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Molecular cloning, expression and characterization ofprotein disulfide isomerase from Conus marmoreusZhi-Qiang Wang1, Yu-Hong Han1,2, Xiao-Xia Shao1, Cheng-Wu Chi1,2and Zhan-Yun Guo11 Institute of Protein Research, Tongji University, Shanghai, China2 Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School ofthe Chinese Academy of Sciences, Shanghai, ChinaIn eukaryotes, all proteins that travel along or residein the secretory pathway are folded in the endoplas-mic reticulum (ER). As one of the most importantpost-translational modifications, the disulfide bondsare formed in the ER lumen, where oxidoreductasescatalyze the reaction and serve as disulfide donors [1].The archetypical oxidoreductase in the ER lumen isprotein disulfide isomerase (PDI). In the oxidizedstate, PDI functions as a disulfide donor for its clientproteins. In the reduced state, PDI catalyzes reductionand isomerization of pre-existing disulfides. The abil-ity of PDI to function as a reductase, an oxidase andan isomerase ensures PDI’s ability to serve as a majorcatalyst for disulfide formation in vivo [2,3]. Moreover,PDI also acts as a chaperone for substrates duringcatalysis [4].Conotoxins are small, cysteine-rich peptides pro-duced by marine cone snails [5]. Although their aminoacid sequences are hypervariable, they can form spe-cific disulfide patterns that are essential for their bio-logical activities. It is believed that cone snails possessevolving mechanisms to ensure efficient folding ofconotoxins in vivo, but these mechanisms are not fullyunderstood yet.Keywordsconotoxin; disulfide isomerization; oxidativefolding; protein disulfide isomeraseCorrespondenceZ Y. Guo, Institute of Protein Research,Tongji University, 1239 Siping Road,Shanghai 200092, ChinaFax: +86 21 65988403Tel: +86 21 65985167E-mail: zhan-yun.guo@mail.tongji.edu.cnC W. Chi, Shanghai Institute ofBiochemistry and Cell Biology, ChineseAcademy of Sciences, 320 YueYang Road,Shanghai 200031, ChinaFax: +86 21 54921011Tel: +86 21 54921165E-mail: chi@sunm.shcnc.ac.cn(Received 2 March 2007, revised 8 July2007, accepted 18 July 2007)doi:10.1111/j.1742-4658.2007.06003.xThe oxidative folding of disulfide-rich conotoxins is essential for theirbiological functions. In vivo, disulfide bond formation is mainly catalyzedby protein disulfide isomerase. To elucidate the physiologic roles of pro-tein disulfide isomerase in the folding of conotoxins, we have cloned anovel full-length protein disulfide isomerase from Conus marmoreus. ItsORF encodes a 500 amino acid protein that shares sequence homologywith protein disulfide isomerases from other species, and 70% homologywith human protein disulfide isomerase. Enzymatic analyses of recombi-nant C. marmoreus protein disulfide isomerase showed that it sharedfunctional similarities with human protein disulfide isomerase. Usingconotoxins tx3a and sTx3.1 as substrate, we analyzed the oxidase andisomerase activities of the C. marmoreus protein disulfide isomerase andfound that it was much more efficient than glutathione in catalyzing oxi-dative folding and disulfide isomerization of conotoxins. We further dem-onstrated that macromolecular crowding had little effect on the proteindisulfide isomerase-catalyzed oxidative folding and disulfide isomerizationof conotoxins. On the basis of these data, we propose that the C. mar-moreus protein disulfide isomerase plays a key role during in vivo foldingof conotoxins.AbbreviationscPDI, Conus marmoreus protein disulfide isomerase; ER, endoplasmic reticulum; hPDI, human protein disulfide isomerase; GSH, reducedglutathione; GSSG, oxidized glutathione; nTx3.1, native Tx3.1; PDI, protein disulfide isomerase; RNase A, bovine pancreatic RNase A;sTx3.1, swap Tx3.1.4778 FEBS Journal 274 (2007) 4778–4787 ª 2007 The Authors Journal compilation ª 2007 FEBSOne mechanism exploited by cone snails to ensureefficient folding of conotoxins is adding necessaryfolding information to the mature polypeptides. Forexample, the C-terminal Gly that is used for amidationof the mature form of x-conotoxin containing an ami-dated C-terminus, as isolated from the venom of Conusmagnus (x-MVIIA) can significantly increase the foldingyield [6]. The carboxylation of Glu residues can alsoimprove the folding yield, because the resultantc-carboxyglutamates can bind Ca2+and facilitate dis-ulfide pairing [5]. Other post-translational modifications,such as O-glycosylation, bromination of tryptophan,hydroxylation of proline, and l-tod-epimerization, mayalso facilitate the folding of conotoxins [7].Another mechanism to improve the folding of cono-toxins in vivo is utilization of the molecular chaperonesand foldases [5,8]. Many chaperones and foldases arepresent in the ER lumen [9,10]. Among them, PDI(EC 5.3.4.1) is a unique and multifunctional enzymethat exhibits disulfide reductase, oxidase and isomeraseactivities, as well as chaperone activity, and its concen-tration in the ER lumen can be as high as  200 lm[2,3]. Previous results indicated that PDI was themajor soluble protein in the ER, and was expressedthroughout the whole length of Conus venom ducts.Two full-length cDNAs encoding two PDI isoformshave been isolated from Conus textile [5,11]. However,the enzymatic properties of Conus PDI have not beenthoroughly investigated, especially using the endoge-nous conotoxins as substrates.In this article, we report gene cloning, recombinantexpression and enzymatic activity analyses of a novelConus marmoreus PDI (cPDI). Our results stronglysuggest that cPDI might play a key role during in vivofolding of conotoxins.ResultsMolecular cloning of a novel PDI fromC. marmoreusPDI is an abundant protein in the venom ducts ofC. textile, from which two PDIs have been cloned[5,11]. In present work, we cloned a novel PDIfrom C. marmoreus (GenBank accession numberDQ486867). The 1742 bp full-length cDNA includes a3¢-UTR and a polyadenylation consensus sequence(AATTATAA) located 12 nucleotides upstream of thepolyA tail. Its 1500 bp ORF encodes a 500 amino acidprotein (Fig. 1A) that shares sequence homology withPDIs from other species. A signal peptide (17 aminoacids) predicted by the signalp program [12] is presentat its N-terminus, and a typical ER retention signal,RDEL, is present at its C-terminus. The maturecPDI protein has a calculated molecular mass of54 913.7 Da and an isoelectric point of 4.6. The cPDIcontains four thioredoxin domains and an acidic C-ter-minal tail (a, b, b¢, a¢ and c). Two thioredoxin activesites (WCGHCK) are found in the a and a¢ domains,respectively. The cPDI shares 94% amino acidsequence identity with its homologs C. textile PDI 1and C. textile PDI 2 [5], 70% identity with humanPDI (hPDI), and 42% with yeast PDI.An unrooted neighbor-joining phylogenetic tree wasobtained by comparing the deduced amino acidsequences of different PDIs from fungi to mammalsusing the mega (Molecular Evolutionary GeneticAnalysis Software, Version 3.1) program, bootstrap:1000 replications (Fig. 1B).Enzymatic activities of cPDIThe cPDI was recombinantly expressed in Escherichiacoli as a soluble cytoplasmic protein, recovered from asoluble cell extract, and purified to homogeneity. Itspurity was approximately 95% as judged by SDS ⁄PAGE. The expression yield was about 50 mgÆL)1.As shown in Table 1, the enzymatic activities ofcPDI were analyzed using various substrates and com-pared with those of hPDI. The recombinant cPDI andhPDI shared similar reductase, oxidase and isomeraseactivities.PDI exhibits both chaperone and antichaperoneactivities when catalyzing the refolding of reduced ⁄denatured lysozyme in Hepes buffer [13]. As shown inFig. 2, we analyzed the chaperone and antichaperoneactivities of cPDI. Without PDI, the final refoldingyield of the reduced ⁄ denatured lysozyme reachedapproximately 40%. At low concentrations, both cPDIand hPDI decreased the refolding yield of lysozyme(antichaperone activity). At high concentrations, bothcPDI and hPDI increased the refolding yield of lyso-zyme (chaperone activity). Thus, cPDI and hPDIshared similar chaperone and antichaperone activities.In summary, the cPDI cloned from C. marmoreushad similar foldase and chaperone activities as hPDI,suggesting that the biological functions of PDI arehighly conserved during evolution.cPDI-catalyzed oxidative folding of tx3aDuring oxidative folding, oxidized PDI catalyzes disul-fide formation through transferring its active site’sdisulfide to dithiols of reduced polypeptide. We ana-lyzed the oxidase activity of cPDI using reduced tx3a(20 lm) as substrate. As shown in Fig. 3, the conotoxinZ Q. Wang et al. Characterization of PDI from Conus marmoreusFEBS Journal 274 (2007) 4778–4787 ª 2007 The Authors Journal compilation ª 2007 FEBS 4779ABFig. 1. Comparison of amino acid sequences of PDIs from C. marmoreus and other species. (A) Multiple sequence alignment of PDIs fromhuman, yeast, C. marmoreus,andC. textile. Identical or similar residues are shaded in black or gray. The potential N-terminal signal peptides areboxed; the thioredoxin active sites are underlined; and the ER-retention motif is indicated by underlined dashes. (B) A phylogenetic tree con-structed on the basis of the amino acid sequences of different PDIs, listed with GenBank accession numbers. The scale bar represents 0.1 units.Table 1. Enzymatic activities of cPDI compared with those of hPDI.PDIReduction activitya[102· (DAÆmin)1ÆlM PDI)1)]%ofcPDIOxidase activityb[102· (lM)1Æmin)1)]%ofcPDIIsomerization activityb(lMÆmin)1ÆlM PDI)1)%ofcPDIConus 5.74 ± 0.36 100 9.24 ± 0.83 100 1.11 ± 0.05 100Human 5.38 ± 0.52 94 10.18 ± 0.57 110 1.17 ± 0.10 105aThe disulfide reduction activity assay (thiol-protein oxidoreductase) was performed in 0.2 M sodium phosphate buffer (pH 7.5) containing8mM GSH, 30 lM insulin, 120 lM NADPH, 0.5 units of glutathione reductase, 5 mM EDTA, and 0.7 lM PDI. The absorbance decrease at340 nm was monitored.bRefolding of the reduced RNase A (final concentration 8.4 lM) was carried out in the refolding buffer (0.1 MTris ⁄ Cl, pH 8.0, 0.2 mM GSSG, 1 mM GSH, 2 mM EDTA, 4.5 mM cCMP) and catalyzed by appropriate concentrations of PDI (0–10 lM). Theabsorbance increase at 296 nm was monitored.Characterization of PDI from Conus marmoreus Z Q. Wang et al.4780 FEBS Journal 274 (2007) 4778–4787 ª 2007 The Authors Journal compilation ª 2007 FEBStx3a is a 16 residue peptide containing three disulfidebonds [14,15]. Figure 4A shows the HPLC profiles ofthe cPDI-catalyzed (1 lm) oxidative refolding ofreduced tx3a at different refolding stages. The refold-ing was finished after 2 h, and the folding yield wasover 90%. As shown in Fig. 4B, when the concentra-tion of cPDI increased, the refolding of reduced tx3aaccelerated accordingly. Thus cPDI can catalyze theoxidative folding of reduced conotoxins in vitro.Itislogical to expect that this process also occurs in vivo.The calculated oxidase activity of cPDI was measuredas the initial rate of decrease of reduced tx3a(Table 2).Besides cPDI, both oxidized glutathione (GSSG) andmolecular oxygen can also oxidize dithiols to formdisulfide bonds [16], and both of them are present inthe ER lumen. To compare the roles of these differentoxidants during the folding of conotoxins, the refoldingof reduced tx3a was carried out in three different sys-tems (Fig. 5). In all these systems, the final refoldingyields were approximately 90 ± 5%. When molecularoxygen dissolved in the buffer was used as an oxidant,the folding of reduced tx3a was barely detectable atthe start and finally reached equilibrium 10 h later(Fig. 5A). As with air oxidation of reduced bovine pan-creatic RNase A (RNase A) [17], a significant lag time( 60 min) caused by formation of partially and⁄ orfully oxidized intermediates was observed when molec-ular oxygen was used as an oxidant (Fig. 5A). Therefolding intermediates were collected, alkylated byN-ethylmaleimide or 4-vinylpyridine, and analyzed byMS, which revealed that these intermediates were com-plex mixtures of one, two or three disulfide isomersFig. 2. The effect of PDIs on the refolding of lysozyme. The oxida-tive refolding of the denatured ⁄ reduced lysozyme was carried outin the refolding buffer (0.1M Hepes, pH 7.0, 2 mM EDTA, 5 mMMgCl2,20mM NaCl, 1 mM GSSG, 2 mM GSH) and catalyzed by dif-ferent concentrations of PDI. In the refolding reaction mixture, thefinal concentration of reduced lysozyme was 10 lM. The refoldingwas carried out at room temperature for 2 h, and then the lyso-zyme activity was measured. The refolding yields were calculatedfrom the activity recovery on the basis of a standard curve.Fig. 3. The amino acid sequences of tx3a and Tx3.1. tx3a andTx3.1 have identical disulfide linkages, indicated by connectionlines. The asterisk indicates C-terminal amidation.ABFig. 4. The oxidase activity of cPDI determined by using reduced tx3a as substrate. (A) The HPLC profiles of the tx3a refolding mixture. Therefolding of reduced tx3a (20 lM) was performed in the refolding buffer (0.1 M Tris ⁄ Cl, 1 mM EDTA, pH 7.5) containing 0.1 mM GSSG and1 lM cPDI. At the indicated times, the reaction mixture was acidified and immediately analyzed by RP-HPLC. N indicates the native tx3a. (B)The time course of tx3a refolding catalyzed by different concentrations of cPDI. The amount of reduced tx3a was calculated from its elutionpeak area. The original data were fitted by Y(t) ¼ e–kt· 100%, where Y is the percentage of the linear form, and t is the refolding time.Z Q. Wang et al. Characterization of PDI from Conus marmoreusFEBS Journal 274 (2007) 4778–4787 ª 2007 The Authors Journal compilation ª 2007 FEBS 4781(data no shown). When GSSG was used as an oxidant,the folding process was expedited, and the lag time wasdiminished ( 6 min), as shown in Fig. 5B. When cPDI(at a final concentration of 2 lm) was added to theGSSG refolding system, the refolding process furtheraccelerated (Fig. 5B). The calculated molar specific oxi-dase activities of cPDI and GSSG are listed in Table 2:cPDI was about 268-fold more effective than GSSG inpromoting disulfide formation.cPDI-catalyzed disulfide isomerizationof swap Tx3.1Besides having an oxidase function, PDI can also cata-lyze the isomerization of non-native disulfide bonds[2,3]. To date, there are no reports of the PDI-cata-lyzed disulfide isomerization of conotoxins. Here, weused a homogeneous non-native conotoxin isomer,swap Tx3.1 (sTx3.1), to study the isomerase activity ofcPDI. As shown in Fig. 3, Tx3.1 is an 18 amino acidconotoxin with three disulfide bonds [14]. During theoxidative refolding of reduced Tx3.1, two major fold-ing products, native Tx3.1 (nTx3.1) and sTx3.1, wereformed at the final folding stage (first trace ofFig. 6A). The molecular mass of sTx3.1 as measuredby MS was identical to that of nTx3.1. After modifica-tion by N-ethylmaleimide under denatured condition(6 m urea), its molecular mass did not change. Thus,sTx3.1 was a fully oxidized isomer that had similarthermodynamic stability to the native form. Wepurified sTx3.1 and used it as the cPDI substrate inthe isomerase activity assay. The traces b–e in Fig. 6ATable 2. Oxidase and isomerase activities of cPDI measured using conotoxins as substrates.Oxidasea,bMoles of reduced tx3aper mole of oxidaseper min (· 10)2) Isomeraseb,cMoles of nTx3.1per mole of isomeraseper min (· 10)3)cPDI (1–8 lM) 115.13 ± 9.08 cPDI (0.5–4 lM) 208.17 ± 21.61GSSG (0.1–1 mM) 0.43 ± 0.04 GSH (0.25–1 mM) 0.12 ± 0.01aThe oxidase activity assay was performed in the refolding buffer (0.1 M Tris ⁄ Cl, 1 mM EDTA, pH 7.5) containing different concentrationsof GSSG or cPDI. The refolding mixture of reduced tx3a was analyzed by HPLC using the conditions described in the legend of Fig. 4.bOxidase or isomerase is a broad definition [16], including any compound that is capable of promoting disulfide formation and isomerization.cThe isomerase activity assay was performed in the refolding buffer (0.1 M Tris ⁄ Cl, 1 mM EDTA, pH 7.5) containing different concentrationsof GSH or cPDI. The refolding mixture of sTx3.1 was analyzed by HPLC as described in the legend of Fig. 6.Fig. 5. The tx3a refolding catalyzed by different oxidants. (A) Refolding carried out in buffer A (0.1 M Tris ⁄ Cl, pH 7.5, 1 mM EDTA). The filledand open circles denote native and linear tx3a, respectively. (B). Refolding carried out in buffer B (buffer A plus 1 mM GSH and 1 mM GSSG)and in buffer C (buffer B plus 2 lM cPDI). Filled circles, native tx3a in the presence of PDI; open circles, linear tx3a in the presence of PDI;filled squares, native tx3a in the absence of PDI; open squares, linear tx3a in the absence of PDI. At different reaction times, the refoldingmixture was acidified and immediately analyzed by RP-HPLC. The amounts of native and linear tx3a were calculated from their elution peakareas. The data are the average of three independent experiments. For the rate of decrease of the linear form, the original data were fittedby Y(t) ¼ e–kt· 100%, where Y is the percentage of the linear form, and t is the refolding time. For the rate of increase of the native formin the presence of cPDI, the original data were fitted by Y(t) ¼ Ymax(1 ) e–kt) · 100%, where Y is the percentage of the native form, andt is the refolding time. For the rate of increase of the native form in the absence of cPDI, the original data were fitted byY(t) ¼ Ymax⁄ [1 + e–k(t ) a)] · 100%, where Y is the percentage of the native form, and t is the refolding time.Characterization of PDI from Conus marmoreus Z Q. Wang et al.4782 FEBS Journal 274 (2007) 4778–4787 ª 2007 The Authors Journal compilation ª 2007 FEBSrepresent the disulfide isomerization process of sTx3.1(20 lm) catalyzed by cPDI (at a final concentration of1 lm). cPDI could accelerate disulfide reshuffle, but itcould not shift the equilibrium between nTx3.1 andsTx3.1, which had similar thermodynamic stability.Thus, cPDI could not convert all of the sTx3.1 to thenative form. When the concentration of cPDI wasincreased, the disulfide isomerization process signifi-cantly accelerated (Fig. 6B). The calculated molar spe-cific isomerase activity of cPDI was expressed as theinitial rate of increase of nTx3.1 (Table 2).Besides PDI, it is known that GSH, an abundantredox molecule in the ER lumen, can also catalyzedisulfide isomerization [16]. As shown in Fig. 7, wecompared the isomerase activities of reduced glutathi-one (GSH) and cPDI, and the result showed that cPDIwas much more efficient than GSH as an isomerase.The half-times of sTx3.1 disappearance in the presenceor absence of cPDI were approximately 3.9 min and13.5 min, respectively. The half-times of nTx3.1appearance in the presence or absence of cPDI wereabout 4.9 min and 21.9 min, respectively. The molarspecific isomerase activity of cPDI was about 1700-foldhigher than that of GSH (Table 2).Effect of macromolecular crowding on thePDI-catalyzed foldingThe intracellular environment is highly crowded, con-sisting of various proteins and other macromolecules,the concentration being about 80–300 g ÆL)1[8]. Thus,the protein folding catalyzed by PDI in vivo occurs ina crowded environment. To mimic the scenario ofin vivo PDI-catalyzed folding, the reactions of PDI-cat-alyzed oxidative folding of reduced tx3a (Fig. 8A) andPDI-catalyzed isomerization of sTx3.1 (Fig. 8B) werecarried out in a crowded environment, using Ficoll 70as crowding agent. As shown in Fig. 8, the crowdinghad little effect on PDI-catalyzed disulfide formationor isomerization of conotoxins. Our results are similarto those obtained with hirudin, which is a 65 aminoacid peptide containing three disulfide bonds [18].DiscussionIn the present work, we cloned a novel PDI fromC. marmoreus. The cPDI shares high sequence homol-ogy with PDIs from C. textile and other organisms. Italso has similar biological functions as hPDI, includingdisulfide reductase, oxidase and isomerase activities, aswell as chaperone and antichaperone activities. Thehigh sequence and function conservations support thehypothesis that all of the current PDIs evolved from acommon ancestral enzyme [19].We further analyzed the enzymatic activities of cPDIusing its potential endogenous substrates, namelyreduced tx3a and sTx3.1. Both tx3a and Tx3.1 belongto the M-1 branch of the M-superfamily. The differentbranches in the M-superfamily possess different disul-fide linkages [14]. The oxidative folding properties ofABFig. 6. The isomerase activity of cPDI determined by using sTx3.1 as substrate. (A) (a) The HPLC profile of the refolding mixture of reducedTx3.1. The refolding was carried out in the refolding buffer (50 mM NH4CO3, pH 8.0, 5 mM GSH, 0.5 mM GSSG) for 8 h. nTx3.1 and sx3.1are designated as N and S, respectively. (b–e) The HPLC profiles of sTx3.1 refolding mixtures. The disulfide isomerization of sTx3.1 (20 lM)was carried out in the refolding buffer (0.1M Tris ⁄ Cl, pH 7.5, 1 mM EDTA, 0.1 mM GSH) and catalyzed by 1 lM cPDI. At different reactiontimes, the reaction mixture was acidified and immediately analyzed by RP-HPLC. (B) The time course of sTx3.1 isomerization catalyzed bydifferent concentrations of cPDI. The isomerization of sTx3.1 was performed in the refolding buffer (0.1M Tris ⁄ Cl, pH 7.5, 1 mM EDTA,0.1 mM GSH) and catalyzed by different concentrations of cPDI. The amount of nTx3.1 was calculated from its elution peak area on HPLC.The original data were fitted by Y(t) ¼ Ymax(1 ) e–kt) · 100%, where Y is the percentage of the native form, and t is the refolding time.Z Q. Wang et al. Characterization of PDI from Conus marmoreusFEBS Journal 274 (2007) 4778–4787 ª 2007 The Authors Journal compilation ª 2007 FEBS 4783four M-4 branch conotoxins have been thoroughlyinvestigated, and two distinct folding mechanisms havebeen unveiled [20]. Through comparison of foldingkinetics and thermodynamics, the folding mechanismof tx3a and Tx3.1 was found to be similar to that ofconotoxins GIIIA and RIIIK [20]. Their refoldingfollows a slow-rearrangement mechanism, where thepartially ⁄ fully oxidized folding intermediates areformed quickly and then converted to the native formslowly.The activity analyses demonstrate that cPDI cangreatly accelerate both oxidative folding and disulfideisomerization of conotoxins. The calculated molar spe-cific oxidase and isomerase activities of cPDI are muchFig. 7. The disulfide isomerization of sTx3.1 catalyzed by GSH or by cPDI. (A) The time course of nTx3.1 appearance. (B) The time course ofsTx3.1 disappearance. The disulfide isomerization of sTx3.1 was performed in buffer A (0.1M Tris ⁄ Cl, pH 7.5, 1 mM EDTA, 1 mM GSH)(open circles) or in buffer B (buffer A plus 2 lM cPDI) (filled circles). At different reaction times, the refolding mixture was acidified andimmediately analyzed by HPLC. The amounts of nTx3.1 and sTx3.1 were calculated from their elution peak areas. The data are the averageof three independent experiments. For the rate of increase of the native form, the original data were fitted by Y(t) ¼ Ymax(1 ) e–kt) · 100%,where Y is the percentage of the native form, and t is the refolding time. For the rate of decrease of the linear form, the original data werefitted by Y(t) ¼ [Ymax+(1) Ymax)e–kt] · 100%, where Y is the percentage of the linear form, and t is the refolding time.Fig. 8. Effects of macromolecular crowding on the PDI-catalyzed folding of conotoxins. (A) The oxidative folding of reduced tx3a in theabsence (open squares) or presence (filled squares) of crowding agent. The refolding was performed in refolding buffer (0.1M Tris ⁄ Cl, pH 7.5,1mM EDTA, 1 mM GSH, 1 mM GSSG, 2 lM cPDI) in the presence or absence of 200 gÆL)1Ficoll 70. At different reaction times, the refoldingmixture was acidified and immediately analyzed by HPLC. The amounts of native and linear tx3a were calculated from their elution peak areas.(B) The isomerization of sTx3.1 in the absence (open squares) or presence (filled squares) of crowding agent. The isomerization was per-formed in refolding buffer (0.1M Tris ⁄ Cl, pH 7.5, 1 mM EDTA, 0.1 mM GSH, 2 lM cPDI) in the presence or absence of 200 gÆL)1Ficoll 70.The amounts of nTx3.1 and sTx3.1 were calculated from their elution peak areas. The data are the average of three independent experiments.For the rate of increase of the native form in the presence of cPDI, the original data were fitted by Y(t) ¼ Ymax(1 ) e–kt) · 100%, where Y isthe percentage of the native form, and t is the refolding time. For the rate of decrease of the linear form, the original data were fitted byY(t) ¼ [Ymax+(1) Ymax)e–kt] · 100%, where Y is the percentage of the linear form, and t is the refolding time.Characterization of PDI from Conus marmoreus Z Q. Wang et al.4784 FEBS Journal 274 (2007) 4778–4787 ª 2007 The Authors Journal compilation ª 2007 FEBShigher than those of glutathione (Table 2). The con-centrations of GSH and GSSG in the ER lumen are inthe millimolar range, whereas the concentration ofPDI is about 200 lm (about 10-fold lower than theconcentration of glutathione). This work providesdirect evidence that the molar specific oxidase andisomerase activities of cPDI are much higher (268-foldand 1500-fold, respectively) than those of glutathione;hence, the total oxidase and isomerase activities in theER lumen should be dominated by cPDI. We thereforepropose the hypothesis that PDI plays a key role dur-ing in vivo folding of conotoxins.Experimental proceduresMaterialsThe plasmid encoding mature hPDI was a generous giftfrom L. W. Ruddock (Biocenter, University of Oulu,Finland). Ni2+-chelating Sepharose Fast Flow resin andQ-Sepharose Fast Flow resin were obtained from Amer-sham Biosciences (Arlington Heights, IL, USA). TheRACE kit was obtained from Invitrogen (Carlsbad, CA,USA). Lysozyme, Micrococcus lysodeikticus and RNase Awere products of Sigma (St Louis, MO, USA). Otherreagents were of analytical grade.Gene cloning of cPDIThe full-length cDNA of cPDI was amplified by RT-PCRfrom total RNAs isolated from the venom ducts ofC. marmoreus. The 3¢-end fragment was amplified using a3¢-RACE adapter primer and a degenerate primer basedon the conserved amino acid sequence (WCGHCK) ofthe thioredoxin-like active site found in other PDIs. The3¢-RACE product was gel-purified, cloned into pGEM-Teasy vector, and sequenced. The nested PCR primers for5¢-RACE amplification were based on the 3¢-end sequence,and the 5¢-end fragment was amplified using the nestedprimer and the 5¢-RACE adapter primer. The 5¢-RACEproduct was also gel-purified, cloned into pGEM-T easyvector, and sequenced. Primers for amplifying the full-length cDNA were designed on the basis of these RACEproducts. The full-length cDNA of the cPDI was insertedinto an expression vector, pET24a, which contains anN-terminal His6tag.Expression and purification of cPDI and hPDIThe expression plasmid of cPDI was transformed into BL21(DE3) cells. The transformed E. coli cells were cultured inLB medium containing 25 lgÆmL)1kanamycin at 37 °C,and the expression was induced by standard procedures.Thereafter, the cells were harvested, resuspended in buffer A(20 mm phosphate buffer, pH 7.5, 0.5 m NaCl) and lysed bysonication. After centrifugation (12 000 g,4°C, 20 min;Hitachi Himac CR22G centrifuge, rotor 46), the superna-tant was loaded onto an Ni2+-chelating Sepharose FastFlow column pre-equilibrated with buffer A. The columnwas extensively washed with buffer A, and then the nonspe-cifically bound proteins were eluted with buffer B (buffer Aplus 20 mm imidazole). Finally, the recombinant cPDI waseluted from the column with buffer C (buffer A plus250 mm imidazole). The eluted cPDI was dialyzed against20 mm phosphate buffer (pH 7.5) at 4 °C, and subsequentlyapplied to a Q-Sepharose Fast Flow column pre-equili-brated with 20 mm phosphate buffer (pH 7.5). cPDI waseluted from the ion exchange column using a linear NaClgradient (0–1 m). The cPDI fraction was collected, analyzedby SDS ⁄ PAGE, dialyzed against distilled water, and storedat ) 80 °C for later use.Expression and purification of human PDI were per-formed as described previously [21], and its purity was ana-lyzed by SDS ⁄ PAGE.Enzymatic activity assays of PDIThe thiol-protein oxidoreductase activity of PDI was mea-sured as described previously, using insulin as substrate[22]. The assay was performed in 0.2 m sodium phosphatebuffer (pH 7.5) containing 8 mm GSH, 30 lm insulin,120 lm NADPH, 0.5 units of glutathione reductase, 5 mmEDTA, and 0.7 lm PDI. The assay mixture (without insu-lin and PDI) was equilibrated at 25 °C, and the NADPHoxidation rate was recorded against a reference cuvette con-taining NADPH, EDTA and buffer only. Subsequently,insulin was added, and a stable nonenzymatic rate wasrecorded. Finally, PDI was added, and the total NADPHoxidation rate was recorded.The oxidase and isomerase activities of PDI were mea-sured using the refolding assay of fully reduced RNase A aspreviously described [17,23,24]. Briefly, it was performed inthe assay solution (0.1 m Tris ⁄ Cl, pH 8.0, 0.2 mm GSSG,1mm GSH, 2 mm EDTA, 4.5 mm cCMP) at 25 °C. Afterpreincubation, the fully reduced RNase A (8.4 lm) and dif-ferent concentrations of PDI (0–10 lm) were added to theassay solution to initiate refolding. The formation of activeRNase A was measured spectrophotometrically by monitor-ing cCMP hydrolysis at 296 nm. During the oxidativerefolding, the reduced RNase A was quickly converted toinactive oxidized forms by the oxidase activity of PDI, andthese inactive oxidized forms were then slowly converted toactive native form by the isomerase activity of PDI [23].The lag time before appearance of the active RNase Aindicates the oxidase activity, which corresponds to thex-intercept of the RNase activity plot. The oxidase activitymatches the slope of the linear plot of reciprocal of lag timesagainst the PDI concentrations in units of lm)1Æmin)1. TheZ Q. Wang et al. Characterization of PDI from Conus marmoreusFEBS Journal 274 (2007) 4778–4787 ª 2007 The Authors Journal compilation ª 2007 FEBS 4785isomerase activity was determined from the linear increaseof enzymatically active RNase A after the lag time.The chaperone and antichaperone activities of PDI wereanalyzed using the renaturation of reduced ⁄ denatured lyso-zyme [13]. The lysozyme activity was measured at 30 °Cbyfollowing the absorbance decrease at 450 nm of the M. lyso-deikticus suspension (0.25 mgÆmL)1in 67 mm sodiumphosphate buffer, pH 6.2, and 0.1 m NaCl).Peptide synthesisConotoxins tx3a and Tx3.1 [14,15] were chemically synthe-sized by using the Fmoc method on an ABI 433 A peptidesynthesizer. The crude reduced peptides were purified byC18reversed-phase HPLC and lyophilized. The identity ofeach peptide was confirmed by MS. The molecular massesof linear tx3a and Tx3.1 were 1660.5 Da (theoretical value:1660.8 Da) and 2158.6 Da (theoretical value: 2158.44 Da).Preparation of homogeneous sTx3.1Refolding of the linear Tx3.1 was carried out in the refold-ing buffer (50 mm NH4CO3,5mm GSH, 0.5 mm GSSG,pH 8.0) at 25 °C for 8 h, and the refolding mixture wasanalyzed by RP-HPLC. Two major disulfide isomers,nTx3.1 and sTx3.1, were collected and lyophilized.PDI-catalyzed oxidative folding and isomerizationof conotoxinsThe oxidative folding or isomerization of conotoxins wasperformed in the refolding buffer (0.1 m Tris ⁄ Cl, pH 7.5,plus appropriate concentrations of GSH, GSSG and cPDIas indicated in the figure legends) at ambient temperature(23–25 °C). The folding reaction was initiated by addingthe peptide stock solution to the final concentration of20 lm. At different reaction times, the folding reaction wasquenched by adding formic acid to the final concentrationof 8%. The reaction mixture was immediately analyzedusing C18analytical RP-HPLC. The amounts of linearform, native form and swap form were calculated fromtheir integrated elution peak areas, and the PDI’s oxidaseand isomerase activities were expressed as the initial rate ofdecrease of linear tx3a and the initial rate of increase ofnTx3.1, respectively. To investigate the effect of macromo-lecular crowding on the PDI-catalyzed oxidative folding ordisulfide isomerization of conotoxins, the folding reactionwas carried out as described above, except that 200 gÆL)1of Ficoll 70 was added to the refolding buffer.AcknowledgementsThe authors wish to acknowledge Professors C. C.Wang, D. F. Cui, Q. Y. Dai and L. W. Ruddock fortheir generous support for this work. This work wassupported by the National Basic Research Program ofChina (2004CB719904).References1 Ellgaard L & Ruddock LW (2005) The human proteindisulphide isomerase family: substrate interactions andfunctional properties. EMBO Rep 6 , 28–32.2 Wilkinson B & Gilbert HF (2004) Protein disulfideisomerase. Biochim Biophys Acta 1699, 35–44.3 Kersteen EA & Raines RT (2003) Catalysis of proteinfolding by protein disulfide isomerase and small-mole-cule mimics. Antioxid Redox Signal 5, 413–424.4 Song JL & Wang CC (1995) Chaperone-like activity ofprotein disulfide-isomerase in the refolding of rhoda-nese. Eur J Biochem 231, 312–316.5 Bulaj G, Buczek O, Goodsell I, Jimenez EC, Kranski J,Nielsen JS, Garrett JE & Olivera BM (2003) Efficientoxidative folding of conotoxins and the radiation ofvenomous cone snails. Proc Natl Acad Sci USA 100(Suppl. 2), 14562–14568.6 Price-Carter M, Gray WR & Goldenberg DP (1996)Folding of omega-conotoxins. 2. Influence of precursorsequences and protein disulfide isomerase. Biochemistry35, 15547–15557.7 Craig AG, Bandyopadhyay P & Olivera BM (1999)Post-translationally modified neuropeptides from Conusvenoms. Eur J Biochem 264, 271–275.8 van den Berg B, Ellis RJ & Dobson CM (1999) Effectsof macromolecular crowding on protein folding andaggregation. EMBO J 18, 6927–6933.9 Gething MJ & Sambrook J (1992) Protein folding in thecell. Nature 355, 33–45.10 Ellis J (1987) Proteins as molecular chaperones. Nature328, 378–379.11 Garrett JE, Buczek O, Watkins M, Olivera BM & BulajG (2005) Biochemical and gene expression analyses ofconotoxins in Conus textile venom ducts. BiochemBiophys Res Commun 328, 362–367.12 Nielsen H, Engelbrecht J, Brunak S & von Heijne G(1997) Identification of prokaryotic and eukaryoticsignal peptides and prediction of their cleavage sites.Protein Eng 10, 1–6.13 Song J, Quan H & Wang C (1997) Dependence of theanti-chaperone activity of protein disulphide isomeraseon its chaperone activity. Biochem J 328 (3), 841–846.14 Han YH, Wang Q, Jiang H, Liu L, Xiao C, Yuan DD,Shao XX, Dai QY, Cheng JS & Chi CW (2006) Charac-terization of novel M-superfamily conotoxins with newdisulfide linkage. FEBS J 273, 4972–4982.15 Corpuz GP, Jacobsen RB, Jimenez EC, Watkins M,Walker C, Colledge C, Garrett JE, McDougal O, Li W,Gray WR et al. (2005) Definition of the M-conotoxinCharacterization of PDI from Conus marmoreus Z Q. 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FEBS J 272, 1727–1738.21 Lappi AK, Lensink MF, Alanen HI, Salo KE, LobellM, Juffer AH & Ruddock LW (2004) A conservedarginine plays a role in the catalytic cycle of theprotein disulphide isomerases. J Mol Biol 335, 283–295.22 Lambert N & Freedman RB (1983) Kinetics andspecificity of homogeneous protein disulphide-isomerase in protein disulphide isomerization and inthiol-protein-disulphide oxidoreduction. Biochem J213, 235–243.23 Lyles MM & Gilbert HF (1991) Catalysis of the oxida-tive folding of ribonuclease A by protein disulfide iso-merase: dependence of the rate on the composition ofthe redox buffer. Biochemistry 30, 613–619.24 Wilkinson B, Xiao R & Gilbert HF (2005) A structuraldisulfide of yeast protein-disulfide isomerase destabilizesthe active site disulfide of the N-terminal thioredoxindomain. J Biol Chem 280, 11483–11487.Z Q. Wang et al. Characterization of PDI from Conus marmoreusFEBS Journal 274 (2007) 4778–4787 ª 2007 The Authors Journal compilation ª 2007 FEBS 4787 . Molecular cloning, expression and characterization of protein disulfide isomerase from Conus marmoreus Zhi-Qiang Wang1, Yu-Hong. physiologic roles of pro-tein disulfide isomerase in the folding of conotoxins, we have cloned anovel full-length protein disulfide isomerase from Conus marmoreus.
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