Using conotoxins tx3a and sTx3.1 as substrate, we analyzed the oxidase and isomerase activities of the C.. Abbreviations cPDI, Conus marmoreus protein disulfide isomerase; ER, endoplasmi
Trang 1protein disulfide isomerase from Conus marmoreus
Zhi-Qiang Wang1, Yu-Hong Han1,2, Xiao-Xia Shao1, Cheng-Wu Chi1,2and Zhan-Yun Guo1
1 Institute of Protein Research, Tongji University, Shanghai, China
2 Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai, China
In eukaryotes, all proteins that travel along or reside
in the secretory pathway are folded in the
endoplas-mic reticulum (ER) As one of the most important
post-translational modifications, the disulfide bonds
are formed in the ER lumen, where oxidoreductases
catalyze the reaction and serve as disulfide donors [1]
The archetypical oxidoreductase in the ER lumen is
protein disulfide isomerase (PDI) In the oxidized
state, PDI functions as a disulfide donor for its client
proteins In the reduced state, PDI catalyzes reduction
and isomerization of pre-existing disulfides The
abil-ity of PDI to function as a reductase, an oxidase and
an isomerase ensures PDI’s ability to serve as a major catalyst for disulfide formation in vivo [2,3] Moreover, PDI also acts as a chaperone for substrates during catalysis [4]
Conotoxins are small, cysteine-rich peptides pro-duced by marine cone snails [5] Although their amino acid sequences are hypervariable, they can form spe-cific disulfide patterns that are essential for their bio-logical activities It is believed that cone snails possess evolving mechanisms to ensure efficient folding of conotoxins in vivo, but these mechanisms are not fully understood yet
Keywords
conotoxin; disulfide isomerization; oxidative
folding; protein disulfide isomerase
Correspondence
Z.-Y Guo, Institute of Protein Research,
Tongji University, 1239 Siping Road,
Shanghai 200092, China
Fax: +86 21 65988403
Tel: +86 21 65985167
E-mail: zhan-yun.guo@mail.tongji.edu.cn
C.-W Chi, Shanghai Institute of
Biochemistry and Cell Biology, Chinese
Academy of Sciences, 320 YueYang Road,
Shanghai 200031, China
Fax: +86 21 54921011
Tel: +86 21 54921165
E-mail: chi@sunm.shcnc.ac.cn
(Received 2 March 2007, revised 8 July
2007, accepted 18 July 2007)
doi:10.1111/j.1742-4658.2007.06003.x
The oxidative folding of disulfide-rich conotoxins is essential for their biological functions In vivo, disulfide bond formation is mainly catalyzed
by protein disulfide isomerase To elucidate the physiologic roles of pro-tein disulfide isomerase in the folding of conotoxins, we have cloned a novel full-length protein disulfide isomerase from Conus marmoreus Its ORF encodes a 500 amino acid protein that shares sequence homology with protein disulfide isomerases from other species, and 70% homology with human protein disulfide isomerase Enzymatic analyses of recombi-nant C marmoreus protein disulfide isomerase showed that it shared functional similarities with human protein disulfide isomerase Using conotoxins tx3a and sTx3.1 as substrate, we analyzed the oxidase and isomerase activities of the C marmoreus protein disulfide isomerase and found 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 protein disulfide isomerase-catalyzed oxidative folding and disulfide isomerization
of conotoxins On the basis of these data, we propose that the C mar-moreus protein disulfide isomerase plays a key role during in vivo folding
of conotoxins
Abbreviations
cPDI, Conus marmoreus protein disulfide isomerase; ER, endoplasmic reticulum; hPDI, human protein disulfide isomerase; GSH, reduced glutathione; GSSG, oxidized glutathione; nTx3.1, native Tx3.1; PDI, protein disulfide isomerase; RNase A, bovine pancreatic RNase A; sTx3.1, swap Tx3.1.
Trang 2One mechanism exploited by cone snails to ensure
efficient folding of conotoxins is adding necessary
folding information to the mature polypeptides For
example, the C-terminal Gly that is used for amidation
of the mature form of x-conotoxin containing an
ami-dated C-terminus, as isolated from the venom of Conus
magnus(x-MVIIA) can significantly increase the folding
yield [6] The carboxylation of Glu residues can also
improve the folding yield, because the resultant
c-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- to d-epimerization, may
also facilitate the folding of conotoxins [7]
Another mechanism to improve the folding of
cono-toxins in vivo is utilization of the molecular chaperones
and foldases [5,8] Many chaperones and foldases are
present in the ER lumen [9,10] Among them, PDI
(EC 5.3.4.1) is a unique and multifunctional enzyme
that exhibits disulfide reductase, oxidase and isomerase
activities, 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 the
major soluble protein in the ER, and was expressed
throughout the whole length of Conus venom ducts
Two full-length cDNAs encoding two PDI isoforms
have been isolated from Conus textile [5,11] However,
the enzymatic properties of Conus PDI have not been
thoroughly investigated, especially using the
endoge-nous conotoxins as substrates
In this article, we report gene cloning, recombinant
expression and enzymatic activity analyses of a novel
Conus marmoreus PDI (cPDI) Our results strongly
suggest that cPDI might play a key role during in vivo
folding of conotoxins
Results
Molecular cloning of a novel PDI from
C marmoreus
PDI is an abundant protein in the venom ducts of
C textile, from which two PDIs have been cloned
[5,11] In present work, we cloned a novel PDI
from C marmoreus (GenBank accession number
DQ486867) The 1742 bp full-length cDNA includes a
3¢-UTR and a polyadenylation consensus sequence
(AATTATAA) located 12 nucleotides upstream of the
polyA tail Its 1500 bp ORF encodes a 500 amino acid
protein (Fig 1A) that shares sequence homology with
PDIs from other species A signal peptide (17 amino
acids) predicted by the signalp program [12] is present
at its N-terminus, and a typical ER retention signal,
RDEL, is present at its C-terminus The mature cPDI protein has a calculated molecular mass of
54 913.7 Da and an isoelectric point of 4.6 The cPDI contains four thioredoxin domains and an acidic C-ter-minal tail (a, b, b¢, a¢ and c) Two thioredoxin active sites (WCGHCK) are found in the a and a¢ domains, respectively The cPDI shares 94% amino acid sequence identity with its homologs C textile PDI 1 and C textile PDI 2 [5], 70% identity with human PDI (hPDI), and 42% with yeast PDI
An unrooted neighbor-joining phylogenetic tree was obtained by comparing the deduced amino acid sequences of different PDIs from fungi to mammals using the mega (Molecular Evolutionary Genetic Analysis Software, Version 3.1) program, bootstrap:
1000 replications (Fig 1B)
Enzymatic activities of cPDI The cPDI was recombinantly expressed in Escherichia coli as a soluble cytoplasmic protein, recovered from a soluble cell extract, and purified to homogeneity Its purity was approximately 95% as judged by SDS⁄ PAGE The expression yield was about 50 mgÆL)1
As shown in Table 1, the enzymatic activities of cPDI were analyzed using various substrates and com-pared with those of hPDI The recombinant cPDI and hPDI shared similar reductase, oxidase and isomerase activities
PDI exhibits both chaperone and antichaperone activities when catalyzing the refolding of reduced⁄ denatured lysozyme in Hepes buffer [13] As shown in Fig 2, we analyzed the chaperone and antichaperone activities of cPDI Without PDI, the final refolding yield of the reduced⁄ denatured lysozyme reached approximately 40% At low concentrations, both cPDI and hPDI decreased the refolding yield of lysozyme (antichaperone activity) At high concentrations, both cPDI and hPDI increased the refolding yield of lyso-zyme (chaperone activity) Thus, cPDI and hPDI shared similar chaperone and antichaperone activities
In summary, the cPDI cloned from C marmoreus had similar foldase and chaperone activities as hPDI, suggesting that the biological functions of PDI are highly conserved during evolution
cPDI-catalyzed oxidative folding of tx3a During oxidative folding, oxidized PDI catalyzes disul-fide formation through transferring its active site’s disulfide 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 conotoxin
Trang 3B
Fig 1 Comparison of amino acid sequences of PDIs from C marmoreus and other species (A) Multiple sequence alignment of PDIs from human, yeast, C marmoreus, and C textile Identical or similar residues are shaded in black or gray The potential N-terminal signal peptides are boxed; 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.
PDI
Reduction activitya
[10 2 · (DAÆmin)1Æl M PDI)1)]
% of cPDI
Oxidase activityb [10 2 · (l M )1Æmin)1)]
% of cPDI
Isomerization activityb (l M Æmin)1Æl M PDI)1)
% of cPDI
a
The disulfide reduction activity assay (thiol-protein oxidoreductase) was performed in 0.2 M sodium phosphate buffer (pH 7.5) containing
8 m M GSH, 30 l M insulin, 120 l M NADPH, 0.5 units of glutathione reductase, 5 m M EDTA, and 0.7 l M PDI The absorbance decrease at
340 nm was monitored b Refolding of the reduced RNase A (final concentration 8.4 l M ) was carried out in the refolding buffer (0.1 M
Tris ⁄ Cl, pH 8.0, 0.2 m M GSSG, 1 m M GSH, 2 m M EDTA, 4.5 m M cCMP) and catalyzed by appropriate concentrations of PDI (0–10 l M ) The absorbance increase at 296 nm was monitored.
Trang 4tx3a is a 16 residue peptide containing three disulfide bonds [14,15] Figure 4A shows the HPLC profiles of the cPDI-catalyzed (1 lm) oxidative refolding of reduced tx3a at different refolding stages The refold-ing was finished after 2 h, and the foldrefold-ing yield was over 90% As shown in Fig 4B, when the concentra-tion of cPDI increased, the refolding of reduced tx3a accelerated accordingly Thus cPDI can catalyze the oxidative folding of reduced conotoxins in vitro It is logical to expect that this process also occurs in vivo The calculated oxidase activity of cPDI was measured
as the initial rate of decrease of reduced tx3a (Table 2)
Besides cPDI, both oxidized glutathione (GSSG) and molecular oxygen can also oxidize dithiols to form disulfide bonds [16], and both of them are present in the ER lumen To compare the roles of these different oxidants during the folding of conotoxins, the refolding
of reduced tx3a was carried out in three different sys-tems (Fig 5) In all these syssys-tems, the final refolding yields were approximately 90 ± 5% When molecular oxygen dissolved in the buffer was used as an oxidant, the folding of reduced tx3a was barely detectable at the 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 ⁄ or fully oxidized intermediates was observed when molec-ular oxygen was used as an oxidant (Fig 5A) The refolding intermediates were collected, alkylated by N-ethylmaleimide or 4-vinylpyridine, and analyzed by
MS, which revealed that these intermediates were com-plex mixtures of one, two or three disulfide isomers
Fig 2 The effect of PDIs on the refolding of lysozyme The
oxida-tive refolding of the denatured⁄ reduced lysozyme was carried out
in the refolding buffer (0.1 M Hepes, pH 7.0, 2 m M EDTA, 5 m M
MgCl 2 , 20 m M NaCl, 1 m M GSSG, 2 m M GSH) and catalyzed by
dif-ferent concentrations of PDI In the refolding reaction mixture, the
final concentration of reduced lysozyme was 10 l M The refolding
was carried out at room temperature for 2 h, and then the
lyso-zyme activity was measured The refolding yields were calculated
from the activity recovery on the basis of a standard curve.
Fig 3 The amino acid sequences of tx3a and Tx3.1 tx3a and
Tx3.1 have identical disulfide linkages, indicated by connection
lines The asterisk indicates C-terminal amidation.
Fig 4 The oxidase activity of cPDI determined by using reduced tx3a as substrate (A) The HPLC profiles of the tx3a refolding mixture The refolding of reduced tx3a (20 l M ) was performed in the refolding buffer (0.1 M Tris ⁄ Cl, 1 m M EDTA, pH 7.5) containing 0.1 m M GSSG and
1 l M 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 elution peak 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.
Trang 5(data no shown) When GSSG was used as an oxidant,
the folding process was expedited, and the lag time was
diminished ( 6 min), as shown in Fig 5B When cPDI
(at a final concentration of 2 lm) was added to the
GSSG refolding system, the refolding process further
accelerated (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 in
promoting disulfide formation
cPDI-catalyzed disulfide isomerization
of swap Tx3.1
Besides 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, we used a homogeneous non-native conotoxin isomer, swap Tx3.1 (sTx3.1), to study the isomerase activity of cPDI As shown in Fig 3, Tx3.1 is an 18 amino acid conotoxin with three disulfide bonds [14] During the oxidative refolding of reduced Tx3.1, two major fold-ing products, native Tx3.1 (nTx3.1) and sTx3.1, were formed at the final folding stage (first trace of Fig 6A) The molecular mass of sTx3.1 as measured
by MS was identical to that of nTx3.1 After modifica-tion by N-ethylmaleimide under denatured condimodifica-tion (6 m urea), its molecular mass did not change Thus, sTx3.1 was a fully oxidized isomer that had similar thermodynamic stability to the native form We purified sTx3.1 and used it as the cPDI substrate in the isomerase activity assay The traces b–e in Fig 6A
Table 2 Oxidase and isomerase activities of cPDI measured using conotoxins as substrates.
Oxidase a,b
Moles of reduced tx3a per mole of oxidase
Moles of nTx3.1 per mole of isomerase per min (· 10)3)
a
The oxidase activity assay was performed in the refolding buffer (0.1 M Tris ⁄ Cl, 1 m M EDTA, pH 7.5) containing different concentrations
of GSSG or cPDI The refolding mixture of reduced tx3a was analyzed by HPLC using the conditions described in the legend of Fig 4.
b Oxidase or isomerase is a broad definition [16], including any compound that is capable of promoting disulfide formation and isomerization c
The isomerase activity assay was performed in the refolding buffer (0.1 M Tris ⁄ Cl, 1 m M EDTA, pH 7.5) containing different concentrations
of 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 m M EDTA) The filled and open circles denote native and linear tx3a, respectively (B) Refolding carried out in buffer B (buffer A plus 1 m M GSH and 1 m M GSSG) and in buffer C (buffer B plus 2 l M 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 refolding mixture was acidified and immediately analyzed by RP-HPLC The amounts of native and linear tx3a were calculated from their elution peak areas The data are the average of three independent experiments For the rate of decrease of the linear form, 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 For the rate of increase of the native form
in the presence of cPDI, the original data were fitted by Y(t) ¼ Y max (1 ) e –kt
) · 100%, where Y is the percentage of the native form, and
t is the refolding time For the rate of increase of the native form in the absence of cPDI, the original data were fitted by Y(t) ¼ Y max ⁄ [1 + e –k(t ) a)]· 100%, where Y is the percentage of the native form, and t is the refolding time.
Trang 6represent the disulfide isomerization process of sTx3.1
(20 lm) catalyzed by cPDI (at a final concentration of
1 lm) cPDI could accelerate disulfide reshuffle, but it
could not shift the equilibrium between nTx3.1 and
sTx3.1, which had similar thermodynamic stability
Thus, cPDI could not convert all of the sTx3.1 to the
native form When the concentration of cPDI was
increased, the disulfide isomerization process
signifi-cantly accelerated (Fig 6B) The calculated molar
spe-cific isomerase activity of cPDI was expressed as the
initial rate of increase of nTx3.1 (Table 2)
Besides PDI, it is known that GSH, an abundant
redox molecule in the ER lumen, can also catalyze
disulfide isomerization [16] As shown in Fig 7, we
compared the isomerase activities of reduced
glutathi-one (GSH) and cPDI, and the result showed that cPDI
was much more efficient than GSH as an isomerase
The half-times of sTx3.1 disappearance in the presence
or absence of cPDI were approximately 3.9 min and
13.5 min, respectively The half-times of nTx3.1
appearance in the presence or absence of cPDI were
about 4.9 min and 21.9 min, respectively The molar
specific isomerase activity of cPDI was about 1700-fold
higher than that of GSH (Table 2)
Effect of macromolecular crowding on the
PDI-catalyzed folding
The intracellular environment is highly crowded,
con-sisting of various proteins and other macromolecules,
the concentration being about 80–300 gÆL)1 [8] Thus, the protein folding catalyzed by PDI in vivo occurs in
a crowded environment To mimic the scenario of
in vivoPDI-catalyzed folding, the reactions of PDI-cat-alyzed oxidative folding of reduced tx3a (Fig 8A) and PDI-catalyzed isomerization of sTx3.1 (Fig 8B) were carried out in a crowded environment, using Ficoll 70
as crowding agent As shown in Fig 8, the crowding had little effect on PDI-catalyzed disulfide formation
or isomerization of conotoxins Our results are similar
to those obtained with hirudin, which is a 65 amino acid peptide containing three disulfide bonds [18]
Discussion
In the present work, we cloned a novel PDI from
C marmoreus The cPDI shares high sequence homol-ogy with PDIs from C textile and other organisms It also has similar biological functions as hPDI, including disulfide reductase, oxidase and isomerase activities, as well as chaperone and antichaperone activities The high sequence and function conservations support the hypothesis that all of the current PDIs evolved from a common ancestral enzyme [19]
We further analyzed the enzymatic activities of cPDI using its potential endogenous substrates, namely reduced tx3a and sTx3.1 Both tx3a and Tx3.1 belong
to the M-1 branch of the M-superfamily The different branches in the M-superfamily possess different disul-fide linkages [14] The oxidative folding properties of
Fig 6 The isomerase activity of cPDI determined by using sTx3.1 as substrate (A) (a) The HPLC profile of the refolding mixture of reduced Tx3.1 The refolding was carried out in the refolding buffer (50 m M NH4CO3, pH 8.0, 5 m M GSH, 0.5 m M GSSG) for 8 h nTx3.1 and sx3.1 are designated as N and S, respectively (b–e) The HPLC profiles of sTx3.1 refolding mixtures The disulfide isomerization of sTx3.1 (20 l M ) was carried out in the refolding buffer (0.1 M Tris ⁄ Cl, pH 7.5, 1 m M EDTA, 0.1 m M GSH) and catalyzed by 1 l M cPDI At different reaction times, the reaction mixture was acidified and immediately analyzed by RP-HPLC (B) The time course of sTx3.1 isomerization catalyzed by different concentrations of cPDI The isomerization of sTx3.1 was performed in the refolding buffer (0.1 M Tris ⁄ Cl, pH 7.5, 1 m M EDTA, 0.1 m M 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 Y max (1 ) e –kt
) · 100%, where Y is the percentage of the native form, and t is the refolding time.
Trang 7four M-4 branch conotoxins have been thoroughly
investigated, and two distinct folding mechanisms have
been unveiled [20] Through comparison of folding
kinetics and thermodynamics, the folding mechanism
of tx3a and Tx3.1 was found to be similar to that of
conotoxins GIIIA and RIIIK [20] Their refolding
follows a slow-rearrangement mechanism, where the
partially⁄ fully oxidized folding intermediates are formed quickly and then converted to the native form slowly
The activity analyses demonstrate that cPDI can greatly accelerate both oxidative folding and disulfide isomerization of conotoxins The calculated molar spe-cific oxidase and isomerase activities of cPDI are much
Fig 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 of sTx3.1 disappearance The disulfide isomerization of sTx3.1 was performed in buffer A (0.1 M Tris ⁄ Cl, pH 7.5, 1 m M EDTA, 1 m M GSH) (open circles) or in buffer B (buffer A plus 2 l M cPDI) (filled circles) At different reaction times, the refolding mixture was acidified and immediately analyzed by HPLC 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, the original data were fitted by Y(t) ¼ Y max (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 were fitted by Y(t) ¼ [Y max + (1 ) Y max )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 the absence (open squares) or presence (filled squares) of crowding agent The refolding was performed in refolding buffer (0.1 M Tris ⁄ Cl, pH 7.5,
1 m M EDTA, 1 m M GSH, 1 m M GSSG, 2 l M cPDI) in the presence or absence of 200 gÆL)1Ficoll 70 At different reaction times, the refolding mixture 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 (0.1 M Tris ⁄ Cl, pH 7.5, 1 m M EDTA, 0.1 m M GSH, 2 l M 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 Y max (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 were fitted by Y(t) ¼ [Y max + (1 ) Y max )e –kt ] · 100%, where Y is the percentage of the linear form, and t is the refolding time.
Trang 8higher than those of glutathione (Table 2) The
con-centrations of GSH and GSSG in the ER lumen are in
the millimolar range, whereas the concentration of
PDI is about 200 lm (about 10-fold lower than the
concentration of glutathione) This work provides
direct evidence that the molar specific oxidase and
isomerase activities of cPDI are much higher (268-fold
and 1500-fold, respectively) than those of glutathione;
hence, the total oxidase and isomerase activities in the
ER lumen should be dominated by cPDI We therefore
propose the hypothesis that PDI plays a key role
dur-ing in vivo folddur-ing of conotoxins
Experimental procedures
Materials
The plasmid encoding mature hPDI was a generous gift
from L W Ruddock (Biocenter, University of Oulu,
Finland) Ni2+-chelating Sepharose Fast Flow resin and
Q-Sepharose Fast Flow resin were obtained from
Amer-sham Biosciences (Arlington Heights, IL, USA) The
RACE kit was obtained from Invitrogen (Carlsbad, CA,
USA) Lysozyme, Micrococcus lysodeikticus and RNase A
were products of Sigma (St Louis, MO, USA) Other
reagents were of analytical grade
Gene cloning of cPDI
The full-length cDNA of cPDI was amplified by RT-PCR
from total RNAs isolated from the venom ducts of
C marmoreus The 3¢-end fragment was amplified using a
3¢-RACE adapter primer and a degenerate primer based
on the conserved amino acid sequence (WCGHCK) of
the thioredoxin-like active site found in other PDIs The
3¢-RACE product was gel-purified, cloned into pGEM-T
easy vector, and sequenced The nested PCR primers for
5¢-RACE amplification were based on the 3¢-end sequence,
and the 5¢-end fragment was amplified using the nested
primer and the 5¢-RACE adapter primer The 5¢-RACE
product was also gel-purified, cloned into pGEM-T easy
vector, and sequenced Primers for amplifying the
full-length cDNA were designed on the basis of these RACE
products The full-length cDNA of the cPDI was inserted
into an expression vector, pET24a, which contains an
N-terminal His6tag
Expression and purification of cPDI and hPDI
The expression plasmid of cPDI was transformed into BL21
(DE3) cells The transformed E coli cells were cultured in
LB medium containing 25 lgÆmL)1 kanamycin at 37C,
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 by sonication After centrifugation (12 000 g, 4C, 20 min; Hitachi Himac CR22G centrifuge, rotor 46), the superna-tant was loaded onto an Ni2+-chelating Sepharose Fast Flow column pre-equilibrated with buffer A The column was extensively washed with buffer A, and then the nonspe-cifically bound proteins were eluted with buffer B (buffer A plus 20 mm imidazole) Finally, the recombinant cPDI was eluted from the column with buffer C (buffer A plus
250 mm imidazole) The eluted cPDI was dialyzed against
20 mm phosphate buffer (pH 7.5) at 4C, and subsequently applied to a Q-Sepharose Fast Flow column pre-equili-brated with 20 mm phosphate buffer (pH 7.5) cPDI was eluted from the ion exchange column using a linear NaCl gradient (0–1 m) The cPDI fraction was collected, analyzed
by SDS⁄ PAGE, dialyzed against distilled water, and stored
at) 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 PDI The 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 phosphate buffer (pH 7.5) containing 8 mm GSH, 30 lm insulin,
120 lm NADPH, 0.5 units of glutathione reductase, 5 mm EDTA, and 0.7 lm PDI The assay mixture (without insu-lin and PDI) was equilibrated at 25C, and the NADPH oxidation rate was recorded against a reference cuvette con-taining NADPH, EDTA and buffer only Subsequently, insulin was added, and a stable nonenzymatic rate was recorded Finally, PDI was added, and the total NADPH oxidation rate was recorded
The oxidase and isomerase activities of PDI were mea-sured using the refolding assay of fully reduced RNase A as previously described [17,23,24] Briefly, it was performed in the assay solution (0.1 m Tris⁄ Cl, pH 8.0, 0.2 mm GSSG,
1 mm GSH, 2 mm EDTA, 4.5 mm cCMP) at 25C After preincubation, the fully reduced RNase A (8.4 lm) and dif-ferent concentrations of PDI (0–10 lm) were added to the assay solution to initiate refolding The formation of active RNase A was measured spectrophotometrically by monitor-ing cCMP hydrolysis at 296 nm Durmonitor-ing the oxidative refolding, the reduced RNase A was quickly converted to inactive oxidized forms by the oxidase activity of PDI, and these inactive oxidized forms were then slowly converted to active native form by the isomerase activity of PDI [23] The lag time before appearance of the active RNase A indicates the oxidase activity, which corresponds to the x-intercept of the RNase activity plot The oxidase activity matches the slope of the linear plot of reciprocal of lag times against the PDI concentrations in units of lm)1Æmin)1 The
Trang 9isomerase activity was determined from the linear increase
of enzymatically active RNase A after the lag time
The chaperone and antichaperone activities of PDI were
analyzed using the renaturation of reduced⁄ denatured
lyso-zyme [13] The lysolyso-zyme activity was measured at 30C by
following the absorbance decrease at 450 nm of the M
lyso-deikticus suspension (0.25 mgÆmL)1 in 67 mm sodium
phosphate buffer, pH 6.2, and 0.1 m NaCl)
Peptide synthesis
Conotoxins tx3a and Tx3.1 [14,15] were chemically
synthe-sized by using the Fmoc method on an ABI 433 A peptide
synthesizer The crude reduced peptides were purified by
C18 reversed-phase HPLC and lyophilized The identity of
each peptide was confirmed by MS The molecular masses
of 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 sTx3.1
Refolding of the linear Tx3.1 was carried out in the
refold-ing buffer (50 mm NH4CO3, 5 mm GSH, 0.5 mm GSSG,
pH 8.0) at 25C for 8 h, and the refolding mixture was
analyzed by RP-HPLC Two major disulfide isomers,
nTx3.1 and sTx3.1, were collected and lyophilized
PDI-catalyzed oxidative folding and isomerization
of conotoxins
The oxidative folding or isomerization of conotoxins was
performed in the refolding buffer (0.1 m Tris⁄ Cl, pH 7.5,
plus appropriate concentrations of GSH, GSSG and cPDI
as indicated in the figure legends) at ambient temperature
(23–25C) The folding reaction was initiated by adding
the peptide stock solution to the final concentration of
20 lm At different reaction times, the folding reaction was
quenched by adding formic acid to the final concentration
of 8% The reaction mixture was immediately analyzed
using C18 analytical RP-HPLC The amounts of linear
form, native form and swap form were calculated from
their integrated elution peak areas, and the PDI’s oxidase
and isomerase activities were expressed as the initial rate of
decrease of linear tx3a and the initial rate of increase of
nTx3.1, respectively To investigate the effect of
macromo-lecular crowding on the PDI-catalyzed oxidative folding or
disulfide isomerization of conotoxins, the folding reaction
was carried out as described above, except that 200 gÆL)1
of Ficoll 70 was added to the refolding buffer
Acknowledgements
The authors wish to acknowledge Professors C C
Wang, D F Cui, Q Y Dai and L W Ruddock for
their generous support for this work This work was supported by the National Basic Research Program of China (2004CB719904)
References
1 Ellgaard L & Ruddock LW (2005) The human protein disulphide isomerase family: substrate interactions and functional properties EMBO Rep 6, 28–32
2 Wilkinson B & Gilbert HF (2004) Protein disulfide isomerase Biochim Biophys Acta 1699, 35–44
3 Kersteen EA & Raines RT (2003) Catalysis of protein folding by protein disulfide isomerase and small-mole-cule mimics Antioxid Redox Signal 5, 413–424
4 Song JL & Wang CC (1995) Chaperone-like activity of protein 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) Efficient oxidative folding of conotoxins and the radiation of venomous 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 precursor sequences and protein disulfide isomerase Biochemistry
35, 15547–15557
7 Craig AG, Bandyopadhyay P & Olivera BM (1999) Post-translationally modified neuropeptides from Conus venoms Eur J Biochem 264, 271–275
8 van den Berg B, Ellis RJ & Dobson CM (1999) Effects
of macromolecular crowding on protein folding and aggregation EMBO J 18, 6927–6933
9 Gething MJ & Sambrook J (1992) Protein folding in the cell Nature 355, 33–45
10 Ellis J (1987) Proteins as molecular chaperones Nature
328, 378–379
11 Garrett JE, Buczek O, Watkins M, Olivera BM & Bulaj
G (2005) Biochemical and gene expression analyses of conotoxins in Conus textile venom ducts Biochem Biophys Res Commun 328, 362–367
12 Nielsen H, Engelbrecht J, Brunak S & von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites Protein Eng 10, 1–6
13 Song J, Quan H & Wang C (1997) Dependence of the anti-chaperone activity of protein disulphide isomerase
on 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 new disulfide 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-conotoxin
Trang 10superfamily: characterization of novel peptides from
mol-luscivorous Conus venoms Biochemistry 44, 8176–8186
16 Lu BY & Chang JY (2005) Assay of disulfide oxidase
and isomerase based on the model of hirudin folding
Anal Biochem 339, 94–103
17 Walker KW, Lyles MM & Gilbert HF (1996) Catalysis
of oxidative protein folding by mutants of protein
disulfide isomerase with a single active-site cysteine
Biochemistry 35, 1972–1980
18 Chang JY, Lu BY & Lai PH (2006) Oxidative folding
of hirudin in human serum Biochem J 394, 249–257
19 Kanai S, Toh H, Hayano T & Kikuchi M (1998)
Molecular evolution of the domain structures of protein
disulfide isomerases J Mol Evol 47, 200–210
20 Fuller E, Green BR, Catlin P, Buczek O, Nielsen JS,
Olivera BM & Bulaj G (2005) Oxidative folding of
conotoxins sharing an identical disulfide bridging
frame-work FEBS J 272, 1727–1738
21 Lappi AK, Lensink MF, Alanen HI, Salo KE, Lobell
M, Juffer AH & Ruddock LW (2004) A conserved arginine plays a role in the catalytic cycle of the protein disulphide isomerases J Mol Biol 335, 283– 295
22 Lambert N & Freedman RB (1983) Kinetics and specificity of homogeneous protein disulphide-isomerase in protein disulphide isomerization and in thiol-protein-disulphide oxidoreduction Biochem J
213, 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 of the redox buffer Biochemistry 30, 613–619
24 Wilkinson B, Xiao R & Gilbert HF (2005) A structural disulfide of yeast protein-disulfide isomerase destabilizes the active site disulfide of the N-terminal thioredoxin domain J Biol Chem 280, 11483–11487