disulfide isomerase by disulfide bond formation with free cysteines in the substrate-binding domain Nina Dickerhof1, Torsten Kleffmann2, Ralph Jack3and Sally McCormick1 1 Department of B
Trang 1disulfide isomerase by disulfide bond formation with free cysteines in the substrate-binding domain
Nina Dickerhof1, Torsten Kleffmann2, Ralph Jack3and Sally McCormick1
1 Department of Biochemistry, University of Otago, Dunedin, New Zealand
2 Centre for Protein Research, University of Otago, Dunedin, New Zealand
3 Seperex Nutritionals, The Centre for Innovation, Dunedin, New Zealand
Introduction
Protein disulfide isomerase (PDI, EC 5.3.4.1) is an
endoplasmic reticulum-resident enzyme in eukaryotic
cells that catalyzes both the oxidation of cysteines to
form disulfide bonds and the reduction and
rearrange-ment of disulfide bonds in proteins, depending on the
redox potential of the cell [1–3] PDI comprises four
structural thioredoxin-like domains, a, b, b¢ and a¢,
and an x-linker region between the b¢-domain and
a¢-domain [4–6] The different activities of PDI are
carried out by different redox states of the catalytic
Cys-Gly-His-Cys motif present in each of the two
a-domains of PDI, which can exist in either the
reduced dithiol or the oxidized disulfide state [7]
The two b-domains are noncatalytic; however, the
b¢-domain displays a large hydrophobic surface, and has been identified as the principal substrate-binding domain of PDI [8,9]
The peptide antibiotic bacitracin was reported to be
an inhibitor of PDI in 1981 [10], and since then has been widely used to demonstrate the role of PDI in cellular processes, including glioma cell invasion [11], melanoma cell death [12], virus entry [13,14], and platelet function [15] In vitro evidence for the specific-ity of bacitracin as a PDI inhibitor, however, is limited, and its mechanism of action remains elusive Karala and Ruddock [16] recently questioned the use
of bacitracin as a specific PDI inhibitor, after they demonstrated a partial effect on the reductive activity
Keywords
bacitracin; cyclic peptide; protein disulfide
isomerase; protein disulfide isomerase (PDI)
inhibition; substrate-binding domain;
thiol–disulfide exchange
Correspondence
S McCormick, Department of Biochemistry,
University of Otago, PO Box 56, Dunedin,
New Zealand
Fax: +64 3 479 7866
Tel: +64 3 479 7840
E-mail: sally.mccormick@otago.ac.nz
(Received 13 January 2011, revised 15
March 2011, accepted 6 April 2011)
doi:10.1111/j.1742-4658.2011.08119.x
The peptide antibiotic bacitracin is widely used as an inhibitor of protein disulfide isomerase (PDI) to demonstrate the role of the protein-folding catalyst in a variety of molecular pathways Commercial bacitracin is a mixture of at least 22 structurally related peptides The inhibitory activity
of individual bacitracin analogs on PDI is unknown For the present study,
we purified the major bacitracin analogs, A, B, H, and F, and tested their ability to inhibit the reductive activity of PDI by use of an insulin aggrega-tion assay All analogs inhibited PDI, but the activity (IC50) ranged from
20 lMfor bacitracin F to 1050 lMfor bacitracin B The mechanism of PDI inhibition by bacitracin is unknown Here, we show, by MALDI-TOF⁄ TOF MS, a direct interaction of bacitracin with PDI, involving disulfide bond formation between an open thiol form of the bacitracin thiazoline ring and cysteines in the substrate-binding domain of PDI
Abbreviations
ACN, acetonitrile; CID, collision-induced dissociation; PDI, protein disulfide isomerase.
Trang 2of PDI, but no effect of bacitracin on PDI-catalyzed
disulfide formation or isomerization
Bacitracin, a dodecapeptide antibiotic produced by
certain strains of Bacillus licheniformis and Bacillus
subtilis, is a mixture of at least 22 structurally related
peptides, which can be separated by RP-HPLC and
characterized by MS [17] The basic structure of
bacitra-cin consists of a cyclic peptide of seven amino acids with
a linear peptide side chain of five amino acids (Fig 1A)
A thiazoline ring is present at the N-terminus of the
pep-tide formed either by l-cysteine and l-isoleucine or by
l-cysteine and l-valine The various analogs result from
substitutions of hydrophobic amino acids within the
peptide sequence and from oxidative transformation of
the thiazoline ring For example, bacitracin A, B1, B2
and B3 are transformed into bacitracin F, H1, H2 and
H3, respectively, by oxidation of the amino-thiazoline
ring to the keto-thiazole ring [18,19] Bacitracin A
makes up approximately 70% of the total mass of
com-mercial bacitracin and, together with bacitracin B1, B2
and B3, accounts for more than 96% of total
antimicro-bial activity in commercial bacitracin products [20] The
analog responsible for the reported inhibitory activity
on PDI is unknown Here, we have purified
bacitra-cin A, B1–3, F and H1–3, and tested their ability to
inhi-bit the reductive activity of PDI Furthermore, we have
investigated the mechanism of action of bacitracin on
PDI, and demonstrated its ability to form disulfide
bonds with cysteines in the substrate-binding domain of
PDI
Results
The separation of commercial bacitracin into
individ-ual analogs was achieved by RP-HPLC (Fig 1B)
Major peaks were collected, pooled and analyzed by
MALDI-TOF⁄ TOF MS to identify isolated bacitracin
analogs (Fig 1C and Fig S1) Bacitracin H and F,
containing the keto-thiazole ring, eluted at a later
retention time than bacitracin A and B, owing to their
increased hydrophobicity The homogeneity of the
iso-lated fractions was subsequently assessed by analytical
RP-HPLC (Fig 1D) The bacitracin B fraction showed
traces of early-eluting compounds, which are likely to
be products formed by breakdown after purification
The ability of each analog to inhibit the reductive
activity of PDI was tested in a turbidometric assay based
on the reduction of insulin by dithiothreitol in the
pres-ence of PDI Upon reduction, insulin forms aggregates,
and the rate of aggregation was followed by turbidity
measurement at 562 nm (Fig 2A–E) The kinetics of
this reaction were biphasic, with an initial lag phase
fol-lowed by an exponential increase in turbidity In each
case, the presence of the bacitracin analog resulted in a longer lag phase and an attenuated increase in turbidity,
in a dose-dependent manner Figure 2F shows a compar-ison of the absorbance reached at 100 min when each analog was present at a concentration of 100 lm Bacitracin F and H were the most effective analogs Dose–response curves were generated to obtain IC50 values for each analog by expressing activity as bance at 100 min as a percentage of the control absor-bance obtained with no inhibitor present (Fig 3) Bacitracin F and H were found to be approximately 25-fold more active as PDI inhibitors than bacitracin A and B (IC50of 20 and 40 lm versus 590 and 1050 lm, respectively) The IC50of the commercial mix was 70 lm The mechanism of action of bacitracin on PDI is unclear In order to determine a direct interaction of bacitracin with PDI, we separated incubations contain-ing bacitracin analogs and PDI by SDS⁄ PAGE Western blot analysis with an anti-PDI serum and an antibody against bacitracin showed that each bacitra-cin analog comigrated with PDI in SDS⁄ PAGE under nonreducing conditions (Fig 4A) This suggests that bacitracin undergoes a robust interaction with PDI that is resistant to the denaturing conditions in SDS⁄ PAGE However, the PDI–bacitracin complex could not be detected under reducing conditions, indi-cating that reducible bond formation is involved in the PDI–bacitracin interaction Furthermore, we showed that the PDI–bacitracin complex could be immunopre-cipitated with an anti-PDI serum (Fig 4B), using pro-tein G beads Several washing steps of the beads with NaCl⁄ Pi containing 0.1% Tween-20 did not diminish the immunoblot signals for bacitracin at the site of PDI (Fig 4C) These data support the view of a robust covalent PDI–bacitracin interaction
We hypothesized that the thiazoline ring of bacitra-cin reacts with cysteines on PDI to form disulfide bonds On the basis of reported disulfide formation resulting from opening of a thiazole ring to a thiol form [21], we proposed a mechanism of ring opening for bacitracin with subsequent disulfide bond forma-tion with free cysteines on PDI (Fig 5A) In order to test this proposed mechanism, we performed in-gel tryptic digestions of the PDI–bacitracin complex, and analyzed the generated peptides by MALDI-TOF⁄ TOF MS Mass spectra were investigated for peaks with a predicted [M + H]+potentially comprising cys-teine-containing PDI peptides crosslinked to the open thiol form of bacitracin Figure 5B shows an example for a predicted crosslink between the Cys345-contain-ing PDI peptide Ile341–Arg347 ([M + H]+: 905.43) and the open thiol form of bacitracin A ([M + H]+: 1440.77) A peak at m⁄ z 2343.09 was detected in the
Trang 3mass spectrum, which matched the predicted
[M + H]+ of 2343.17 for this crosslinked peptide
(Fig 5C) The precursor m⁄ z 2343.09 was selected for
collision-induced dissociation (CID)-TOF⁄ TOF MS
analysis The MS⁄ MS spectrum acquired in positive
ion mode showed a cluster of three peaks at
m⁄ z 871.36, 905.36 and 937.33 (Fig 5D), which are 34 and 32 mass units apart, respectively This peak cluster
is indicative of the Ile341–Arg347 peptide being involved in a disulfide bond The three peaks represent CID-based cleavage events at the cysteine b carbon– sulfur bond, with double bond formation between the
Fig 1 Separation of bacitracin analogs (A) Structures of the most abundant bacitracin analogs of commercial bacitracin mixtures including the amino-thiazoline ring (a) and (b) or keto-thiazole ring (c) and (d) (B) Representative chromatogram for the separation of bacitracin by RP-HPLC, with a gradient of 10–90% ACN over 40 min and detection at 252 nm Bacitracin A and F and bacitracin B and H were purified as pooled fractions of B 1–3 and H 1–3 (C) MALDI-TOF ⁄ TOF MS spectrum of bacitracin F: The most significant fragment ions, their structures and m ⁄ z values that were used to discriminate different bacitracin analogs by MALDI-TOF ⁄ TOF MS are shown (D) The purity of each frac-tion was assessed by analytical RP-HPLC, with similar condifrac-tions as used for the preparative RP-HPLC.
Trang 4a and b carbons (871.36), the sulfur–sulfur bond
(905.36) and the sulfur–carbon bond of the open
thiaz-oline ring of bacitracin A (937.33)
Negative ion mode CID-TOF⁄ TOF MS analysis of
the same precursor yielded the corresponding signature
peaks (m⁄ z 1404.88, 1438.82 and 1470.80) of the other
part of the disulfide bond, i.e the negatively charged
open-ring bacitracin A ion without sulfur, free thiol
and two sulfurs, respectively (Fig 5E)
All three sulfur bonds are prone to cleavage upon
ionization by increased laser energy, which generates a
small proportion of fragments in the ion source The
combination of in-source decay of the precursor
m⁄ z 2343.09 with CID on the in-source fragment
m⁄ z 937.22 in positive ion mode confirmed the location
of the disulfide (Fig 5F) The in-source decay⁄ CID
fragment spectrum yielded unambiguous sequence
information for the Cys345-containing PDI peptide,
with an additional 32 mass units at position 345
(Fig 5F)
In addition to Cys345, Cys314 was also found to be crosslinked to bacitracin (Fig S2)
Discussion
The peptide antibiotic bacitracin is widely used experi-mentally in vivo as a specific PDI inhibitor, although evidence for its specificity is scarce, and the activities
of its different analogs are unknown Our study dis-sected the activities of the various major bacitracin analogs on the reductive activity of PDI, and showed that the H and F analogs are 25-fold more active than the A and B analogs
As Karala and Ruddock [16] could not demonstrate
a significant effect of 1 mm commercial bacitracin on the oxidase and isomerase activities of PDI, we con-centrated on the reductive activity of PDI in this study When Roth [10] originally identified bacitracin
as an inhibitor of PDI, they studied the reduction of insulin by rat liver lysates, and found that 250 lm
Fig 2 Insulin reduction by PDI in the presence or absence of bacitracin analogs (A–E) Insulin (1 mgÆmL)1) was incubated in 100 m M potas-sium phosphate and 1 m M EDTA (pH 7.4) in the absence (uncatalyzed) or presence of 10 lgÆmL)1PDI and increasing amounts of commer-cial bacitracin or individual bacitracin analogs The reaction was initiated with 0.1 m M dithiothreitol (at time 0) (F) Comparison of A at
100 min after incubation with 100 l M of each analog Data are presented as mean ± standard error of independent triplicate experiments.
*P < 0.05 and ***P < 0.001 for comparison with the control.
Trang 5inhibited 90% of the activity Smith et al [22] reported
an IC50value of 152 lm for bacitracin for the
inhibi-tion of PDI reductive activity The IC50 value
deter-mined in our study for the inhibition of PDI reductive
activity by bacitracin was 70 lm The experimental
conditions in all studies varied with respect to
dith-iothreitol, PDI and insulin concentrations, which may
in part account for differences in the IC50 values for
bacitracin-mediated inhibition of PDI reductive
activ-ity Furthermore, on the basis of our results, the
inhib-itory activity of the bacitracin mixture will depend on
the concentrations of individual bacitracin analogs
Indeed, we found that bacitracin sourced from
differ-ent suppliers varies in analog composition
The commercial bacitracin that we used here
consisted of 65% bacitracin A and B as analyzed by
analytical RP-HPLC We therefore expected the IC50
of the commercial bacitracin to be close to the IC50
values for bacitracin A and B (590 and 1050 lm,
respectively) However, the IC50of commercial
bacitra-cin (70 lm) was much lower, and closer to the IC50of
the less abundant bacitracin H and F (40 and 20 lm,
respectively) We believe that, along with bacitracin H
and F, there are other more active but low-abundance
analogs within the commercial bacitracin mix that
con-tribute to the lower than expected IC50 This is evident
from the many minor peaks observed in the HPLC
separation of commercial bacitracin (Fig 1B), and
supported by reports identifying up to 22 different bac-itracin analogs [17] We have purified and tested only the four major analogs here, as purification of the low-abundance analogs would yield insufficient quantities for activity studies
The mechanism of the inhibitory action of bacitracin
on PDI is unclear Bacitracin acts as an antibiotic by forming a complex between divalent cations and the bacterial C55-isoprenyl lipid carrier, ultimately resulting
in the inhibition of cell wall biosynthesis A free amino group adjacent to a thiazoline ring has been shown to
be essential for bacitracin to form a complex with divalent cations [23] Bacitracin A and B fulfill this requirement, making them the active antimicrobial compounds of the bacitracin mix The oxidation of the amino-thiazoline ring to the keto-thiazole ring to form bacitracin H and F results in a loss of metal binding and subsequent antimicrobial activity [20] However,
we show here that the ability to inhibit PDI is higher for bacitracin H and F, which excludes the coordina-tion of metal ions as a potential mechanism of accoordina-tion
on PDI
Fig 3 Dose–response curves of bacitracin analogs PDI activity
was expressed as A562 nm at 100 min as a percentage of the
A 562 nm of the control reaction containing no inhibitor after
subtrac-tion of the A562 nmof the uncatalyzed reaction Data are presented
as mean ± standard error of independent triplicate experiments.
Nonlinear regression analysis revealed IC 50 values, which are given
along with the 95% confidence level range in parentheses.
Fig 4 Bacitracin binding to PDI (A) Incubations of 10 lgÆmL)1PDI and 250 l M bacitracin analog were subjected to separation by SDS ⁄ PAGE, under either reducing or nonreducing conditions, and western blot analysis Blots were probed with both rabbit poly-clonal anti-PDI serum and peroxidase-conjugated anti-rabbit IgG, or with a peroxidase-conjugated sheep anti-bacitracin IgG (B) Incuba-tions of 10 lgÆmL)1 PDI and 250 l M commercial bacitracin were also subjected to immunoprecipitation with a polyclonal anti-PDI serum, using protein G beads Immunoprecipitates were eluted from the beads after three washes with NaCl ⁄ P i containing 0.1% Tween-20, separated by SDS ⁄ PAGE, and subjected to western blot analysis under nonreducing conditions (C) Immunoprecipitates were eluted from the beads after three, four and five washes with NaCl ⁄ P i containing 0.1% Tween-20.
Trang 6To further investigate the mechanism of inhibition
of PDI by bacitracin, we tested for a direct interaction
between PDI and bacitracin We were able to
demon-strate a direct and covalent interaction of each bacitra-cin analog with PDI by colocalization in immunoblot analyses after SDS⁄ PAGE, as well as by
coimmuno-Fig 5 Disulfide bond formation between bacitracin and Cys345 on PDI (A) Scheme of thiol formation of the thiazoline ring of bacitracin A with subsequent formation of a mixed disulfide with PDI (B) Proposed crosslink between the PDI peptide Ile341–Arg347 and bacitracin A through a disulfide bond between Cys345 and the thiol form of bacitracin A The thiol form of the thiazoline ring of bacitracin A and Cys345 are shown as chemical structures, the rest of bacitracin A as ‘Bacitracin A’, and all other amino acids not involved in the crosslink by the sin-gle-letter code The PDI peptide side of the disulfide bond is named R1, and the bacitracin side R2 (C) MALDI-TOF MS spectrum of pep-tides generated by tryptic digestion of the PDI–bacitracin complex containing the crosslinked peptide Ile341–Arg347 ⁄ bacitracin A (arrow) (D) Area of the CID-TOF ⁄ TOF MS spectrum of the precursor ion m ⁄ z 2343.09 acquired in positive ion mode, showing signature peaks for the peptide [M + H] + 905.36 involved in a disulfide bond (E) Area of the CID-TOF ⁄ TOF MS spectrum of the precursor ion m ⁄ z 2341.09 acquired
in negative ion mode, showing signature peaks for the open thiol form of bacitracin A [M – H])1438.82 involved in a disulfide bond (F) In-source decay of the precursor ion m ⁄ z 2343.09 combined with CID on the in-source fragment m ⁄ z 937.33 reveals an additional 32 mass units at Cys345 (arrow), confirming the disulfide bond at this position.
Trang 7precipitation of bacitracin and PDI with an anti-PDI
serum We showed that the interaction was disrupted
under reducing conditions, indicating the involvement
of disulfide bond formation We hypothesized that
bacitracin may react with cysteines on PDI to form a
disulfide bond Using MALDI-TOF⁄ TOF MS
analy-sis, we were able to show that disulfide bond
forma-tion occurs between an open thiol form of bacitracin
and Cys314 and Cys345 of PDI
PDI comprises four domains, a, b, b¢ and a¢, and an
x-linker region between the b¢-domain and a¢-domain
The a-domains are catalytically active, the b¢-domain
functions as a substrate-binding domain displaying a
large hydrophobic surface area, and the x-linker
func-tions to gate access to the b¢-domain [8,9,24] The
cysteines involved in disulfide bond formation
with bacitracin, Cys314 and Cys345, are present in the
b¢-domain and x-linker, respectively The crystal
struc-ture of this region of PDI shows that these cysteines
are free [9] We propose that bacitracin binds to the
hydrophobic surface of the substrate-binding region of
PDI, allowing subsequent disulfide bond formation
with the free cysteines Cys314 and Cys345 The
pres-ence of the covalently bound bacitracin in this region
would impair the binding of the substrate insulin,
ulti-mately inhibiting its reduction Although all bacitracin
analogs seem to interact covalently with PDI (Fig 4),
there is a 25-fold increase in inhibitory activity
between bacitracin A and B and bacitracin F and H,
respectively If we assume that bacitracin interacts with
the hydrophobic surface of the substrate-binding site
prior to the disulfide bond formation with Cys314 or
Cys345, bacitracin H and F, which are more
hydro-phobic than bacitracin A and B, might be more potent
binding partners
Karala and Ruddock [16] tested the effect of
bacitra-cin on the reductive activity of a truncated PDI
con-taining the catalytic a-domain, but lacking the
independent substrate-binding b¢-domain Bacitracin
did not seem to affect the rate of catalysis of the
trun-cated PDI, whereas full-length PDI showed a
signifi-cantly lower rate of catalysis in the presence of
bacitracin These findings can be explained by our
results showing that bacitracin targets the
substrate-binding domain of PDI, but not the catalytic domain
Furthermore, Karala and Ruddock [16] showed no
effect of bacitracin on the oxidase activity of PDI,
which can be carried out by either of the catalytic
domains, a or a¢, with no requirement for the
sub-strate-binding domain [25]
Although bacitracin has been commonly used as a
specific PDI inhibitor, its ability to react with cysteines
is unlikely to be limited to PDI Indeed, we have tested
the binding of bacitracin to various proteins, and found it to bind BSA and IgG (Fig S3) Interestingly,
it did not bind apolipoprotein A1 or pepsin (Fig S3), neither of which has any free cysteines We assume that the same mechanism of bacitracin binding as shown here for PDI applies to other proteins contain-ing free cysteines
In conclusion, we show a mechanism of action of bacitracin on PDI that involves covalent binding of an open thiol form of bacitracin to free cysteines in the substrate-binding domain of PDI However, the inter-action between bacitracin and PDI is nonspecific, and applies to other proteins containing free cysteines
Experimental procedures
Materials
As commercial bacitracin has been reported to contain pro-teases [26], we incubated 5 mm bacitracin from different sources with 1 mg of BSA, and tested for albumin degrada-tion by SDS⁄ PAGE No evidence of degradation was found with bacitracin sourced from Calbiochem (Gibbs-town, NJ, USA), and this was used for subsequent experi-ments PDI from bovine liver was purchased from Sigma (St Louis, MO, USA), porcine monocomponent insulin from Nova Research (Copenhagen, Denmark), dithiothrei-tol from Roche (Mannheim, Germany), peroxidase-conju-gated goat anti-rabbit IgG from Thermo Fisher Scientific (Rockford, IL, USA), and Pure Proteome Protein G Mag-netic Beads from Millipore (Billerica, MA, USA) The anti-bacitracin IgG was purchased from GenWay Biotech (San Diego, CA, USA), and conjugated with peroxidase by use
of a Lightning Link labelling kit (Innova Biosciences, Cam-bridge, UK) Polyclonal rabbit anti-rat PDI serum, which crossreacts with bovine PDI [27], was a generous gift from
M Hubbard (University of Melbourne)
Purification of bacitracin analogs and MALDI-TOF⁄ TOF MS analysis
The nomenclature of Ikai et al [20] for the different baci-tracin analogs was used in this study Bacibaci-tracin A, B1–3, H1–3 and F (referred to as bacitracin A, B, H and F, respectively) were isolated by semipreparative RP-HPLC on C-18 resin, using a Jasco (Great Dunmow, UK) HPLC sys-tem with an LG 2080-02 Ternary Gradient pump, a
DG 2080 Degasser, and an MD 2010-plus detector Baci-tracin was dissolved at a concentration of 50 mgÆmL)1 in 10% acetonitrile (ACN) containing 0.1% trifluoroacetic acid, and filtered through a 0.2 lm filter; 10 mg was then injected for separation on a preparative XTerra MS C-18 column (5 lm, 10· 150 mm; Waters, Milford, MA, USA)
A gradient was run from 10% to 90% ACN containing
Trang 80.1% trifluoroacetic acid over 40 min at a flow rate of
7 mLÆmin)1 to elute the various analogs, which were
moni-tored by absorbance at 252 nm Fractions were
subse-quently analyzed for purity by analytical RP-HPLC, with
the same gradient, at a flow rate of 0.6 mLÆmin)1, on an
analytical XTerra MS C-18 column (3.5 lm, 3· 10 mm)
The structural identity of the isolated analogs was
con-firmed by TOF⁄ TOF MS, with a 4800
MALDI-TOF⁄ TOF Analyzer (AB SCIEX, Framingham, MA,
USA) MALDI-TOF and TOF⁄ TOF MS spectra were
acquired with 800–1000 and 2000–2800 laser shots per
sam-ple spot, respectively For based MS⁄ MS, the 2-kV
opera-tion mode was used, with air as collision gas at a pressure
of 1· 106Torr
Purified fractions were dried by rotary evaporation under
vacuum, dissolved in 80% 2-methyl-propan-2-ol containing
0.05% HCl, and subsequently lyophilized The weight of
the dry bacitracin analog was determined, and it was
dissolved in NaCl⁄ Pifor activity assays
Testing the effect of bacitracin analogs on PDI
reductive activity
PDI activity was measured with an assay that measures the
catalytic reduction of insulin, as described by Holmgren
[28] Insulin (1 mgÆmL)1) was incubated in 100 mm
potas-sium phosphate and 1 mm EDTA (pH 7.4), in the presence
of 10 lgÆmL)1PDI and varying amounts of bacitracin
ana-log at room temperature The reaction was initiated after
10 min by the addition of 0.1 mm dithiothreitol, and the
increase in turbidity was monitored at 562 nm on an
Elx808 Ultra Microplate Reader (Bio-Tex Instruments,
Winooski, VT, USA) over 100 min Dose–response curves
were generated, with expression of PDI activity as
absor-bance at 100 min as a percentage of the absorabsor-bance of the
control reaction containing no inhibitor, after subtraction
of the absorbance of the uncatalyzed reaction containing
no PDI IC50 values were determined by applying a
nonlinear least squares fit of the equation Y =
bot-tom + (top – botbot-tom)⁄ {1 + 10^[(log IC50– X)*Hill slope]}
to activity versus the log of inhibitor concentration, using
graph pad prism Version 5.0 for MacOSX (San Diego,
CA, USA) Although the Hill slope was variable, the
bottom and top values were constrained to 0 and 100,
respectively
Analysis of PDI–bacitracin interaction by
SDS⁄ PAGE and immunoprecipitation
PDI at 10 lgÆmL)1 was incubated with 250 lm bacitracin
analog in a total volume of 100 lL for 30 min at room
temperature The reaction mixtures were separated by 10%
SDS⁄ PAGE under either reducing or nonreducing
condi-tions, and subsequently transferred to nitrocellulose
membrane Blots were probed with both rabbit polyclonal anti-PDI serum and peroxidase-conjugated anti-rabbit IgG
or with peroxidase-conjugated sheep antibody against bacitracin
For immunoprecipitation studies, 5 lL of polyclonal anti-PDI serum was added to the PDI–bacitracin reactions and incubated overnight at 4C The reaction mixture was then added to 50 lL of magnetic protein G beads and incu-bated for 90 min at room temperature The beads were washed three times with NaCl⁄ Picontaining 0.1%
Tween-20, before elution of the complex from the beads by addi-tion of SDS buffer and heating at 90C for 10 min To demonstrate a robust interaction between bacitracin and PDI, the beads were also washed four and five times with NaCl⁄ Pi containing 0.1% Tween-20 before elution The imunoprecipitates were separated by 10% SDS⁄ PAGE under nonreducing conditions, and subjected to western blot analysis as described above
Analysis of PDI–bacitracin interaction by MALDI-TOF⁄ TOF MS
PDI was incubated with commercial bacitracin, and reac-tion mixtures were separated by 10% SDS⁄ PAGE under nonreducing conditions, as described above After staining with Coomassie Blue, the protein band was excised from the SDS polyacrylamide gel and subjected to in-gel diges-tion with trypsin to generate crosslinked peptides Proteins were digested with trypsin at a ratio of 1 lg of protease to
10 lg of protein at 37C for 15 h Tryptic fragments were eluted from the gel matrix and analyzed by MALDI-TOF⁄ TOF MS
The PDI–bacitracin was characterized by a combination
of in-source decay and CID-TOF⁄ TOF MS, based on the method described by Kleffmann et al [29] Briefly, for a selected precursor analysis, spectra were investigated for peaks with a predicted [M + H]+ potentially containing PDI peptides with a cysteine crosslinked to bacitracin Diagnostic crosslinked peptide ions were subjected to MALDI-TOF⁄ TOF MS analysis Spectra were acquired in the 2-kV positive and 1-kV negative ion mode, with 2000–
2800 laser shots per sample spot For unambiguous deter-mination of the amino acids involved in the crosslink for-mation, peptide ions generated by in-source decay of the crosslinked peptide were selected for further fragmentation
by CID
Acknowledgements
We are very grateful to S Huettenhain for the use of his HPLC equipment and for sending bovine PDI We thank S Wilbanks and G Jameson for helping us to interpret data and providing insights into potential inhibitory mechanisms We would also like to thank
Trang 9A von Zychlinksy-Kleffmann and D Petras for their
kind assistance regarding the purification, as well as
M Hubbard for the polyclonal antibody against PDI
This work was supported in part by the National
Heart Foundation, New Zealand (Grant No 1321)
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Supporting information
The following supplementary material is available: Fig S1 Assignment of MALDI-TOF⁄ TOF MS signa-ture peaks for bacitracin analogs
Fig S2 Disulfide bond formation between bacitracin and Cys314 on PDI
Fig S3 Bacitracin binding to different proteins This supplementary material can be found in the online version of this article
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