteolytic Digestion-- Isolated SP cells were solubilized in CT/T digest buffer (50 mM Tris-HCl, pH 7.4, containing 0.15 M NaCl, 10 mM EDTA, 1% (v/v) Triton X-100) and centrifuged at 13,000 × g for 5 min to remove cell debris. The supernatant was recovered and then digested with a combination of chymotrypsin (250 µg/ml) and trypsin (100 µg/ml) (Sigma) for 1 min at 30 °C. The reactions were stopped by the addition of soybean trypsin inhibitor (Sigma) to a final concentration of 500 µg/ml and acidified by the addition of HCl to a final concentration of 100 mM. Samples were incubated with pepsin (100 µg/ml) for 2 h at 30 °C. The reactions were stopped by neutralization with Tris base (100 mM) and prepared for electrophoresis as described below.
Chemical Cross-linking-- After translation, SP cell pellets were resuspended in a final volume of 50 µl of KHM and chemical cross-linkers added from a 50 mM stock (prepared fresh in DMSO) to a final concentration of 1 mM for both DSP and BMH cross-linking experiments. Cross-linking of samples was performed for 10 min at 25 °C followed by a further 10 min incubation after addition of 100 mM glycine or 5 mM DTT to quench the DSP or BMH reactions, respectively.
Immunoprecipitation--
Cross-linked samples were denatured by
boiling for 5 min in SDS/Nonidet P-40 denaturation buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl containing 1%
(w/v) SDS and 1% (v/v) Nonidet P-40). Insoluble material was removed
by centrifugation at 13,000 × g for 10 min, and the
supernatant was adjusted to a final volume of 1 ml of
immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, containing 0.15 M NaCl, 10 mM EDTA, 1% (v/v)
Triton X-100). Immunoprecipitations were preincubated at 4 °C for 40 min in IP buffer containing 50 µl of protein A-Sepharose (10% (w/v)
in PBS) (Zymed Laboratories Inc., San Francisco, CA),
and the samples were centrifuged for 1 min at 10,000 × g to remove protein A-binding components. Supernatants were
recovered and made up to a volume of 1 ml with IP buffer. Immunoprecipitation of cross-linked products was carried out at 4 °C
in the presence of antibodies and 50 µl of protein A-Sepharose (10%
(w/v) in PBS). The polyclonal antisera to bovine PDI and rat P4-H subunit were used as described previously (2, 27). Immune complexes
were retrieved by brief centrifugation (13,000 × g for
30 s) and washed twice in IP buffer, once in IP buffer containing
500 mM NaCl, and finally in IP buffer alone.
SDS-Polyacrylamide Gel Electrophoresis-- Samples prepared for electrophoresis by the addition of SDS-PAGE loading buffer (0.0625 M Tris/HCl, pH 6.8, SDS (2% w/v), glycerol (10% v/v), bromphenol blue) in the presence or absence of 50 mM DTT and boiled for 5 min. After electrophoresis, gels were dried, processed for autoradiography, and exposed to Kodak X-Omat AR film.
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RESULTS |
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Assembly of Procollagen Mini-chains in SP Cells--
The main aim
of this study was to investigate the role of resident proteins within
the endoplasmic reticulum in the folding and assembly of procollagen.
To facilitate these studies we constructed a variety of different
procollagen "mini-chains" that contain deletions within the triple
helical domain. These deletions preserve the
Gly-X-Y triplet consensus and are not predicted
to alter the folding of the chains. We also prepared a
C-propeptide-minus (CP-minus) construct that contains all of the
pro1(III)
1 chain apart from the C-propeptide, the last amino acid
being at the C-proteinase cleavage site (Fig.
1). Previous experiments have shown that
procollagen mini-chains translated in the presence of SP cells are
efficiently translocated, modified, and, in the case of
pro
1(III)
1, assembled into a correctly aligned triple helix (20,
25).
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Interaction of Prolyl 4-Hydroxylase with Unfolded Procollagen Chains-- Previous work on the substrate specificity of P4-H has demonstrated that the conformation of the procollagen triple helix determines enzyme activity (15). Thus, the enzyme will readily hydroxylate unhydroxylated procollagen chains as long as the chains have not formed a triple helix (29). It has also been shown that malfolded chains may be isolated as stable complexes with P4-H (30). This suggests that in addition to its enzymatic role, P4H may interact specifically with unfolded procollagen chains in a chaperone-like manner.
The approach we adopted to investigate this possibility was to allow the hydroxylation of newly synthesized procollagen chains to occur posttranslationally and assay the formation of a stable triple helix. We then determined whether P4-H or any other ER protein was associated with the folding chains. Translation reactions were carried out in the presence of
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Interaction of PDI with Monomeric Chains--
Having established
that P4-H interacts with the triple helical domain of procollagen
chains during their assembly, we then wanted to address the possibility
that ER resident proteins may interact with the C-propeptide of chains
that remain monomeric. For these experiments, we expressed the
individual chains of type I procollagen, which remain monomeric prior
to the formation of heterotrimeric molecules. We translated the pro1
and pro
2 mini-chains of type I and the pro
1 mini-chain of type
III procollagen individually and then added the thiol-specific,
noncleavable, bi-functional cross-linking reagent BMH. BMH was used
instead of DSP for these studies to identify proteins interacting at
regions of the protein other than the triple helical domain. When
separated under reducing conditions, the appearance of a radiolabeled
higher molecular weight product would indicate cross-linking of
procollagen to the immunoprecipitated protein. Clear cross-linked
products were seen for the pro
2(I)
1 chain (Fig.
5, compare lanes 3 and
6). More diffuse cross-linked products were also seen for
the pro
1(III)
1 and pro
1(I)
1 chains (Fig. 5, compare
lane 1 with lane 4 and lane 2 with
lane 5). After immunoprecipitation, only the PDI antibody was able to precipitate cross-linked products (Fig. 5, lanes
8 and 9). The cross-linked products from the
pro
1(I)
1 and pro
2(I)
1 translations were resolved as
distinct bands after immunoprecipitation (Fig. 5, lanes 8 and 9). The type III pro
-chain cross-linked products were
not immunoprecipitated by the PDI antibody (Fig. 5, lane 7).
We investigated this result further by expressing just the C-propeptide
of type III procollagen (24) and cross-linking with BMH. Here again, no
cross-links to PDI were observed (results not shown). These results
suggest that PDI was able to interact with type I procollagen chains
but not type III procollagen chains.
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DISCUSSION |
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The biosynthesis of multisubunit proteins entering the secretory pathway is regulated at the ER, where the individual subunits are synthesized and their assembly is coordinated. This regulation ensures that unassembled subunits are prevented from being transported out of the ER and are either degraded or maintained in an assembly competent state by interacting with ER resident proteins (31). The mechanism underlying this "quality control" appears to involve the binding of unassembled subunits to a variety of ER proteins until assembly occurs. The assembled complex is then released and can be transported from the ER. Such a mechanism has been likened to affinity chromatography, with the "matrix" being the resident proteins in the ER and the selective interactions occurring via oligosaccharide side chains (32), hydrophobic regions in the protein (33), of free thiol residues (34) or with specific molecular chaperones (35, 36). This study has demonstrated that protein-disulfide isomerase also plays a role in this regulation by binding to procollagen chains either as a subunit of prolyl 4-hydroxylase or independently.
The procollagen trimer is folded and assembled through a series of distinct intermediates; the coordination of this assembly is crucial to produce a correctly folded, thermally stable triple helical molecule. One consequence of the sequence of events occurring during procollagen folding is that the individual chains have to be maintained in a soluble form prior to assembly occurring. The triple helical domain is inherently insoluble and must be prevented from self-association for several minutes, (37) because the chains associate at their C-propeptides and the triple helical domain folds in the C to N direction (16). We show here that P4-H plays a key role in ensuring that the triple helical domain remains soluble by binding to unhydroxylated chains. The binding of P4-H to its substrate in the absence of the co-factors iron and ascorbate has been shown previously using purified proteins (38). Our observation that this interaction also occurs during biosynthesis within the ER provides convincing evidence that enzymes involved in posttranslational modification can under certain circumstances also play a crucial role in maintaining polypeptides in an assembly competent state. That P4-H can also bind to hydroxylated chains provides a clue to how procollagen chains hydroxylated at the N-terminal end of the triple helical domain are prevented from associating prior to folding and association of the C-propeptides. Interestingly, mutant procollagen chains, which contain a deletion in their triple helical domain and which form trimers with wild type chains, have also been shown to form a stable interaction with P4H (30). Such a stable interaction has been suggested to prevent secretion of these molecules by retention in the ER.
Monomeric procollagen chains that are destined to be incorporated into
heterotrimers also need to remain soluble and be prevented from
nonpermissive associations prior to assembly. When we expressed the
type I pro-chains individually, we observed an interaction of the
C-propeptides of these chains with PDI. The pro
1 chain of type I
procollagen has previously been shown to be able to form homotrimers at
a low efficiency (39). This was also the case here, with the majority
of the synthesized chains remaining monomeric. The pro
1 chain of
type III efficiently form homotrimers and, significantly, did not
interact with PDI at its C-propeptide. However, when this chain was
altered to prevent association by changing its recognition site to that
of the pro
2-chain of type I, an interaction with PDI could be
detected. We and others have previously shown that point mutations
within the C-propeptide can cause misfolding of this domain (21), which
leads to binding to ER proteins, such as immunoglobulin heavy
chain-binding protein (BiP) (40). However, in these cases, the
pro
-chains were unable to form correct intrachain disulfide bonds
and migrated as a diffuse smear when separated under nonreducing
conditions. The pro
-chain we have constructed here with an altered
recognition site was able to form intrachain disulfide bonds. The
polypeptide synthesized migrated as a sharp band when separated under
nonreducing conditions, which co-migrated with the wild type protein.
This indicates that the C-propeptide folded correctly but was unable to
assemble due to its altered recognition site. These results clearly
demonstrate that PDI plays a crucial role in binding to the
C-propeptide, thereby coordinating heterotrimer assembly.
A growing body of evidence is accumulating that suggests a key role for the collagen binding protein HSP47 in the biosynthesis of procollagen (36). Co-immunoprecipitation experiments have shown that procollagen within the ER is associated with HSP47 (41) and can dissociate upon transport to the Golgi apparatus (42). It has been reported that HSP47 binds to the N-propeptide of type I procollagen (43), yet other workers have also reported binding to the triple helical domain (42). During our studies, we were unable to immunoprecipitate our procollagen constructs with anti-HSP47 antibodies after cross-linking. It could be that our shortened triple helical domains do not contain the binding site for this molecule or could simply reflect inefficient cross-linking. The folding and assembly of procollagen is a complex process and may require a number of different chaperone proteins to ensure efficient folding, assembly, and intracellular transport. There is also likely to be redundancy in the involvement of accessory proteins, as has been illustrated by the successful expression and assembly of procollagen in insect cells (44), which are unlikely to contain HSP47. Clearly, PDI, P4H, and HSP47 may have overlapping functions as molecular chaperones during procollagen biosynthesis. The interactions described here provide the first direct evidence for a chaperone role for PDI (and P4-H) during assembly of procollagen chains by preventing their premature and hence nonproductive interaction. The interaction of procollagen intermediates with PDI may also, by virtue of its KDEL-retention sequence, provide a mechanism for retention of non-triple helical procollagen chains.
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ACKNOWLEDGEMENTS |
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We thank Steve McLaughlin for critical reading of the manuscript and Darwin Prockop for the cDNA clones to the procollagen molecules.
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FOOTNOTES |
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* This work was supported by The Royal Society.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.
Holder of a Medical Research Council studentship award.
§ To whom correspondence should be addressed. Tel.: 44-0161-275-5103; Fax: 44-0161-275-5082; E-mail: neil.bulleid{at}man.ac.uk.
1 The abbreviations used are: PDI, protein-disulfide isomerase; BMH, Bismaleimidohexane; bp, base pair(s); SP, semipermeabilized; KHM, 110 mM KOAc, 2 mM MgOAc, 20 mM HEPES, pH 7.2; DSP, dithiobis(succinimidyl propionate); PAGE, polyacrylamide gel electrophoresis; C-propeptide, COOH-terminal propeptide; CP-minus, C-propeptide-minus; P4-H, prolyl 4-hydroxylase; ER, endoplasmic reticulum.
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REFERENCES |
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