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• W2030752885 abstract "A mutant human protein disulfide isomerase with the COOH-terminal 51 amino acid residues deleted (abb′a′) has been expressed in Escherichia coli. Its secondary structures are very similar to those of the native bovine enzyme. The mutant enzyme shows neither peptide binding ability nor chaperone activity in assisting the refolding of denaturedd-glyceraldehyde-3-phosphate dehydrogenase but keeps most of the catalytic activities for reduction of insulin and isomerization of scrambled ribonuclease. It assists the reactivation of denatured and reduced proteins containing disulfide bonds, acid phospholipase A2, and lysozyme to different levels, which are significantly lower than those by the native bovine enzyme. A mutant human protein disulfide isomerase with the COOH-terminal 51 amino acid residues deleted (abb′a′) has been expressed in Escherichia coli. Its secondary structures are very similar to those of the native bovine enzyme. The mutant enzyme shows neither peptide binding ability nor chaperone activity in assisting the refolding of denaturedd-glyceraldehyde-3-phosphate dehydrogenase but keeps most of the catalytic activities for reduction of insulin and isomerization of scrambled ribonuclease. It assists the reactivation of denatured and reduced proteins containing disulfide bonds, acid phospholipase A2, and lysozyme to different levels, which are significantly lower than those by the native bovine enzyme. Protein disulfide isomerase (PDI), 1The abbreviations used are: PDI, protein disulfide isomerase; GAPDH, d-glyceraldehyde-3-phosphate dehydrogenase; APLA2, acidic phospholipase A2; abb′a′, mutant human PDI with the COOH-terminal 51 amino acid residues deleted; hPDI, human PDI; bPDI, bovine PDI; mPDI, modified (S-carboxymethylated) PDI; ANS, 8-anilino-1-naphthalenesulfonic acid; BSA, bovine serum albumin; GdnHCl, guanidine hydrochloride; TPOR, thiol-protein oxidoreductase. as one of the two foldases thus far characterized (1Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3607) Google Scholar), has attracted much attention in studies on protein folding. The enzyme catalyzes the formation, reduction, or isomerization of disulfide bonds of proteins depending on the redox potentials in in vitro systems (2Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (656) Google Scholar) and is responsible for the formation of native disulfide bonds of nascent peptides in vivo (3Noiva R. Protein Exp. Purif. 1994; 5: 1-13Crossref PubMed Scopus (58) Google Scholar, 4Bullied N.J. Freedman R.B. Nature. 1988; 335: 649-651Crossref PubMed Scopus (261) Google Scholar). Although the three-dimensional structure has not yet been reported, the PDI molecule has been suggested to be constructed of several domains in the order of -a-b-b′-a′-c as inferred from its cDNA deduced sequence (5Edman J.C. Ellis L. Blacher R.W. Roth R.A. Rutter W.J. Nature. 1985; 317: 267-270Crossref PubMed Scopus (477) Google Scholar) and structural studies (6Kemmink J. Darby N.J. Dijkstra K. Scheek R.M. Creighton T.E. Protein Sci. 1995; 4: 2587-2593Crossref PubMed Scopus (49) Google Scholar, 7Darby N.J. Kemmink K. Creighton T.E. Biochemistry. 1996; 35: 10517-10528Crossref PubMed Scopus (89) Google Scholar). The sequences of -CGHC- in domain a and a′ and a peptide fragment of 26 amino acid residues in the COOH-terminal c domain have been identified, respectively, to be the active sites (8Hawkins H.C. Freedman R.B. Biochem. J. 1991; 275: 335-339Crossref PubMed Scopus (124) Google Scholar,9Vuori K. Myllyla R. Pihlajaniemi T. Kivirikko K.I. J. Biol. Chem. 1992; 267: 7211-7214Abstract Full Text PDF PubMed Google Scholar) and the peptide binding site (10Noiva R. Kimura H. Roos J. Lennarz W.J. J. Biol. Chem. 1991; 266: 19645-19649Abstract Full Text PDF PubMed Google Scholar, 11Noiva R. Freedman R.B. Lennarz W.J. J. Biol. Chem. 1993; 268: 19210-19217Abstract Full Text PDF PubMed Google Scholar, 12Morjana N.A. Gilbert H.F. Biochemstry. 1991; 30: 4985-4990Crossref PubMed Scopus (79) Google Scholar). PDI is also a remarkable multifunctional protein (13Noiva R. Lennarz W.J. J. Biol. Chem. 1992; 267: 3553-3556Abstract Full Text PDF PubMed Google Scholar). In addition to its isomerase activity it is the essential β subunit of prolyl-4-hydroxylase (14Pihlajaniemi T. Helaakoski T. Tasanen K. Myllyla R. Huhtala M.-L. Koivu J. Kivirikko K.I. EMBO J. 1987; 6: 643-649Crossref PubMed Scopus (330) Google Scholar, 15Koivu J. Myllyla R. Helaakoski T. Pihlajaniemi T. Tasanen K. Kivirikko K.I. J. Biol. Chem. 1987; 262: 6447-6449Abstract Full Text PDF PubMed Google Scholar) and the small subunit of microsomal triglyceride transfer protein complex (16Wetterau J.R. Combs K.A. Spinner S.N. Joiner B.J. J. Biol. Chem. 1990; 265: 9801-9807Abstract Full Text PDF Google Scholar). It has recently been suggested that PDI is not only an isomerase but also has chaperone activity (3Noiva R. Protein Exp. Purif. 1994; 5: 1-13Crossref PubMed Scopus (58) Google Scholar, 17Wang C.C. Tsou C.L. FASEB J. 1993; 7: 1515-1517Crossref PubMed Scopus (142) Google Scholar, 18Wang C.C. Guzman N.A. Prolyl Hydroxylase, Protein Disulfide Isomerase, and Other Structurally Related Proteins. Marcel Dekker Inc., New York1997Google Scholar). This was supported by its assistance in the in vitro refolding of denatured proteins with no disulfide bond, such asd-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (19Cai H. Wang C.C. Tsou C.L. J. Biol. Chem. 1994; 269: 24550-24552Abstract Full Text PDF PubMed Google Scholar) and rhodanese (20Song J.L. Wang C.C. Eur. J. Biochem. 1995; 231: 312-316Crossref PubMed Scopus (136) Google Scholar) as well as disulfide-containing proteins such as lysozyme (21Puig A. Gilbert H.F. J. Biol. Chem. 1994; 269: 7764-7771Abstract Full Text PDF PubMed Google Scholar) and acidic phospholipase A2(APLA2) (22Yao Y. Zhou Y.C. Wang C.C. EMBO J. 1997; 16: 651-658Crossref PubMed Scopus (112) Google Scholar), although Lilie et al. (23Lilie H. McLaughlin S. Freedman R.B. Buchner J. J. Biol. Chem. 1994; 269: 14290-14296Abstract Full Text PDF PubMed Google Scholar) had reported that PDI showed no chaperone effect on the refolding of denatured immunoglobulin Fab with intact disulfides. A PDI mutant, inactive as an isomerase, has the same function as an essential subunit for the assembly of the fully active tetramer of prolyl-4-hydroxylase α2β2 (24Vuori K. Pihlajaniemi T. Myllyla R. Kivirikko K.I. EMBO J. 1992; 11: 4213-4217Crossref PubMed Scopus (128) Google Scholar) and the dimer of microsomal triglyceride transfer protein complex (25Lamberg A. Jauhiainen M. Metso J. Ehnholm C. Shoulders C. Scott J. Pihlajaniemi T. Kivirikko K.I. Biochem. J. 1996; 315: 533-536Crossref PubMed Scopus (55) Google Scholar), suggesting that the role of PDI in the above two proteins is independent of its isomerase activity but related to its chaperone-like peptide binding function. The deletion of the NH2-terminal 3 amino acid residues of the peptide binding region of human PDI indeed prevents prolyl-4-hydroxylase tetramer formation (25Lamberg A. Jauhiainen M. Metso J. Ehnholm C. Shoulders C. Scott J. Pihlajaniemi T. Kivirikko K.I. Biochem. J. 1996; 315: 533-536Crossref PubMed Scopus (55) Google Scholar, 26Koivunen P. Helaakoski T. Annunen P. Veijola J. Raisanen S. Pihlajaniemi P. Kivirikko K.I. Biochem. J. 1996; 316: 599-605Crossref PubMed Scopus (41) Google Scholar). It was shown that a mutant PDI with Ser substituted for Cys at the -CGHC- active sites and devoid of isomerase activity can increase the folding and secretion of lysozyme coexpressed in yeast (27Hayano T. Hirose M. Kikuchi M. FEBS Lett. 1995; 377: 505-511Crossref PubMed Scopus (84) Google Scholar). The yeast bearing a mutant PDI shortened from the COOH terminus and devoid of the putative peptide binding region in the middle part of the molecule can hardly survive (28LaMantia M. Lennarz W.J. Cell. 1993; 74: 899-908Abstract Full Text PDF PubMed Scopus (185) Google Scholar). It has been shown in our previous work that the chaperone activity of PDI in assisting GAPDH refolding is suppressed by competitive peptide binding (29Quan H. Fan G.B. Wang C.C. J. Biol. Chem. 1995; 270: 17078-17080Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), which also inhibits its enzymatic activity (12Morjana N.A. Gilbert H.F. Biochemstry. 1991; 30: 4985-4990Crossref PubMed Scopus (79) Google Scholar, 29Quan H. Fan G.B. Wang C.C. J. Biol. Chem. 1995; 270: 17078-17080Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In this paper a mutant human PDI, abb′a′, with the deletion of the COOH-terminal 51 amino acid residues responsible for peptide binding, has been expressed in Escherichia coli. The mutant shows neither peptide binding ability nor chaperone activity in assisting the refolding of denatured GAPDH but displays most of the catalytic activities of PDI and assists the reactivation of denatured and reduced APLA2 and lysozyme to an extent significantly lower than those by the native bovine enzyme. The plasmid pBR322-PDI, containing the full-length of human PDI (hPDI) cDNA (11Noiva R. Freedman R.B. Lennarz W.J. J. Biol. Chem. 1993; 268: 19210-19217Abstract Full Text PDF PubMed Google Scholar), is a generous gift from Prof. K. Kivirikko, University of Oulu, Finland. GAPDH was from rabbit muscle (30Liang S.J. Lin Y.Z. Zhou J.M. Tsou C.L. Wu P. Zhou Z. Biochim. Biophys. Acta. 1990; 1038: 240-246Crossref PubMed Scopus (70) Google Scholar). APLA2 was from Agkistrodon blomhoffii brevicaudus (Agkistrodon halys Pallas) (31Wu X.F. Jiang Z.P. Chen Y.C. Acta Biochim. Biophys. Sin. 1984; 16: 664-671Google Scholar). PDI was prepared from bovine liver (bPDI) essentially according to Lambert and Freedman (32Lambert N. Freedman R.B. Biochem. J. 1983; 213: 225-234Crossref PubMed Scopus (124) Google Scholar) and showed one band on SDS-polyacrylamide gel electrophoresis with a specific activity of more than 800 units/g.S-Carboxymethylated A-chain of insulin was prepared according to Zheng et al. (33Zheng W.D. Quan H. Song J.L. Yang S.L. Wang C.C. Arch. Biochem. Biophys. 1997; 337: 326-331Crossref PubMed Scopus (38) Google Scholar). Modified PDI (mPDI) carboxymethylated at thiols in the -CGHC- sequence of active sites, was prepared as described previously (29Quan H. Fan G.B. Wang C.C. J. Biol. Chem. 1995; 270: 17078-17080Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Restriction endonucleases, T4 DNA ligase, isopropyl 1-thio-β-d-galactopyranoside, and dithiothreitol were from Promega. VentR DNA polymerase and the large (Klenow) fragment of DNA polymerase I were from New England Biolabs, Inc. The prokaryotic gene fusion expression vector pGEX-4T-1, E. coliBL21 strain (F−, ompT, RB−, mB−), the T7 sequencing kit, and glutathione-Sepharose 4B were from Pharmacia Biotech Inc. α-35S-dATP was obtained from NEN Life Science Products. 5,5′-Dithiobis(2-nitrobenzoic acid) was from Fluka. Glutathione (GSH), glutathione disulfide (GSSG), NAD+ (98%), and NADPH (type III) were from Boehringer Mannheim. Thrombin, glutathione reductase (yeast, type III), glyceraldehyde 3-phosphate, 8-anilino-1-naphthalenesulfonic acid (ANS), bovine serum albumin (BSA, 98–99% albumin, fraction V), guanidine hydrochloride (GdnHCl),Micrococcus lysodeikticus dried cells, and insulin were from Sigma. Hen egg white lysozyme was from Serva. All other chemicals were local products of analytical grade. The concentrations of GdnHCl-denatured GAPDH, and denatured and reduced APLA2 were determined by the method of Bradford (34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) with BSA as a standard. The concentrations of other proteins were determined spectrophotometrically at 280 nm with the following absorption coefficients (A 1 cm0.1%): 0.94 for PDI, 0.66 for BSA, 0.98 for GAPDH, 1.03 for insulin, 1.3 for APLA2, 1.14 for S-carboxymethylated A-chain of insulin (35Hua Q.X. Qian Y.Q. Tsou C.L. Biochim. Biophys. Acta. 1984; 189: 234-240Crossref Scopus (13) Google Scholar), 2.63 for native lysozyme, and 2.37 for denatured lysozyme (21Puig A. Gilbert H.F. J. Biol. Chem. 1994; 269: 7764-7771Abstract Full Text PDF PubMed Google Scholar). The A 1 cm0.1% value of abb′a′ was determined to be 0.83. For the convenience of comparison, both GAPDH and PDI are considered as protomers in the calculation of molar ratios. Thiol groups of proteins were determined with 5,5′-dithiobis(2-nitrobenzoic acid) (36Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-78Crossref PubMed Scopus (21624) Google Scholar). The primer I (5′-CGGGATCCGACGCCCCCGAGGAG-3′) was designed to hybridize with the first 15 nucleotides at the 5′-terminus of the hPDI cDNA sequence and contains a BamHI site (underlined) just before the sequence. The reverse primer II (5′-GGGTTAGTAATCGATGAC-3′) with a stop codon (underlined) hybridizes with the sequence between 1309 and 1320 base pairs of the hPDI cDNA. The double-stranded 1.3-kilobase DNA fragment coding for the sequence of the Asp1 to Tyr440 of PDI (abb′a′) was generated by polymerase chain reaction using VentR DNA polymerase with the above two primers and pBR322-PDI as a template. The polymerase chain reaction product was elongated to blunt ends in both termini with the large (Klenow) fragment of DNA polymerase I, digested with BamHI at the 5′-terminus, and ligated into pGEX-4T-1 digested with BamHI and SmaI in-frame with the glutathioneS-transferase fusion codons to construct the expression plasmid pGEX-abb′a′. The foreign DNA sequences cloned into the expression plasmid was verified by nucleotide sequencing. Protein production was carried out in E. coli strain BL21 containing the pGEX-abb′a′ plasmid. Cells grown overnight at 37 °C in LB medium were diluted 100-fold and grown at 28 °C with vigorous shaking to anA 600 of 0.8, and then grown for another 4 h after adding 0.1 mm isopropyl 1-thio-β-d-galactopyranoside. Cell pellet was disrupted by sonication and then mixed gently with 1% Triton X-100 by stirring for 30 min at room temperature. The supernatant of cell lysate was loaded onto a 2-ml glutathione-Sepharose 4B RediPack column, and the fusion protein on the matrix was cleaved with 100 units of thrombin at 22 °C for 16 h. The eluted abb′a′ fraction was loaded onto a Mono Q HR (5/5) column equilibrated with 20 mm phosphate buffer, pH 6.3, and eluted with a linear gradient from 0 to 0.3m NaCl. The abb′a′ fraction was identified by 10% SDS-polyacrylamide gel electrophoresis analysis, dialyzed thoroughly, and lyophilized. The disulfide isomerase and thiol-protein oxidoreductase (TPOR) activities were assayed according to Lambert and Freedman (32Lambert N. Freedman R.B. Biochem. J. 1983; 213: 225-234Crossref PubMed Scopus (124) Google Scholar). Lysozyme activity was determined at 30 °C by following the absorbance decrease at 450 nm of a 0.25 mg/ml M. lysodeikticus suspension in 67 mm sodium phosphate buffer, pH 6.2, containing 100 mm NaCl (21Puig A. Gilbert H.F. J. Biol. Chem. 1994; 269: 7764-7771Abstract Full Text PDF PubMed Google Scholar, 37Goldberg M. Rudolph R. Jaenicke R. Biochemistry. 1991; 30: 2790-2797Crossref PubMed Scopus (410) Google Scholar). Denaturation and assisted reactivation of GAPDH and APLA2 by bPDI and/or abb′a′ were carried out according to Cai et al. (19Cai H. Wang C.C. Tsou C.L. J. Biol. Chem. 1994; 269: 24550-24552Abstract Full Text PDF PubMed Google Scholar) and Yaoet al. (22Yao Y. Zhou Y.C. Wang C.C. EMBO J. 1997; 16: 651-658Crossref PubMed Scopus (112) Google Scholar), respectively. Lysozyme at 20 mg/ml was completely denatured and reduced in 0.1 m sodium phosphate, pH 8.0, containing 8 m GdnHCl and 150 mmdithiothreitol at room temperature for 4 h. The reaction mixture was brought to pH 2.0 with 6 n HCl, dialyzed first against 0.01 n HCl for 3 h and then against 0.1 nacetic acid at 4 °C thoroughly. The denatured and reduced lysozyme was aliquoted and stored at −20 °C. Oxidative refolding of reduced and denatured lysozyme was carried out by dilution into phosphate buffer containing 0.1 m sodium phosphate, 2 mmEDTA, 1 mm GSSG, and 2 mm GSH, pH 7.5, to a final concentration of 10 μm. The activity recovery was completed and determined 2 h after dilution. CD spectrum determinations in the far ultraviolet region from 200 to 250 nm were carried out with a Jasco J720 spectropolarimeter at 25 °C. ANS fluorescence spectra were measured at 25 °C in a Hitachi F-4010 spectrofluorometer with an excitation wavelength of 365 nm. The inserted coding sequence for abb′a′ in the expression plasmid pGEX-abb′a′ was verified by DNA sequence analysis (data not shown). The constructed protein should contain residues 1–440 and an additional Gly-Ser at the NH2 terminus of hPDI. Experimental conditions for sonication, growth temperature, and isopropyl 1-thio-β-d-galactopyranoside concentration were optimized by 10% SDS-polyacrylamide gel electrophoresis analysis (data not shown). Fig. 1 shows the high yield of abb′a′ expression and the homogeneity of the purified product. The molecular weight of abb′a′ is about 48,000 as expected. As shown in Fig.2, the CD spectrum of abb′a′ is almost the same as that of bPDI, indicating that the COOH-terminal shortened enzyme has secondary structures closely similar to that of bPDI. The same ANS fluorescence spectra of bPDI and abb′a′ indicated that the truncation of COOH-terminal 51 residues of PDI has little effect on the surface hydrophobicity of the intact molecule (data not shown). The mutant abb′a′ has about 69 ± 3% of isomerase activity and 80 ± 2% of TPOR activity compared with that of bPDI (TableI), suggesting that the truncation of the COOH-terminal 51 amino acid residues of PDI has only a minor effect on these enzymatic activities.Table IActivities of PDI, abb′a′ and mPDIChaperoneIsomerizationReduction%%PDI+100100abb′a′−69 ± 380 ± 2mPDI+−− Open table in a new tab As shown in Fig.3, the presence ofS-carboxymethylated A-chain of insulin inhibits the TPOR activity of PDI; in sharp contrast, it has no effect on the TPOR activity of abb′a′. The above result is highly suggestive that theS-carboxymethylated A-chain of insulin inhibits the TPOR activity of PDI by binding at its peptide binding site, which is lacking in the mutant abb′a′. Fig.4 shows that the reactivation of GAPDH in the presence of bPDI increases from 4 to 19% with the increase of the molar ratio of PDI to GAPDH from 0 to 10. In contrast, with BSA used for comparison, abb′a′ at the same range of ratios shows no effect on the reactivation of denatured GAPDH. This result indicates the necessity of the peptide binding site of PDI for its chaperone activity in the reactivation of GAPDH. It has been proposed that the foldase activity of PDI consists of both its isomerase and chaperone activities and the latter activity can be fully replaced by mPDI (22Yao Y. Zhou Y.C. Wang C.C. EMBO J. 1997; 16: 651-658Crossref PubMed Scopus (112) Google Scholar), which is devoid of isomerase activity but nearly as active as native PDI in its chaperone activity in assisting the reactivation of GAPDH (29Quan H. Fan G.B. Wang C.C. J. Biol. Chem. 1995; 270: 17078-17080Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The reactivation of GdnHCl-denatured and reduced APLA2, containing seven disulfide bonds, upon dilution in the presence and absence of abb′a′ and/or mPDI was determined to examine the foldase activity of abb′a′. Fig. 5 Ashows that the spontaneous reactivation of APLA2 at 12 μm is only about 1%, and the reactivation in the presence of abb′a′ takes 10 h to reach completion as in the presence of PDI. In the presence of abb′a′ the reactivation yield increases with increasing concentrations of abb′a′ to a maximal level of 15% when the molar ratio of abb′a′ to APLA2 approaches 2 as shown in Fig. 5 B. Higher ratios of abb′a′ has little further effect on the reactivation of APLA2. mPDI alone has no effect on the reactivation of APLA2; however, the simultaneous presence of both mPDI and abb′a′ increases markedly the reactivation yield of APLA2 compared with the same amount of abb′a′ alone in the refolding buffer. In presence of mPDI at 36 μm together with abb′a′ at 120 μm the reactivation yield of APLA2 approaches 48%, about the same as the maximal level obtainable by native bPDI. Further increases in the concentrations of either mPDI or abb′a′ or both have no further effect on the reactivation of APLA2. As shown in Fig. 6 the very low spontaneous reactivation of denatured and reduced lysozyme in phosphate buffer increases with increasing concentrations of bPDI in the refolding buffer to a maximal level of around 71% at a stoichiometric amount of PDI. However, the maximal reactivation yield of lysozyme in presence of abb′a′ is reduced to 58% under the same condition. From the nearly identical CD spectra and ANS binding, in addition to similar enzyme activities between bPDI and abb′a′, it appears that the addition of 2 extra residues at the NH2 terminus and the deletion of the COOH-terminal 51 amino acid residues show little effect on the secondary structures, the surface hydrophobicity, and the enzymatic activity of PDI. The NH2-terminal sequence of PDI seems to be of little importance as it has also been found that even an extension of 10 residues at the NH2 terminus showed no deleterious effect on the properties of PDI (38Gao Y. Quan H. Jiang M.Y. Dai Y. Wang C.C. J. Biotechnol. 1997; 54: 105-112Crossref PubMed Scopus (4) Google Scholar). The mutant abb′a′ is structurally stable and retains most of the isomerase and oxidoreductase activities of PDI, suggesting that it is more or less independent from the c domain, but the involvement of the c domain in the above activities cannot be excluded, apart from the peptide binding site; it could also contribute to substrate binding. Alternatively, deletion of the c domain could alter somewhat the functional conformation of PDI, leading to decreased enzymatic activities. The peptide inhibition of the TPOR activity of PDI suggests its binding at the peptide binding site of PDI (12Morjana N.A. Gilbert H.F. Biochemstry. 1991; 30: 4985-4990Crossref PubMed Scopus (79) Google Scholar, 29Quan H. Fan G.B. Wang C.C. J. Biol. Chem. 1995; 270: 17078-17080Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 33Zheng W.D. Quan H. Song J.L. Yang S.L. Wang C.C. Arch. Biochem. Biophys. 1997; 337: 326-331Crossref PubMed Scopus (38) Google Scholar). In this respect, it is not surprising that the TPOR activity of abb′a′ is not inhibited by the presence of an excess of the peptide as abb′a′ no longer has the c domain, which is the major if not the only peptide binding site (11Noiva R. Freedman R.B. Lennarz W.J. J. Biol. Chem. 1993; 268: 19210-19217Abstract Full Text PDF PubMed Google Scholar). The above results indicate that, contrary to the previous suggestion (29Quan H. Fan G.B. Wang C.C. J. Biol. Chem. 1995; 270: 17078-17080Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), the c domain does not appear to be essential for the enzymatic activities of PDI, and the peptide binding site and the substrate binding site are not identical. However, the above does not exclude the possibility that occupation at the peptide binding site could interfere with substrate binding to the substrate binding site, and the c domain could partially contribute to the substrate binding site, thus the peptide and substrate binding sites could be close to and overlapping with each other. This appears to be different from the E. coli trigger factor, in which the active site of peptidyl-prolylcis/trans-isomerase is separated widely enough from the peptide binding site so that the binding of an unfolded protein does not interfere with the catalysis of prolyl cis/transisomerization in a small peptide (39Scholz C. Stoller G. Zarnz T. Fischer G. Schmid F.X. EMBO J. 1997; 16: 54-58Crossref PubMed Scopus (176) Google Scholar). As shown previously, the presence of a peptide in the refolding buffer suppresses GAPDH reactivation assisted by PDI, indicating that peptide binding in competition with the GAPDH folding intermediates prevents and suppresses the PDI-assisted refolding of GAPDH (29Quan H. Fan G.B. Wang C.C. J. Biol. Chem. 1995; 270: 17078-17080Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In this respect, it is to be expected and is actually found that abb′a′ is unable to assist the reactivation of denatured GAPDH and thus provides a straightforward demonstration that the peptide binding site at the c domain of PDI is directly responsible for its chaperone activity. It is widely accepted that PDI plays a critical role in nascent peptide folding by catalyzing the formation of native disulfide(s). However, PDI as a foldase not only catalyzes disulfide isomerization but is also intrinsically involved in peptide chain folding through its peptide binding site(s). Therefore PDI most likely binds with folding intermediates at different folding stages (see SchemeFS1). The two processes of disulfide formation and peptide folding are intimately interdependent and work in cooperation in the generation of the native conformation of disulfide-containing proteins. The chemically modified PDI alkylated at active site cysteine residues is devoid of isomerase activity but retains its chaperone activity almost fully (29Quan H. Fan G.B. Wang C.C. J. Biol. Chem. 1995; 270: 17078-17080Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In this report, the COOH-terminal truncated PDI shows most of the catalytic activity but is devoid of any chaperone activity. Comparisons of the properties and the possible roles of these derivatives in assisting protein folding are summarized in Table I and Scheme FS1, respectively. For spontaneous refolding (the central lines ina, b, and c) the denatured and reduced protein (U) would undergo a fast conformational change upon dilution to form intermediate (I1), which could fold first to I2 and finally to native molecule (N) through both folding steps and oxidative disulfide formation. Both intermediates I1 and I2 face two alternative folding pathways, correct folding to the native molecule (N) and misfolding leading to aggregation (A). The presence of PDI and/or its derivatives affect the relative proportion between the alternative pathways. As shown in Scheme FS1 a, PDI, being a chaperone, binds with I1 at its peptide binding site of domain c and prevents the aggregation of I1. On the other hand, PDI also probably binds at the substrate binding site with I2, which is assumed to represents a folding intermediate better folded than I1 with thiols properly paired to be oxidized to the native disulfide(s) and also decrease aggregation and increase reactivation. As shown in Scheme FS1 b, abb′a′, devoid of a peptide binding site, no longer binds with I1 but binds indeed with I2 through its substrate binding sites and catalyzes disulfide formation, leading to the native molecule (N) with significantly lower efficiency than PDI does. This could also explain the fact that the reactivation of APLA2 assisted by abb′a′ at all concentrations shows a lag phase for the first 30 min (Fig.5 A) in contrast to the apparent first order kinetics of PDI-assisted reactivation (22Yao Y. Zhou Y.C. Wang C.C. EMBO J. 1997; 16: 651-658Crossref PubMed Scopus (112) Google Scholar). Moreover, abb′a′ does not show anti-chaperone activity of PDI in assisting the reactivation of lysozyme in HEPES buffer. 2J. L. Song, H. Quan, and C. C. Wang, unpublished data. As shown in Scheme FS1 c, mPDI, with full peptide binding activity of PDI, binds with both I1 and I2, but without isomerase activity it is unable to catalyze the formation of disulfide and hence the native molecule from I2. After dissociated from the respective complexes with mPDI, the intermediate would aggregate irreversibly, and therefore mPDI does not increase the reactivation yield. However, with both abb′a′ (as enzyme only) and mPDI (as chaperone only) simultaneously present in the refolding buffer, the cooperative action of the two PDI derivatives could assist the reactivation of APLA2 to the same maximal level as native PDI does, but the amount of abb′a′ required in this case for the maximal reactivation is much more than by native PDI alone. This could be accounted for by a higher efficiency of PDI with both functions in the same molecule compared with the combined action of two molecules in abb′a′ and mPDI. It is also significant that compared with thioredoxin, PDI acquired an additional -CXXC- containing domain, domain a′, as well as the domain c during evolution so as to function as a foldase with both isomerase and chaperone activities, whereas thioredoxin shows neither peptide binding ability (3Noiva R. Protein Exp. Purif. 1994; 5: 1-13Crossref PubMed Scopus (58) Google Scholar, 33Zheng W.D. Quan H. Song J.L. Yang S.L. Wang C.C. Arch. Biochem. Biophys. 1997; 337: 326-331Crossref PubMed Scopus (38) Google Scholar) nor chaperone activity (29Quan H. Fan G.B. Wang C.C. J. Biol. Chem. 1995; 270: 17078-17080Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and has a much lower isomerase activity than PDI (2Freedman R.B. Hirst T.R. Tuite M.F. Trends Biochem. Sci. 1994; 19: 331-336Abstract Full Text PDF PubMed Scopus (656) Google Scholar). Similarly, the trigger factor composed of peptidyl-prolylcis/trans-isomerase and peptide binding sites has an efficiency for assisting protein folding 1,000-fold higher than that of peptidyl-prolyl cis/trans-isomerase (39Scholz C. Stoller G. Zarnz T. Fischer G. Schmid F.X. EMBO J. 1997; 16: 54-58Crossref PubMed Scopus (176) Google Scholar). As a foldase, PDI assists folding of different target proteins containing disulfide bond with efficiency different from that shown for APLA2 (from 1 to 45%) and lysozyme (from 1 to 71%). This can be explained by the difference in the folding pathway and properties of folding intermediates of the substrates on the one hand and in the specificity of PDI itself as a chaperone or an isomerase on the other. For APLA2, the maximal reactivation level decreased from 45 to 15% when PDI was replaced by abb′a′, whereas for lysozyme, the level decreased from 71 to 58%. It appears that for APLA2 refolding the chaperone function of PDI is more important compared with the refolding of lysozyme. We are grateful to Prof. K. Kivirikko, University of Oulu, Finland, for the generous gift of the cloned human PDI gene and Prof. Y. C. Zhou, Shanghai Institute of Biochemistry, Academia Sinica, for the kind gift of APLA2. We also thank X. L. Li in this laboratory for kindly providing rabbit muscle GAPDH. We sincerely thank Prof. C. L. Tsou for continuous encouragement, helpful advice, and critical reading of this manuscript." @default.
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• W2030752885 title "A Mutant Truncated Protein Disulfide Isomerase with No Chaperone Activity" @default.
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