Domains b' and a' of Protein Disulfide Isomerase Fulfill the Minimum Requirement for Function as a Subunit of Prolyl 4-Hydroxylase

THE N-TERMINAL DOMAINS a AND b ENHANCE THIS FUNCTION AND CAN BE SUBSTITUTED IN PART BY THOSE OF ERp57*

Annamari PirneskoskiDagger §, Lloyd W. Ruddock§, Peter Klappa, Robert B. Freedman, Kari I. KivirikkoDagger , and Peppi KoivunenDagger ||

From the Dagger  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



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta  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 alpha 2beta 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 Delta -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 alpha  subunit has at least two isoforms, alpha (I) and alpha (II), which form with the PDI polypeptide the (alpha (I))2beta 2 and (alpha (II))2beta 2 tetramers. We report here that all the PDI domain constructs and PDI/ERp57 hybrid polypeptides tested were more effectively associated with the alpha (II) subunit than the alpha (I) subunit.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta  subunit in the animal prolyl 4-hydroxylase alpha 2beta 2 tetramers and alpha beta dimers (7-9) and in the microsomal triglyceride transfer protein alpha beta 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 alpha  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).

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 beta  subunit of prolyl 4-hydroxylase (32).

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 alpha  subunit has at least two isoforms, alpha (I) and alpha (II), which form with the PDI polypeptide the (alpha (I))2beta 2, type I, and (alpha (II))2beta 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 alpha (I) and alpha (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 Delta -somatostatin and the misfolded protein "scrambled" RNase.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha (I) and alpha (II), coding for the corresponding prolyl 4-hydroxylase alpha  subunits (22, 32, 34).

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 alpha (I) subunit and mouse alpha (II) subunit were used.

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 Delta -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 (Delta -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 Delta -somatostatin was performed as recommended by the manufacturer.

Binding and Cross-linking of Delta -Somatostatin and Scrambled RNase-- After precipitation with trichloroacetic acid, the radiolabeled Delta -somatostatin was dissolved in distilled water. Labeled Delta -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 <FR><NU>1</NU><DE>10</DE></FR> volume of cross-linking solution (10 mM DSG in Buffer A). The reaction was carried out for 60 min at 0 °C. Cross-linking was stopped by the addition of SDS-PAGE sample buffer (26). The samples were subjected to electrophoresis in 12.5% SDS-PAGE with subsequent autoradiography. Western blotting was performed using a polyclonal antibody raised against PDI. The detection was carried out with enhanced chemiluminescence (ECL).


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).



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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.

Coexpressions of PDI Domain Constructs with Prolyl 4-Hydroxylase alpha  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 alpha (I) or alpha (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 alpha (I) or alpha (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 alpha (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 alpha (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 alpha  subunits and the PDI domain constructs. Migration of the free monomers is indicated by M.


                              
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Table I
Prolyl 4-hydroxylase activity of Triton X-100-soluble proteins from insect cells expressing the alpha (I) or alpha (II) subunit of human prolyl 4-hydroxylase together with either the wild-type PDI, ERp57, PDI domain constructs, or PDI/ERp57 hybrids
Values are given in dpm/100 µg of extractable cell protein, mean ± S.D., for at least three experiments. Values obtained with the alpha (I) (typically about 600 dpm) and alpha (II) (typically about 800 dpm) subunit alone were subtracted from all values.

When PDIabb' was coexpressed with either the alpha (I) or alpha (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 alpha (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 alpha (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 alpha (I) subunit (Fig. 3, lane 7). However, assembly was still detected with the alpha (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 alpha (I) or alpha (II) subunit.

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 alpha (I) or alpha (II) subunit. No assembly was obtained with this mutant polypeptide with either alpha  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.

Coexpression of PDI/ERp57 Hybrid Polypeptides with Prolyl 4-Hydroxylase alpha  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 alpha (I) or alpha (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 alpha (I) or alpha (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 alpha (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 alpha (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 alpha (I) and alpha (II) subunits even though detection of the complex with the alpha (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 alpha (I) subunit and gave less enzyme activity with the alpha (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-alpha (I) and anti-alpha (II) subunit antibodies (Fig. 4B, lanes 3 and 6).



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Fig. 4.   Analysis of coexpression of the human prolyl 4-hydroxylase alpha (I) or alpha (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 alpha (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 alpha (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 alpha (I) subunit and PDI (lane 1), ERp57 (lane 2), and PDIaERp57bb'a'c (lane 3); coinfections with baculoviruses coding for human alpha (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-alpha (I) subunit antibody in lanes 1-3 and an anti-alpha (II) subunit antibody in lanes 4-6. Asterisks indicate migration of the tetramers.

Binding of Radiolabeled Delta -Somatostatin to PDI Domain Constructs and PDI/ERp57 Hybrids-- 125I-Labeled Bolton-Hunter labeled Delta -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 Delta -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 Delta -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 Delta -somatostatin with PDI domain constructs and PDI/ERp57 hybrid polypeptides expressed in insect cells. Radiolabeled Delta -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).


                              
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Table II
Correlation between binding of Delta -somatostatin and scrambled RNase and the assembly of a functional prolyl 4-hydroxylase tetramer

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).



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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.

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha  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 alpha  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 alpha  subunit or may come from a contribution due to interactions between the beta  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 alpha (I) and alpha (II) subunit tetramerization and the use of PDI/ERp57 hybrids.

The two types of human prolyl 4-hydroxylase alpha  subunit, the alpha (I) and alpha (II) subunits, readily form the (alpha (I))2beta 2 and (alpha (II))2beta 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 alpha  subunit, in that the alpha (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 alpha (I) and alpha (II) subunits.

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 alpha (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.

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, Delta -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 Delta -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 alpha  subunit during the assembly process.

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 alpha (I) or alpha (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.

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 alpha  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 alpha  subunit in prolyl 4-hydroxylase.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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