The hemolytic activity of VVA2 is totally inhibited by VVA1 at a molar ratio of 2 [4,5].. VVA1 completely inhibited the hemolytic and cytotoxic activities of VVA2 at VVA2⁄ VVA1 molar rat
Trang 1on pore-forming cardiotoxic protein volvatoxin A2
by interaction with amphipathic a-helix
Pei-Tzu Wu1, Su-Chang Lin2, Chyong-Ing Hsu1, Yen-Chywan Liaw2and Jung-Yaw Lin1
1 Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan
2 Institute of Molecular Biology Academia Sinica, Taipei, Taiwan
Volvatoxin A (VVA) has been isolated from
Volvari-ella volvacea, and consists of volvatoxin A2 (VVA2)
and volvatoxin A1 (VVA1) [1] VVA has several
biolo-gical activities, such as: (a) lysis of human red blood
cells; (b) swelling tumor cells and the mitochondria
of liver cells; (c) inhibition of protein biosynthesis;
and (d) causing cardiac arrest via activation of the
Ca2+-dependent ATPase enzyme in the ventricular microsomal fraction [1–3] The hemolytic activity of VVA2 is totally inhibited by VVA1 at a molar ratio of
2 [4,5] Previous studies have shown that VVA2 is a b-pore-forming toxin, with a heparin-binding site (HBS) encoded within the C-terminal b-strands (b6, b7 and b8) This HBS structure is indispensable for the
Keywords
amphipathic a-helix; co-pull-down
experiment; tandem repeat protein;
volvatoxin A1; volvatoxin A2
Correspondence
J.-Y Lin, Institute of Biochemistry and
Molecular Biology, College of Medicine,
National Taiwan University, F9, no 1,
Section 1, Jen-Ai Road, Taipei 10051,
Taiwan
Fax: +886 2 23415334
Tel: +886 2 23123456 (ext 8206 ⁄ 8207)
E-mail: jylin@ha.mc.ntu.edu.tw
Database
The nucleotide sequence reported in this
paper has been submitted to the
DDBJ ⁄ EMBL ⁄ GenBank databases under the
accession number AY952461
(Received 22 March 2006, revised 1 May
2006, accepted 17 May 2006)
doi:10.1111/j.1742-4658.2006.05325.x
Volvatoxin A2, a pore-forming cardiotoxic protein, was isolated from the edible mushroom Volvariella volvacea Previous studies have demonstrated that volvatoxin A consists of volvatoxin A2 and volvatoxin A1, and the hemolytic activity of volvatoxin A2 is completely abolished by volvatoxin A1 at a volvatoxin A2⁄ volvatoxin A1 molar ratio of 2 In this study, we investigated the molecular mechanism by which volvatoxin A1 inhibits the cytotoxicity of volvatoxin A2 Volvatoxin A1 by itself was found to be nontoxic, and furthermore, it inhibited the hemolytic and cytotoxic activit-ies of volvatoxin A2 at molar ratios of 2 or lower Interestingly, volvatoxin A1 contains 393 amino acid residues that closely resemble a tandem repeat
of volvatoxin A2 Volvatoxin A1 contains two pairs of amphipathic a-heli-ces but it lacks a heparin-binding site This suggests that volvatoxin A1 may interact with volvatoxin A2 but not with the cell membrane By using confocal microscopy, it was demonstrated that volvatoxin A1 could not bind to the cell membrane; however, volvatoxin A1 could inhibit binding
of volvatoxin A2 to the cell membrane at a molar ratio of 2 Via peptide competition assay and in conjunction with pull-down and co-pull-down experiments, we demonstrated that volvatoxin A1 and volvatoxin A2 may form a complex Our results suggest that this occurs via the interaction of one molecule of volvatoxin A1, which contains two amphipathic a-helices, with two molecules of volvatoxin A2, each of which contains one amphi-pathic a-helix Taken together, the results of this study reveal a novel mechanism by which volvatoxin A1 regulates the cytotoxicity of volvatoxin A2 via direct interaction, and potentially provide an exciting new strategy for chemotherapy
Abbreviations
FITC, fluorescein isothiocyanate; GSH, glutathione; GSP, gene-specific primer; HBS, heparin-binding site; RBC, red blood cell; VVA,
volvatoxin A; VVA1, volvatoxin A1; VVA2, volvatoxin A2; VVA1-CTD, volvatoxin A1 C-terminal domain (198–391 residues); VVA1-NTD, volvatoxin A1 N-terminal domain (1–197 residues).
Trang 2membrane interaction of VVA2 [6] Furthermore, the
VVA2-binding receptor on the cell membrane has been
shown to be a sulfated glycosaminoglycan, as
demon-strated by affinity column chromatography [6] Binding
of VVA2 to the cell membrane induced a protein
con-formational change of the VVA2 amphipathic a-helices
to form a prepore complex [7–10] Therefore, the
amphipathic a-helices play an important role in VVA2
oligomerization and pore formation [6]
Pore-forming toxins are essentially naturally
occur-ring biological weapons produced by both prokaryotes
and eukaryotes, and include well-known toxins such as
diphtheria and anthrax toxins, as well as the less
well-known a-hemolysin and equinatoxin II [11–16] An
important objective is to provide an effective inhibitor
of these virulence factors, and naturally occurring
sub-stances represent one potential source Recently, there
has been much interest in the potential application of
these toxins to chemotherapy and the delivery of drugs
[17,18]
In an attempt to determine the inhibitory
mechan-ism of VVA1 on VVA2, we deduced the amino acid
sequence of VVA1 from the cDNA nucleotide
sequence The primary structure of VVA1 is similar to
that of a tandem repeat form of VVA2, and the
pre-dicted secondary structure showed that it contains
two pairs of amphipathic a-helices VVA1 completely
inhibited the hemolytic and cytotoxic activities of
VVA2 at VVA2⁄ VVA1 molar ratios of 2 or lower
Taken together, our results provide evidence that
VVA1 interacts with VVA2 and regulates the cytotoxic
pore-forming activity of VVA2
Results and Discussion
Characteristics of VVA1 structure
To study the structure of VVA1, we cloned VVA1
cDNA by the RACE method, as described previously
(supplementary Table S1) [19] The coding region of
the cloned VVA1 cDNA contained 1179 nucleotides,
and the deduced amino acid sequence was identical to
that determined previously by protein sequencing
(sup-plementary Fig S1) [5] Interestingly, the amino acid
sequence of VVA1 was very similar to that of a
tan-dem repeat of VVA2 The N-terminal half fragment of
VVA1, designated volvatoxin A1 N-terminal domain
(VVA1-NTD) (1–197 residues), had 46.3% similarity
with VVA2 (Fig 1A), whereas the C-terminal
frag-ment, designated volvatoxin A1 C-terminal domain
(VVA1-CTD) (198–391 residues), displayed 49.2%
similarity to VVA2 (Fig 1A) The similarity between
VVA1-NTD and VVA1-CTD is 42.6% The tertiary
structure of VVA2 shows that it has a pair of amphi-pathic a-helices, denoted a-helix-C and a-helix-D [24], which are essential for VVA2 dimerization [6] Interest-ingly, VVA1 also contains a pair of amphipathic a-helices similar to VVA2 (Fig 1A) (supplementary Fig S2) It has been shown that the amphiphilicity of the amphipathic a-helix of VVA2 is indispensable for protein interaction and oligomerization [6] Secondary structure analysis of VVA1 suggests that there might
be two pairs of amphipathic a-helices in both the N-terminal and C-terminal domains The hydrophobic moments of amphipathic a-helix-C and a-helix-D of VVA1-NTD were calculated to be 0.4 and 0.57, respectively, while those of amphipathic a-helix-D¢ and a-helix-E¢ of VVA1-CTD were 0.49 and 0.57 [25] VVA2 has a basic HBS at its C-terminus that is located within its b-strand, is indispensable for binding
to cell membranes, and has a pI value of 9.6, similar
to that of the snake venom cardiotoxin [6,20–22] Nei-ther VVA1-NTD nor VVA1-CTD has a basic HBS at their C-terminus as VVA2 does Additionally, the pI values of the corresponding C-terminal regions of VVA1-NTD and VVA1-CTD were found to be 4.3 and 4.6, respectively, suggesting that VVA1 has very weak, if any, affinity for the anionic surface of cell membranes [23] Furthermore, we demonstrate here that VVA1 has no noticeable affinity for simple lipid membranes (Fig 1B) When VVA1 was incubated with liposomes and then centrifuged and electrophoresed, analysis of the supernatant and pellet fractions showed that VVA1 cannot bind to these simple membranes (Fig 1B, lanes 3 and 4) Additionally, VVA2 binds liposome-containing membranes, as demonstrated by the exclusive presence of VVA2 in the pellet fraction (Fig 1B, lanes 5 and 6) Intriguingly, the presence of VVA1 inhibited the oligomerization of VVA2 and thus its binding to simple lipid membranes (Fig 1B, lanes 1 and 2) Therefore, this investigation of the structural and binding characteristics of VVA1 indicates that VVA1 may have the capacity for protein–protein inter-action with VVA2 via its amphipathic a-helices, but it
is unlikely that VVA1 would be able to bind to the membrane of cells
Inhibitory effects of VVA1 on the hemolytic and cytotoxic activity of VVA2
The effects of VVA1 on the hemolytic activity of VVA2 were studied by incubation of human red blood cells (RBCs) with the purified proteins VVA1 itself had no hemolytic activity when incubated with human RBCs (Fig 2A, column 2) Strikingly, VVA1 com-pletely abolished the hemolytic activity of VVA2 at
Trang 3B
liposomes
VVA2
+
– –
– – – –
+ S
1 2 3 4 5 6 7 8
P S P S P +
+ VVA1
(MkDa)
(4µg) (4µg)
200
116
97
VVA1
VVA2 S:supernatant
P:pellet
(monomer)
66
45
31
21
Fig 1 Characteristics of volvatoxin A1 (VVA1) structure (A) Alignment of the deduced amino acid sequence of VVA1 N-terminal domain (VVA1-NTD) and VVA1 C-terminal domain (VVA1-CTD) with that of VVA2 (GenBank accession number AY362729) The secondary structural elements of VVA1-NTD predicted by the PROF SEC program are illustrated at the top of the sequence (orange), and those of VVA2 (green) from the X-ray crystallographic analysis are shown below; the arrows represent b-strands, and the rods represent a-helices Secondary struc-tural elements of VVA1-NTD with PROF scores below 5 are shown in light orange The completely conserved residues are shaded in dark green, and similar aligned residues are shaded in pink The residue numbers are indicated on the right The ‘+’ symbol represents the amino acid residues of the heparin-binding site (HBS) of VVA2 (166–194) (B) Inhibitory effects of VVA1 on the binding of VVA2 to liposomes After incubation of VVA1, VVA2 or the mixture of VVA2 and VVA1 at a molar ratio of 2 with liposomes (5 m M ) at 37 C for 30 min, the reaction
mixtures were subjected to centrifugation at 100 000 g at 4C for 1 h The presence of VVA1 or VVA2 in the supernatant and pellet were analyzed by 10% SDS ⁄ PAGE and visualized by Coomassie Blue staining.
Trang 4VVA2⁄ VVA1 molar ratios of 2 or lower (Fig 2A,
col-umns 3–5), while at a molar ratio of 4 the hemolytic
activity of VVA2 was reactivated (Fig 2A, column 6)
To examine whether VVA1 affects the cytotoxicity
of VVA2, HeLa cells were treated with VVA2 (17 nm,
IC50 of VVA2) and various amounts of VVA1 at
37 C for 24 h Similar to the results obtained in
hemolytic experiments, VVA1 itself had no cytotoxicity
(Fig 2B, column 2), but was able to inhibit the
cyto-toxicity of VVA2 completely at a VVA2⁄ VVA1 molar
ratios of 2 or lower (Fig 2B, columns 3–5)
Further-more, the cytotoxicity of VVA2 was reactivated at a
molar ratio of 4 when it was incubated with HeLa cells (Fig 2B, column 6)
Additionally, confocal microscopy was employed to study the inhibitory effects of VVA1 on VVA2 The results showed that VVA1 by itself was unable to bind
to cell membranes (Fig 3, panel FITC-VVA1 and pan-els a–d) Moreover, preincubation of VVA2 and VVA1 (at a molar ratio of 2) inhibited VVA2 binding to the cell membrane (Fig 3, panels e–h) These results strongly suggest that VVA1 inhibits the cytotoxicity of VVA2 by preventing the binding of VVA2 to the cell membrane
Interactions between VVA1 and VVA2
To find whether direct interaction between VVA1 and VVA2 is required for the inhibitory effects of VVA1 on the pore-forming activity of VVA2, pull-down experi-ments were performed As a preliminary experiment, we investigated the effects of different buffer constituents
on the oligomerization of VVA2 Only Triton X-100, and not deoxycholate as had been reported for Bcl-2 family members, was able to induce the oligomerization
of VVA2 (supplementary Fig S3A) [26] Interestingly, not even the harsh, denaturing environment of electro-phoresis through the SDS⁄ PAGE system could affect VVA2 oligomer formation Furthermore, the Triton X-100 induction of oligomerization of VVA2 was able
to mimic the amphipathic environment of an artificial cell membrane of liposomes (supplementary Fig S3B) [26–29] Additionally, incubation of VVA2 without lipo-somes inhibited their oligomerization (supplementary Fig S3B, lane 4) Thus the environment of detergent micelles set up by buffering with Triton X-100 was very similar to the natural environment and was used for further experiments to determine interactions between VVA2 and VVA1
The input controls for the representative pull-down experiment shown in Fig 4A are lanes 11 and 12, where the oligomerization of VVA2 in lane 11 is clearly shown Intriguingly, when VVA2 and VVA1 at
a molar ratio of 2 : 1 were preincubated together and then electrophoresed, no oligomerization of VVA2 was evident (Fig 4A, lane 12) This result seemed to sug-gest that there was indeed some form of interaction between VVA1 and VVA2
Furthermore, when beads linked to VVA2 were incubated with VVA1 and then washed, eluted and run on a 10% SDS⁄ PAGE gel, VVA1 had clearly bound to VVA2 (Fig 4A, lane 1) Furthermore, when
a 2 : 1 molar mixture of VVA2 and VVA1 was incuba-ted with VVA2-linked beads, both proteins were adsorbed, and after elution both proteins were detected
A
B
Fig 2 Effects of volvatoxin A1 (VVA1) on the hemolytic and
cyto-toxic activity of volvatoxin A2 (VVA2) (A) The hemolytic activity of
VVA2 regulated by VVA1 VVA2 (45 n M ) and various concentrations
of VVA1 were preincubated as indicated, and the percentage of
hemolysis was calculated as described in Experimental procedures.
Each value represents the mean ± SD of three independent
experi-ments (B) VVA2 cytotoxicity was affected by VVA1 HeLa cells
were treated with VVA2 (17 n M ) and various amounts of VVA1 at
37 C for 24 h Cell death was assayed by using a trypan blue
exclusion assay [41] Means ± SD are shown for three independent
experiments.
Trang 5via SDS⁄ PAGE analysis (Fig 4A, lane 2)
Interest-ingly, again no oligomers of VVA2 were detected after
incubation of VVA2 with VVA1 at a molar ratio of 2
(Fig 4A, lane 2)
When VVA1 beads were incubated with VVA2,
VVA2 oligomers were adsorbed and eluted (Fig 4A,
lane 4) Additionally, when VVA1 beads were
incuba-ted with the mixture of VVA2 and VVA1 (molar ratio
2 : 1), VVA2 and VVA1 were detected, but again, no
VVA2 oligomer was found (Fig 4A, lane 5) Taken
together, these results strongly support the notion that
there is a direct interaction between VVA1 and VVA2,
and that at a VVA2⁄ VVA1 molar ratio of 2, VVA1 is
able to inhibit the oligomerization of VVA2 Extending
these results, we hypothesized that the inhibition of VVA2 cytotoxic pore formation by VVA1 can only occur at the ideal ratio of 2 : 1 or lower, due to the ability of one molecule of VVA1 to interact with two molecules of VVA2 At a higher ratio of VVA2 to VVA1, the latter is not able to prevent VVA2 oligo-merization and thus cannot inhibit VVA2 cytotoxicity
Number of VVA1-binding sites for VVA2
To further identify the binding characteristics and to investigate the dynamic interaction between VVA1 and VVA2, we carried out co-pull-down experiments The amphipathic a-helix of VVA2 had previously been
h g
f e
Fig 3 Volvatoxin A1 (VVA1) inhibited volvatoxin A2 (VVA2) binding to the cell membrane Binding of VVA2 to the cell membrane was abol-ished by the presence of VVA1 HeLa cells were treated with fluorescein isothiocyanate (FITC)–VVA1, VVA2 and both at a molar ratio of 2,
as described in Experimental procedures VVA1 was conjugated with FITC (green fluorescence, panel FITC–VVA1), and VVA2 was stained with Alexa-568 (red fluorescence, panels a and e), while the nucleus was stained with Hoechst 33258 (blue color, panels b and f) The over-lay of both images is shown in panels c and g The phase-contrast image shows cellular morphology (phase panel) Bar, 40 lm.
Trang 6B
Fig 4 Interaction between volvatoxin A1 (VVA1) and volvatoxin A2 (VVA2) in the presence of Triton X-100 (A) Pull-down experiments VVA1 (45 n M ), VVA2 (45 n M ) or the mixture (VVA2, 45 n M , and VVA1, 22.5 n M ) were incubated with VVA2 beads, VVA1 beads or BSA beads
at 37 C for 30 min in 0.02% Triton X-100 The beads were washed, and the bound proteins were eluted The protein eluents were identi-fied by 10% SDS⁄ PAGE and visualized by silver staining (B) Co-pull-down experiments Linear oligomeric VVA2 (VVA1 beads) was prepared from VVA1 beads, which were treated with VVA2 in 0.02% Triton X-100 buffer, and VVA1 (VVA2 beads) was prepared from VVA2 beads, which were treated with VVA1 in the same buffer as described in Experimental procedures The linear oligomeric VVA2 (VVA1 beads) was then incubated with various amounts of VVA1, while the VVA1 (VVA2 beads) was incubated with various amounts of VVA2 The reaction products were eluted with 0.5% SDS loading buffer, and the proteins in the eluents were analyzed by 10% SDS⁄ PAGE and visualized by silver staining.
Trang 7identified as necessary for the oligomerization of
VVA2 [6] Furthermore, we had determined that
VVA1 encoded two regions that displayed a
reason-ably high degree of conservation to the VVA2
olig-omerization domain Thus, we hypothesized that
VVA1 may contain two binding sites for complex
formation with VVA2 To address this issue,
co-pull-down experiments were carried out First,
VVA1-linked beads (VVA1 beads) were incubated with 45 nm
VVA2 [referred to as linear oligomeric VVA2 (VVA1
beads)] This mixture was then incubated with various
amounts of VVA1 in the presence of 0.02% Triton
X-100, and eluted with 0.5% SDS buffer (Fig 4B)
The results demonstrated that no VVA1 could be
bound to a VVA1 bead that was saturated with VVA2
oligomers (Fig 4B, lanes 1–4), which may imply that
one molecule of VVA2 has one binding site for
interacting with either VVA1 or VVA2 Additional
investigation of the characteristics of binding of VVA2
to VVA1 will be necessary to further understand this
important interaction
Next, we utilized VVA2-linked beads (VVA2 beads)
and incubated them with 4 nm VVA1 protein [referred
to as VVA1 (VVA2) beads] (Fig 4B) VVA1 (VVA2
beads) was incubated with increasing amounts of
VVA2, and visualization on an SDS⁄ PAGE gel showed
that the adsorbed VVA2 had oligomerized As the
amount of VVA2 in the reaction was increased, more
VVA2 was bound to the VVA1 (VVA2 beads) in the
form of oligomers (Fig 4B, lanes 7–10) Collectively,
these data indicate that one molecule of VVA1 has two
binding sites for interaction with two molecules of
VVA2, and that large amounts of free VVA2 can use
VVA1 as a basis for the formation of VVA2 oligomers
Interaction of VVA1 and VVA2 by amphipathic
a-helix
To identify the binding sites in VVA1 responsible for
direct interaction with VVA2, peptide competition
assays, pull-down experiments and western blots were
carried out For the peptide competition assays, the
amphipathic a-helices of VVA1 were generated as
recombinant peptide fragments denoted as
NTD-aH-C-D (amino acids 72–109) and
reVVA1-CTD-aH-D¢-E¢ (amino acids 260–302) (Fig 5A) These
fragments were then used to compete with bead-linked
VVA1 for binding to VVA2 The effectiveness of
com-petition was interpreted via the amount of VVA2
bind-ing to the bead-linked VVA1 after pull-down and
SDS⁄ PAGE electrophoresis The results showed that
the interaction of VVA2 with VVA1 beads was subject
to competition by the N-terminal helix pair
(reVVA1-NTD-aH-C-D) at a reVVA1-NTD-aH-C-D⁄ VVA2 molar ratio of 10 (Fig 5B, lanes 1–3) Interestingly, the peptide fragment containing the C-terminal helix pair (reVVA1-CTD-aH-D¢-E¢) was able to efficiently compete with binding of VVA2 to the bead-linked VVA1 at a reVVA1-CTD-aH-D¢-E¢ ⁄ VVA2 molar ratio
of 2.5 (Fig 5B, lanes 4–6) Furthermore, the reHBSF peptide fragment could not compete with the interac-tion of VVA2 with VVA1 beads (Fig 5B, lanes 7–9) This was an expected result, as the HBS fragment in VVA2 was identified as the membrane-binding domain [6] These results suggest that the N-terminal and the C-terminal pair of a-helices of VVA1 can bind to VVA2 independently of each other and thus enable the direct binding by one molecule of VVA1 of two mole-cules of VVA2 This further complements our previous results suggesting an optimal molar ratio of 2 for bind-ing of VVA2 to VVA1 The anti-VVA2 IgG used in this experiment only detects VVA2 and does not cross-react with VVA1 (supplementary Fig S4)
In the present study, we have shown that VVA1 com-pletely inhibits the biological activity of VVA2 in vitro
at VVA2⁄ VVA1 molar ratios 2 or lower This begs the question of why a mushroom would produce a toxin and at the same time an antidote We believe that the major reason why Volvariella volvacea produces VVA1
is so that it can associate with and, at the right ratio, enhance the toxicity of VVA2 As shown previously, the
LD50 of VVA1 or VVA2 individually is higher than
40 mgÆ(kg body weight))1 At a molar ratio of 2, the
LD50 of VVA2⁄ VVA1 is reduced to 6 mgÆ(kg body weight))1 However, at a molar ratio of 6, which is similar to that in the mushroom, a still lower LD50was evident This intriguing phenomenon requires further investigation [1]
On the basis of the present findings, we propose that one molecule of VVA1 interacts with two molecules of VVA2 and thus inhibits the formation of the mature pore complex Furthermore, we suggest that manipula-tion of the levels of VVA1 may be utilized to inhibit VVA2 oligomerization and pore formation until cer-tain conditions are present to make it biologically valuable For example, it has recently been proposed that native or recombinant pore-forming toxin may be used as a biotherapeutic agent [30–32]
The novel approach of using pore-forming toxins for the treatment of solid tumors, which have proven to be quite resistant to conventional toxins [33–35], shows great promise One of the major drawbacks of using these toxins is that they must be able to preserve the main characteristics of the toxin during the transport process in vivo [32,36] Therefore, a targeted VVA1– VVA2 complex may be introduced to the host as a
Trang 8protoxin, thus having no toxicity to the animal, but with
the ability to target a tumor Once at the appropriate
site, the VVA1 molecule could be dissociated, allowing
the VVA2 molecules to oligomerize and reactivate their
cytotoxic pore-forming activity This has been
demon-strated previously, when a mutated anthrax protoxin
was cleaved by urokinase plasminogen activator and
selectively killed a subset of cancer cells that highly
expressed plasminogen activator [31,37–39] Thus, this
description of a naturally occurring inhibitor of VVA2
represents a significant discovery, although its
import-ance in a clinical setting remains to be investigated
Experimental procedures
Materials
Taq DNA polymerase and the pGEM-T vector were
obtained from Promega (Madison, WI) Restriction
endo-nucleases and T4 DNA ligase were from New England Biolabs Inc (Beverly, MA) The Marathon cDNA amplifi-cation kit was from Clontech (Palo Alto, CA) Fluorescent Alexa-568-labeled goat anti-rabbit and fluorescein isothio-cyanate (FITC) were purchased from Chemicon Inter-national (Temecula, CA) CNBr-activated Sepharose 4B, glutathione (GSH)-agarose-4B column and pGEX-2T vec-tor were from Amersham Biosciences (Uppsala, Sweden) All other chemicals were of analytical grade
Purification, and cDNA cloning of VVA1 VVA1 was purified from mushroom, V volvacea, and the amino acid sequence of VVA1 was determined by protein techniques as reported previously [1,19] The peptides
of VVA1 generated by N-tosyl-l-phenylalanine chloro-methylketone treated-trypsin, Streptococcus aureus V8 endoproteinase or Lys-C endoproteinase digestion were fractionated by HPLC with a C18 reverse-phase column
A
B
Fig 5 Peptide competition assay (A) Schematic representation of peptide competitors (B) Binding of volvatoxin A2 (VVA2) to volvatoxin A1 (VVA1) was inhibited by the amphipathic a-helices of VVA1 The VVA2 and VVA1 mixture (molar ratio 2) was incubated with VVA1 beads; the interaction was examined in the presence of increasing amounts of competitors The adsorbed protein was analyzed by western blots using anti-VVA2 IgG; this indicated that the two amphipathic a-helices competed for the interaction between VVA2 and VVA1 beads.
Trang 9(4.6· 250 mm) that was eluted with a linear gradient of
acetonitrile (0–80%) in 0.1% trifluoroacetic acid The
polypeptides obtained by HPLC were subjected to amino
acid sequence analysis using an ABI 476 A Applied
Biosystems (Foster City, CA) automated amino acid
sequencer [19] The amino acid sequence of VVA1 was
used for the design of degenerate primers and cloning the
cDNA of VVA1 All primers used in this study are
repor-ted in supplementary Table S1
Poly(A+) RNA was isolated from the total RNA
frac-tion on an oligo (dT)-cellulose column, and poly(A+)-rich
mRNA was reverse-transcribed with a Marathon cDNA
amplification kit [19,40] The cDNAs were ligated to
Marathon adaptors for 5¢ and 3¢ rapid amplification of
cDNA ends (RACE), and the products were used as the
template for subsequent PCR In the first PCR, VVA1
cDNA was amplified with the sense degenerate primer A
and the antisense degenerate primer B, corresponding to
amino acid residues 1–6 and 385–391 of VVA1,
respect-ively The amplified first PCR products were used as
tem-plate for nested PCR with the sense degenerate primer A
and the antisense degenerate primer C, which corresponds
to amino acid residues 163–168 of VVA1 The products
of this second PCR were sequenced and used to design
the specific antisense primers GSP-1, corresponding to
amino acid residues 40–47 of VVA1, and GSP-3,
corres-ponding to amino acid residues 24–31 of VVA1 In the
third PCR, GSP-1 was used along with the Marathon
primer AP-1, and the products were used as the template
for the fourth PCR, in which GSP-3 was used along with
the sense primer AP-2 to obtain the 5¢-end of the VVA1
cDNA
The products of the second PCR were used to design
GSP-2 and GSP-4 specific sense primers corresponding to
amino acid residues 128–136 and 155–162 of VVA1,
respectively The two primers were used along with the
Marathon primers AP-1 and AP-2 to yield the 3¢-end of the
VVA1 cDNA
The full-length VVA1 cDNA was obtained by amplifying
V volvacea cDNAs with the sense primer GSP-5, which
encodes the start codon and the first eight N-terminal
amino acid residues of VVA1, and the antisense primer
GSP-6, which encodes the last eight C-terminal amino acid
residues and the stop codon of VVA1 (GenBank accession
number AY952461) The PCR products were then ligated
into the T vector
Liposome-binding assay
Liposomes were prepared as described previously [6]
After incubation of VVA1 (4 lg), VVA2 (4 lg) or the
mixture of VVA2 and VVA1 (at a molar ratio 2) with
liposomes (5 mm) at 37C for 30 min, the reaction
mix-tures were subjected to centrifugation at 100 000 g at
4C for 1 h (Beckman TLA 100.2; Beckman Coulter,
Taipei, Taiwan) Then, the supernatant and pellet were analyzed by 10% SDS⁄ PAGE and visualized by Coomas-sie Blue staining
Hemolytic activity assay Human RBCs were prepared by washing three times with NaCl⁄ Pi (137 mm NaCl, 1.5 mm KH2PO4, 2.7 mm KCl, 8.1 mm Na2HPO4, pH 7.4) [6] VVA2 (45 nm, inducing 50% hemolysis) and various amounts of VVA1 were pre-mixed in NaCl⁄ Pi, and then 0.1 mL of human RBCs (3· 107
cellsÆmL)1) was added The reaction mixtures were further incubated at 37C for 30 min, and the reaction was terminated by centrifuging at 13 000 g for 5 min (KUBOTA RA-155; Kubota, Osaka, Japan) The superna-tant was measured at 540 nm to determine the degree of hemolysis One hundred per cent hemolysis was defined as the same volume of the human red blood cells in the pres-ence of 0.1% Triton X-100 [6]
Cytotoxicity assay HeLa cells were grown in DMEM supplemented with 10% FBS, 2 mm l-glutamine, 100 unitsÆmL)1 penicillin, and
100 lgÆmL)1 streptomycin (Life Technologies, Inc.) under 5% CO2 at 37C HeLa cells (3 · 105) were then treated with mixtures of VVA2 (17 nm, causing 50% cytotoxicity) and various amounts of VVA1 for 24 h The cells were then trypsinized, collected, and treated with 2% trypan blue dye
in NaCl⁄ Pi at 37C for 5 min The surviving cells were counted with a hemocytometer [41,42]
Confocal microscope analysis FITC–VVA1 was prepared by coupling 1.5 mgÆmL)1VVA1 with 40 lgÆmL)1FITC in 0.1 m NaHCO3at 4C for 16 h The free FITC was removed with a Sephadex G25 column, and the effect of FITC–VVA1 on the hemolytic activity of VVA2 was shown to be the same as that of VVA1 HeLa Cells (4· 105) grown on coverslips were treated with FITC-conjugated VVA1 (17 nm), VVA2 (17 nm) or the mixture of VVA2 (17 nm) and VVA1 (8.5 nm) Immunostaining was performed by fixing the cells with 4% paraformaldehyde in
1· NaCl ⁄ Pion coverslips To detect VVA2 protein, nonper-meabilized fixed cells were blocked in blocking buffer (10% FBS in NaCl⁄ Pi) for 30 min The cells were then probed with anti-VVA2 (1 : 1000) at room temperature for 1 h After extensive washing in NaCl⁄ Pi, the washed cells were stained with Alexa-568-conjugated goat anti-rabbit IgG (1 : 1000) for 60 min During the last 15 min of secondary antibody staining, Hoechst 33258 (5 lgÆmL)1) was applied for observa-tion of the nucleus After washing with NaCl⁄ Pi, slides were mounted in mounting solution (80% glycerol in NaCl⁄ Pi), and sealed with nail polish The cells were subjected to
Trang 10immunostaining for observation of the VVA1 and VVA2 as
described previously [41]
Pull-down experiment
VVA2, VVA1 or BSA (8 mg) was coupled to
CNBr-Seph-arose beads (Amersham Pharmacia Biotech) in coupling
buffer (100 mm NaHCO3, pH 8.3, and 500 mm NaCl) and
incubated at 4C overnight Residual active groups were
blocked with 1 m ethanolamine, pH 8.0, at room
tempera-ture for 2 h The beads were then washed four times with
alternating pH buffers Each wash cycle consisted of Tris
buffer (0.1 m Tris, pH 8.0, and 500 mm NaCl) and acid
wash buffer (0.1 m sodium acetate, pH 4.0, and 500 mm
NaCl) [43] Thirty microliters of protein-conjugated beads
was incubated with 45 nm VVA1, VVA2 or a mixture of
VVA2, 45 nm, and VVA1, 22.5 nm, at 37C for 30 min in
0.02% Triton X-100, 50 mm Tris, pH 8.0 The beads were
washed three times with 50 mm Tris buffer and eluted with
0.5% SDS loading buffer (50 mm Tris, pH 8.0, 0.5% SDS,
10% glycerol and 0.03% bromophenol blue) The eluent
was analyzed by 10% SDS⁄ PAGE and visualized by silver
staining
Co-pull-down experiment
For further study of the protein–protein interaction, we
performed a co-pull-down experiment to analyze the
num-ber of binding sites of VVA1 and VVA2 VVA1 (VVA2
beads) was prepared by incubating VVA2 beads with 45 nm
VVA1 at 37C for 30 min Adsorbed proteins were
ana-lyzed as mentioned above After removal of the unbound
VVA1, the VVA1 (VVA2 beads) was incubated with
var-ious amounts of VVA2 in 0.02% Triton X-100 at 37C for
30 min Linear oligomeric VVA2 (VVA1 beads) was
pre-pared by incubating VVA1 beads with 45 nm VVA2 at
37C for 30 min After removal of the unbound VVA2 by
washing with 50 mm Tris buffer, the linear oligomeric
VVA2 (VVA1 beads) was incubated with various amounts
of VVA1 in 0.02% Triton X-100 at 37C for 30 min The
reaction was terminated by centrifugation, and the beads
were washed three times with 50 mm Tris buffer, and eluted
with 0.5% SDS loading buffer The eluents were subjected
to 10% SDS⁄ PAGE analysis
Peptide competition assay
To determine the protein–protein interaction site for VVA1
and VVA2, the amphipathic a-helix regions of VVA1-NTD
(72–109 residues of VVA1) and VVA1-CTD (260–302
resi-dues of VVA1) were PCR amplified (supplementary Table
S1), and then ligated into the pGEX-2T vector for protein
expression The HBS fragment of VVA2 (165–199 residues
of VVA2) was constructed as described previously [6] The
GST fusion proteins were expressed in Escherichia coli and purified by affinity chromatography on a GSH-agarose-4B column, and this was followed by thrombin digestion to obtain pure peptide fragments of VVA1-NTD-aH-C-D, VVA1-CTD-aH-D¢-E¢ or VVA2 (HBS fragment) For the competition assay, the various amounts of peptide compet-itor were added to the mixture of VVA2 and VVA1 (at a molar ratio of 2), and the mixture was incubated with VVA1 beads at 37C for 30 min, and then washed and eluted as described above The eluent was separated by SDS⁄ PAGE and transferred to the polyvinylidene difluo-ride membrane, and western blots were prepared using anti-VVA2 as a standard protocol [6]
Acknowledgements
We would like to thank Professor Ta-Hsiu Liao for his valuable suggestions, and Professor Zee-Fen Chang, Laura Heraty and Dr Brett Hosking for their critical reading of this manuscript This work was sup-ported in part by Grant NSC 89-2320-B-002-238 and Grant NSC 93-2320-B-002-107 from the National Science Council, Republic of China
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