- Email: [email protected]
Purification and biochemical characterization of VesT1s, a novel phospholipase A1 isoform isolated from the venom of the greater banded wasp Vespa tropica
Accepted Manuscript Purification and biochemical characterization of VesT1s, a novel phospholipase A1 isoform isolated from the venom of the greater banded wasp Vespa tropica Prapenpuksiri Rungsa, Steve Peigneur, Sakda Daduang, Jan Tytgat PII:
S0041-0101(18)30127-2
DOI:
10.1016/j.toxicon.2018.03.015
Reference:
TOXCON 5849
To appear in:
Toxicon
Received Date: 8 January 2018 Revised Date:
16 March 2018
Accepted Date: 28 March 2018
Please cite this article as: Rungsa, P., Peigneur, S., Daduang, S., Tytgat, J., Purification and biochemical characterization of VesT1s, a novel phospholipase A1 isoform isolated from the venom of the greater banded wasp Vespa tropica, Toxicon (2018), doi: 10.1016/j.toxicon.2018.03.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Purification and biochemical characterization of VesT1s, a novel phospholipase A1
2
isoform isolated from the venom of the greater banded wasp Vespa tropica
3
Prapenpuksiri Rungsa1,2, Steve Peigneur3, Sakda Daduang1,2*, Jan Tytgat3*
4 5
1
6
Khon Kaen University, Khon Kaen 40002, Thailand
7
2
8
Kaen University, Khon Kaen 40002, Thailand
9
3
Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon
Herestraat 49, 3000 Leuven, Belgium
11
*corresponding author:
[email protected] (J. Tytgat)
*These authors contributed equally.
14
Abstract
M AN U
[email protected] (S. Daduang)
12
15
SC
Toxicology & Pharmacology, University of Leuven (KU Leuven), O&N 2, PO Box 992,
10
13
RI PT
Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI),
Vespa tropica, a social wasp locally found in Thailand is responsible for many out off
17
the record accidental stings due to close encounters with human activities and because of the
18
animal’s highly potent venom. Phospholipase (PLA) is one of the major proteins commonly
19
found in insect venom. In this work, V. tropica phospholipase was successfully isolated,
20
purified and characterized. Three isoforms PLAs have been purified using reversed phase
21
HPLC, and are named VesT1s (VesT1.01a, VesT1.01b and VesT1.02). They are not
22
glycoproteins. VesT1.01s has a molecular weight of 33.72 kDa while for VesT1.02 a mass of
23
34 kDa was found.
EP
TE D
16
The deduced sequence of the mature VesT1.02 protein is composed of 301 amino acid
25
residues (1005 bp), including the catalytic triad (Ser-His-Asp), which is similar to other wasp
26
venom PLAs. The 12 cysteine residues found are conserved among venom PLA1. They form
27
six disulfide bonds, and therefore have no free sulfhydryl groups. Based on homology
28
modelling, VesT1.02 belongs to the α/β hydrolase fold family. Its structure is composed of
29
10 β-sheets and 11 α-helixes, characterized by a β-strand/εSer/α-helix structural motif, which
30
contains the Gly-X-Ser-X-Gly consensus sequence. The shortened lid and shortened β9 loop,
31
which play important roles in substrate selectivity, cause this enzyme to only exhibit PLA
32
activity. Moreover, these PLAs have been shown to be highly thermally stable after heating at
AC C
24
ACCEPTED MANUSCRIPT 33
100 °C for 5 mins. We propose that an inserted Pro residue might be involved in this high
34
thermo-stability.
35
37
Highlights: •
The Vespa tropica phospholipase A1 (VesT1s), one of major allergens is found as three isoforms in the venom.
38
RI PT
36
39
•
The VesT1s are non-glycoproteins.
40
•
The shortened lid and shortened β9 loop, playing important roles in substrate selectivity, cause this enzyme to only exhibit PLA activity.
41
43
•
The VesT1s have been shown to be highly thermally stable. The insertion of a Pro
SC
42
residue might be involved in this thermo-stability.
M AN U
44 45
Key words: greater banded wasp, hornet, wasp, molecular modelling, phospholipase A1,
46
venom, vespid, Vespa tropica
47 48
1. Introduction
Arthropod venom such as bees, wasps, hornets and ants are responsible for causing
50
several allergic reactions in approximately 1% of children and 3% of adults (Golden, 2007;
51
Srisong et al., 2016). The increasing number of insect stings is causing serious problem in
52
tropical regions. Arthropod venoms contain several pharmacologically active molecules, such
53
as proteins and peptides, which are used by the host as venom in order to repel predators, for
54
self-defense, or for protection of their nest (Dias et al., 2015). These stings cause pain,
55
edema, local tissue damage, hemolysis, circulatory failure and immunogenic reactions
56
usually leading to anaphylactic shock (Hou et al., 2016; Yang et al., 2008).
EP
AC C
57
TE D
49
Previous studies have shown important allergic toxins in vespid venom such as
58
hyaluronidase, phospholipase and antigen 5 (An et al., 2012; Caruso et al., 2016; Rungsa et
59
al., 2016b; Sukprasert et al., 2013). Vespid hyaluronidase (Hyase), with a molecular mass of
60
45 kDa, is a glycosylated enzyme that hydrolyses hyaluronic acid (HA), one of the primary
61
components of the extracellular matrix of vertebrates and mainly acts as a “spreading factor”
62
to enhance venom distribution after stinging (Justo Jacomini et al., 2013; Rungsa et al.,
63
2016a). Antigen 5 (Ag 5), a 23 kDa protein, is a non-glycosylated protein identified as an
64
allergic protein that strongly induces an acute hypersensitivity response in human after being
65
stung (Henriksen et al., 2001; Lu et al., 1995). Phospholipase A (PLA1or PLA2), with a
ACCEPTED MANUSCRIPT molecular mass of 10-34 kDa, is a glycosylated or non-glycosylated protein, known as an
67
allergic toxin which primarily is responsible for IgE-mediated allergic reactions (Borodina et
68
al., 2011; dos Santos et al., 2011; Seismann et al., 2010; Sukprasert et al., 2013). PLA1 is an
69
enzyme that hydrolyzes the sn-1 fatty acids from phospholipids resulting in the formation of
70
2-acyl-lysophospholipids. PLA1 is believed to be able to disrupt the phospholipids packing of
71
several kinds of biological membranes, causing severe haemolysis which leads to cardiac
72
dysfunction and hereby is responsible for lethality in animals and human (Chou and Hou,
73
2008; Santos et al., 2007; Yang et al., 2008).
RI PT
66
Eighteen species of wasps belonging to the order of Hymenoptera and genera
75
Vespidae are commonly found in Thailand, especially the Thai banded wasps, Vespa affinis
76
and Vespa tropica. These two wasps are mostly distributed in the forests and are generally
77
considered harmful to humans, but many out off the record stinging accidents in Thailand do
78
occur. Notwithstanding this, in the Asia pacific region several cases of intoxication have been
79
reported after stings by V. affinis. Stings can result in serious symptomology including
80
haemolysis, hepatic dysfunction, oligoanuria and azotaemia (Das and Mukherjee, 2008;
81
Kularatne et al., 2014; Kularatne et al., 2003). V. tropica specimens are bigger than V. affinis
82
with bodies having a length of 3-4 cm. Stinging of these wasps generally induces severe pain,
83
local tissue damage and occasionally results in death in large vertebrates and sometimes
84
humans (Yang et al., 2013). Wasp venom is highly potent and also lethal in insects (PD50 of
85
crude venom approximately 3 µg/g body weight in crickets) and mice (LD50 of approximately
86
2.8 mg/kg) (Rungsa et al., 2016b; Schmidt et al., 1986). A higher phospholipase protein
87
activity is seen for V. tropica venom compared to V. affinis. This study describes the
88
purification, identification, biochemically characterization and homology modelling analysis
89
of PLA from V. tropica venom.
M AN U
TE D
EP
AC C
90
SC
74
91
2. Materials and Methods
92
2.1 Venom collection
93
About 5,000 specimens of V. tropica were collected from Nakorn Pranom Province,
94
Thailand. Fresh venom was collected according to our previously reported method (Rungsa et
95
al., 2016b).
96
2.2 Partial purification of phospholipase
97
The freeze-dried venom (10 mg total venom proteins) was solubilized in 0.1%
98
trifluoroacetic acid (TFA) and fractionated using high performance liquid chromatography
99
(HPLC) on a C18 reversed-phase column (dimensions 4.6 × 250 mm, 5 µm Vydac 218 MS
ACCEPTED MANUSCRIPT C18). The column was equilibrated with two solvents, 0.1% trifluoroacetic acid (TFA) in
101
water (solution A) and 0.085% TFA in CH3CN (solution B). The elution was performed on a
102
linear gradient of solution B from 0% to 80%, with a flow rate of 1 mL/min for 100 min
103
using a Liquid Chromatography system (Gilson, USA). The absorbance was monitored at 214
104
and 280 nm. Individual fractions were collected manually and freeze dried under vacuum
105
(Diego-García et al., 2013).
106
2.3 N-terminal sequence determination
RI PT
100
The N-terminal amino acid sequence of V. tropica PLA was determined by automated
108
Edman degradation using a Shimadzu PPSQ-30 protein sequencer (Peigneur et al., 2013).
109
The obtained sequence was compared with other protein sequences by using BLAST
110
(http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi).
111
2.4 Polyacrylamide gel electrophoresis (PAGE)
SC
107
A one-dimensional polyacrylamide gel electrophoresis was performed following a
113
standard method using 13% (w/v) separating gel and 4% (w/v) stacking gels. The two-
114
dimension polyacrylamide gel electrophoresis (2D-PAGE) was run according to the methods
115
described elsewhere (Incamnoi et al., 2013). The purified protein was separated using 7 cm
116
immobilized dry trips (pH3-11 NL, GE Healthcare). For the first-dimension, the strip was
117
rehydrated for 12 h, after which the isoelectric was focused for a total 9250 voltage-hours
118
using the EttanTM IPGphor system (GE Healthcare Bio-Sciences, Sweden). For the second-
119
dimension a 13% (w/v) separating gel was used, with staining in Coomassie blue G-250.
120
2.5 Protein identification using liquid chromatography
TE D
M AN U
112
The protein bands or spots were excised from the PAGE gel and digested with 20 ng
122
trypsin (Promega, USA) as described previously (Rungsa et al., 2016b). The digested
123
peptides were separated by Nano scale LC separation of tryptic peptides, performed with a
124
NanoAcquity system (Waters Corp., MA) equipped with a Symmetry C18 Trap column and a
125
BEH130 C18 analytical reversed phase column (Waters Corp., MA). All samples were
126
analyzed in triplicate. Analysis of tryptic peptides was performed using a SYNAPT™ HDMS
127
mass spectrometer (Waters Corp., UK). The peptide sequences were submitted to a database
128
search using a local MASCOT sever with the following search parameters: a specified trypsin
129
enzymatic cleavage with one possible missed cleavage, +/−0.6 Da mass tolerances for MS
130
and MS/MS, a peptide tolerance of 1.2 Da, 1+, 2+, 3+ ions, methionine oxidation variable
131
modification, carbamidomethyl (C) fixed modification, monoisotopic mass, and 20 numbers
132
of responses (Uawonggul et al., 2007).
133
2.6 Agar Phospholipase activity
AC C
EP
121
ACCEPTED MANUSCRIPT 134
PLA activity was measured using a lecithin agar assay as described previously
135
(Incamnoi et al., 2013). Briefly, crude venom or purified samples were loaded to 3 mm/wells
136
in an agarose gel containing 1.2% lecithin with 10 mM CaCl2. After 24 h of incubation at 37
137
°C, the plates were observed for the presence of a clear zone to appear in the medium.
138
2.7 Phospholipase A1 activity and thermal stability assay The PLA1 activity was carried out using the EnzCheck® Phospholipase A1 Assay kit
140
(Invitrogen, USA) according to the manufacturer’s protocol. The Leacitase and buffer were
141
used as the positive and negative control. The thermal stability of these enzymes was
142
determined by dilution in 50 mM Tris–HCl (pH 8.0), heated at 100 ˚C for 5 min and kept on
143
ice for 5 min. After cooling down, the samples were tested for PLA1 activity.
144
2.8 Pro-Q® Emerald 300 glycoprotein gel stain
SC
RI PT
139
After separation of the protein by SDS-electrophoresis, the gel was stabilized by a
146
fixing solution and incubated at room temperature for 30 min. These steps were repeated to
147
ensure that SDS was fully removed from the gel. The carbohydrates were oxidized in an
148
oxidizing solution and then washed again prior to staining. The gels were stained with a Pro-
149
Q® Emerald 300 staining solution for 120 min (protected from light) and washed before
150
scanning. The gels were scanned using 300 nm UV transilluminator in Gel Doc™ 2000
151
system (Bio-Rad, USA).
152
2.9 SYPRO® Ruby protein gel stain
TE D
M AN U
145
The gels were immersed in a 10% methanol and 7% acetic acid solution for fixation
154
and they were then stained overnight in SYPRO® Ruby (protected from light) for maximum
155
signal strength. The gels were washed to reduce the fluorescence background and increase
156
sensitivity, and they were scanned using a UV imaging system (optimal with UV
157
transillumination at 300 nm) in a Gel Doc™ 2000 system (Bio-Rad, USA).
158
2.10 Preparation of total RNA and cDNA synthesis
AC C
159
EP
153
Total RNA was extracted from the sting apparatus using the TRIzol® reagent
160
(Invitrogen, USA). RT-PCR was applied according to a protocol of the SMARTer cDNA
161
Library Construction Kit (Clontech Lab Bio, USA). RACE was performed using the In-
162
fusion RACE system of Rapid amplification of cDNA end (Invitrogen, USA) as described in
163
the instruction manual. The gene specific primers and degenerated primers were designed
164
based on the conserved region of vespid phospholipase and the V. tropica N-terminal
165
sequence. The PCR of the cDNA was used to obtain the 3’-end using the specific primers
ACCEPTED MANUSCRIPT 166
(Table 1). The RACE PCR products were cloned into the pGEM® -T easy vector (Promega,
167
USA) for sequencing.
168
2.11 Sequencing analysis and homology modelling The amino acid sequence obtained from the nucleotide sequence was analysed using
170
the basic local alignment search tool BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and
171
compared with other PLA using the ClustalW program (http://www2.ebi.ac.uk/clustalw/).
172
Homology modelling was performed through the SWISS-MODEL program using the
173
automated protein homology modelling template at the ExPASY (Switzerland) and a
174
template search with the Alignment Mode program from the Protein Data Base (PDB;
175
http://swissmodel.expasy.org/) (Arnold et al., 2006; Bordoli et al., 2009). The model was
176
previewed
177
(http://www.expasy.org/spdbv) and Chimera software (https://www.cgl.ucsf.edu/chimera/).
178
To compute pI/MW of V. tropica PLA used tool of ExPASy Bioinformatics tool
179
(http://web.expasy.org/compute_pi/).
180
(http://clavius.bc.edu/clotelab/DiANNA/) was used to predict the formation of disulfide
181
bonds.
analyzed
using
182
Swiss-Pdb
Viewer
Deep
M AN U
and
SC
RI PT
169
The
3. Results
184
3.1 The partial purification and characterization
1.1
v4
web
software
server
TE D
183
DiANNA
View
The soluble venom was fractionated by reverse-phase HPLC and revealed 21
186
fractions as shown in Fig. 1. All fractions were screened for lecithin agar phospholipase
187
activity. The purified fractions P16 and P17 exhibited phospholipase activity on lecithin agar
188
after incubation at 37 ˚C for 24 hr (Fig. 2). Since the peptides were isolated from V. tropica
189
venom, they were designated “VesT 1”. The P16 and P17 were called VesT1.01 and
190
VesT1.02, respectively.
AC C
191
EP
185
Prior to purification, crude venom had been assayed for PLA activity using
192
EnzCheck® Phospholipase A1 Assay kit. The crude venom exhibited 5.54 U/µg protein of
193
specific activity. After purification, fractions P16 and P17 had been checked for their specific
194
activity. They were 185.6 and 214.6 U/µg, respectively. Then, the phospholipase fractions
195
(VesT1.01 and VesT1.02) were subjected to a 13% SDS-PAGE to evaluate the purified
196
products. The VesT1.02 fraction showed a single band under reducing condition, which was
197
approximately 34 kDa (Fig. 3). The peptide sequencing of this fraction, using a Beckman LF
198
300 Protein Sequencer (Palo Alto, CA, USA) revealed the first 14 amino acids of the N-
ACCEPTED MANUSCRIPT 199
terminal sequence (FLPIPYSDMTVKMI). This N-terminal sequence was used to search for
200
homologous
201
(http://www.ncbi.nlh.gov/BLAST/). The homology search indicated that VesT1.02 shares
202
homology with other PLA1 in vespid venom (Table 2).
sequences
in
the
protein
search
engine
tool
BLAST
The VesT1.01 fraction showed two bands under reducing conditions and
204
corresponded to 2 spots at same molecular mass of approximately 33.71 kDa with a slightly
205
different pI, 8.80 and 8.82 respectively (Fig. 3 and Fig. 4). The different and purified bands or
206
spots were trypsinized and subsequently identified using a LC-MS/MS. The peptides were
207
identified by the Protein MASCOT Search Engine using the NCBI Protein Database (Table
208
3). The P16 spots (named VesT1.01a and VesT1.01b) and the band of P17 (VesT1.02)
209
contained peptides which are similar to the PLA in Vespa carbo (P0CH87.1).
210
3.2 Phospholipase activity and the thermal stability assay.
SC
RI PT
203
The quantitative analysis of PLA1 activity was determined using a fluorometric PLA1
212
specific substrate. Five µg/mL of crude venom exhibited an activity of 11.0844 U/mL, as
213
compared with 5 U/mL activity of lecitase (Fig. 5). The results indicate that VesT1.01s (9.28
214
U/mL) and VesT1.02 (10.73 U/ mL) are PLA1s which cleave the sn-1 position. The thermo-
215
stability of V. tropica PLA and VesT1s were investigated. Both the venom and VesT1.01s
216
showed to contain PLA1 activity after exposure at 100 ˚C for 5 min (3.705 U/ mL).
217
Interestingly, VesT1.02 had no PLA1 activity (Fig. 5).
218
3.3 Glycoprotein detection
TE D
M AN U
211
VesT1.01s and VesT1.02 were investigated to detect possible carbohydrates
220
attachments, using the fluorescence probe (Pro-Q Emerald 300). The PLA fractions were
221
separated by SDS-PAGE. The fluorescent substance is assumed to react with the glycoprotein
222
after being oxidized to aldehydes. The VesT1.01s and VesT1.02 did not exhibit any
223
carbohydrates attachment. This gel was post-stained with SYPRO® Ruby solution to evoke
224
total protein (data not shown). The obtained result suggest VesT1s (VesT1.01s and
225
VesT1.02) enzymes to be non-glycoproteins.
226
3.4 cDNA cloning and sequencing analysis
AC C
EP
219
227
The mature sequence encoding VesT1.02 was determined using RT-PCR and 3′ rapid
228
amplification of cDNA ends (3′RACE). The VesT1.02 gene is composed of an open reading
229
frame of 1005 bp and 287 bp of 3′ untranslated region (3′ UTR) (Fig. 6A). VesT1.02 mature
230
protein contains 301 amino acid residues (1005 bp) including stops codon. The sequence of
231
VesT1.02 is rich in the amino acids Lys, Ile, Leu and Gly, with a theoretical pI of 8.80 and a
ACCEPTED MANUSCRIPT 232
predicted molecular mass of 33,243.3 Da. The VesT1.02 amino acid sequence deduced from
233
the nucleotide sequence obtained from RT-PCR was identical to the N-terminal sequence of
234
VesT1.02 (red box, Fig. 6A) and corroborated with the results obtained by LC-MS/MS (blue
235
boxes, Fig. 6A). An amino acid sequence similarity search revealed that the toxin shares similarities
237
with wasp venom PLAs: VesT1.02 contains the conserved catalytic triad (Ser137, Asp165
238
and His230). This sequence is also conserved in members of the pancreatic lipase family
239
(Fig. 8) (Aoki et al., 2007). As expected for wasp venom PLA1, the six disulfide bonds were
240
conserved. These six disulfide bonds are responsible for the structure stabilization of the
241
VesT1.02 protein (red arrows, Fig. 6A). The disulfide bonds are formed as follow Cys87/294,
242
Cys176/245, Cys181/266, Cys219/228, Cys240/246 and Cys267/269 (Fig. 6B). No free
243
sulfhydryl group were detected.
244
3.5 Structure homology modelling and molecular phylogeny analysis
M AN U
SC
RI PT
236
The 3D-structure homology modelling of V. tropica PLA1 (VesT1.02) was created
246
using the SWISS-MODEL program. The X-ray crystal structure of human pancreatic lipase
247
(1n8s.1.A) was used as template for the computational homology modelling (Fig.7B). The
248
VesT1.02 protein presents 29.82 % sequence identity with human pancreatic lipase.
249
Crystallography studies of human pancreatic lipase shows that each lipase is composed of
250
two domains, an N-terminal domain and a C-terminal domain (Roussel et al., 1998). The
251
phospholipase A1 from V. tropica venom (VesT1.02) was identified as a member of the
252
pancreatic lipase family, which shares 19.06 % sequence homology with the N-terminal
253
catalytic domain of human pancreatic lipase. Based on this model, VesT1.02 represents a
254
structure containing 10 β-sheets and 11 α-helixes (Fig. 7A). Comparative analysis of the
255
modelled structures among the sequence of VesT1.02 and human pancreatic lipase, clearly
256
showed that VesT1.02 lacks 24 amino acid residues at the N terminal domain (Fig. 7 and 8).
257
The region containing the α5 helix and its preceding loop (α5 loop) is 10 amino acids longer
258
in VesT1.02 compared to the human pancreatic lipase (1n8s.1.A). However, the C-terminal
259
domain of VesT1.02, which is required for colipase binding is completely missing. The
260
shorter lid domain and β9 loop allows PLA to be more selective for phospholipids (Fig. 7C).
261
The VesT1.02 model indicated the presence of highly conserved N-terminal domains that are
262
essential for catalytic PLA activity (Fig. 8) (Aoki et al., 2007).
263
4. Discussion
AC C
EP
TE D
245
ACCEPTED MANUSCRIPT In general, vespid venom are composed of mainly three bioactive type of compounds:
265
(1) high molecular weight proteins, enzymes and allergens such as phospholipase,
266
hyaluronidase and antigen5 (Abe et al., 2000; Ho and Ko, 1988; Justo Jacomini et al., 2014;
267
Rosenberg et al., 1977; Rungsa et al., 2016a, b; Sukprasert et al., 2013); (2) biologically
268
active amines, including histamine; and (3) small peptides and bioactive molecules, such as
269
mastoparan, kinins and chemolytic peptides (Higashijima et al., 1988; Nakajima et al., 1985;
270
Yang et al., 2013), which, upon envenomation, cause severe pain, local damage, allergic
271
reaction and even death in a very low concentrations. This suggest that these components are
272
potentially interesting for the development of novel pharmaceutical compounds. One such
273
category of components with a potential pharmaceutical application are phospholipases. The
274
wasp venom PLA and PLB are major allergens and they also act as toxins. They are found in
275
the venom of several species from the Asian-Pacific region, Europe and America. The PLA is
276
reported to be one of the most abundant proteins in V.tropica and V.affinis venom (Rungsa et
277
al., 2016b; Sookrung et al., 2014; Sukprasert et al., 2013). PLA1 were classified to be a major
278
allergen of the V.affinis venom. It reacted with IgE in all wasp sera when tested in allergic
279
pateints, indicating 100% allergenic properties in human (Sookrung et al., 2014). Also in the
280
Brazilian social wasp, Polybia paulista, PLA was identified to be the major allergen after
281
being detected in the allergenic response of sensitized patients (Pereez-Riverol et al., 2016).
282
The PLA hydrolyses sn-1 and/or sn-2 acyl group(s) of phospholipids, specifically of
283
membrane phospholipids. This results in membrane damage, severe hemolysis including
284
diverse pharmacological effects like hemorrhage, edema, neurotoxicity, cardiotoxicity,
285
myotoxicity, necrosis, anticoagulant and hypotension (Santos et al., 2007). On the basis here
286
of, PLAs are identified as a lethal factor of the venom (dos Santos et al., 2011; Ho et al.,
287
1998; Ho and Ko, 1988; Liu et al., 2015; Yoon et al., 2015). V. tropica venom is highly
288
potent against several animals. Its venom contains a higher PLA activity but apparently lower
289
protein proportion than PLA of V. affinis (Rungsa et al., 2016b; Schmidt et al., 1986). The
290
purification and characterization reveals at least three isoform of PLAs in V. tropica venom,
291
named VesT1.01a, VesT1.01b and VesT1.02. The PLA activity of VesT1.01s and VesT1.02
292
were determined using the lecithin agar phospholipase activity. The result showed cloudy
293
haloes of degradation of lecithin on the plates. The action of phospholipase results in the
294
formation of a calcium complex with free fatty acids released from phospholipids present in
295
soybean. These complexes will cause cloudy haloes (Chrisope et al., 1976; Incamnoi et al.,
296
2013; Sukprasert et al., 2013). The specific PLA1 activities were investigated using the
AC C
EP
TE D
M AN U
SC
RI PT
264
ACCEPTED MANUSCRIPT 297
fluorometric PLA1 substrate. The activity is comparable with previously described activities
298
(Rungsa et al., 2016b; Sukprasert et al., 2013). VesT1.01a and VesT1.01b are iso-enzymes with the same molecular mass (33.72
300
kDa) and a closely related pI of 8.80 and 8.82. This result was similar to other wasp PLA1s
301
such as those found in the venom of V. affinis (Ves a 1.01; 33.4415 kDa and Ves a 1.02;
302
33.4744 kDa) or V. verutina (vertoxin 2a; 33.360 kDa and 2b; 33.374 kDa) (Ho et al., 1999;
303
Sukprasert et al., 2013). The spots from 2D-PAGE were digested and the proteins were
304
identified using LC-MS/MS. It was shown that several peptide sequences are closely related.
305
VesT1.02 is a PLA1 with a molecular mass of approximately 34 kDa, which corresponds to
306
the experimental mass of PLA from V. tropica venom (Rungsa et al., 2016b). Comparison of
307
the first 14 amino acid residues, obtained by Edman degradation, of VesT1.02 with known
308
vespid phospholipase A1 yielded 79 % identity with proteins of the venoms of V. velutina
309
and V. affinis (Ho et al., 1999; Sukprasert et al., 2013). For VesT1.02 PLA, the presence of a
310
phenylalanine at the first position of N-terminus is quite common in many wasp venom PLAs
311
(Hou et al., 2016). The peptides form LC-MS/MS analysis were similar to many wasp venom
312
PLA1 and corresponded to the VesT1.02 sequences. The six disulphide bonds of VesT1.02
313
are conserved (Chou and Hou, 2008; Ho and Ko, 1988; Soldatova et al., 1993; Sukprasert et
314
al., 2013). The lack of a cysteine at position 4, normally conserved among vespid PLA, may
315
cause less tendency of miss folding and a higher stability of the protein structure. The lower
316
activity of PLA1 from the venom of V. tropica might be due to the free cysteine residue
317
which allows miss-paring of cysteines leading to the unstable conformation of the substrate
318
binding site and catalytic domain.
EP
TE D
M AN U
SC
RI PT
299
Although the PLA from these 2 species (V. tropica and V. affinis) showed high
320
homology (91.69%) in primary structure, the activity of the crude venoms was surprisingly
321
different. A three-fold higher potency was observed for V. tropica venom compared to the V.
322
affinis venom. This three-fold difference in venom potency of V. tropica may follow from the
323
higher hyaluronidase activity in his venom since this hyaluronidase activity might synergize
324
the action of other venom toxins.
AC C
319
325
Sukprasert et al. (2013) reported that V. affinis crude venom showed thermos-stability
326
after heating at 100 °C for 5 min. However, after purification, V. affinis PLA completely lost
327
activity. In this study, V. tropica crude venom and VesT1.01s showed thermo-stability after
328
heating at 100 °C for 5 min. This thermo-stability results from the insertion of proline
329
residues at the mini-lid domain and might protect the enzyme from the high temperature or
ACCEPTED MANUSCRIPT 330
possibly plays an important role in the enzyme-substrate stabilization. These proline residues
331
improved the flexibility of the structure resulting in the high thermo-stability. Furthermore,
332
the absence of possible mismatching in disulfide bond formation may be an additional effect
333
in strengthening the structure stability after heating (Niu et al., 2016). It was suggested that carbohydrate attachment contributes to the biological activity
335
and immunogenicity (Rungsa et al., 2016a). Santos (2011) investigated glycosylation and
336
identified 4 out of 7 isoforms of PLA in Polybia paulista with several having carbohydrates
337
attached to the structure. The VesT1s do not contain (a) carbohydrate(s), similar to what was
338
reported previously (Santos et al., 2007; Sukprasert et al., 2013).
RI PT
334
Since the PLA is normally one of the major venom components, the activity of PLA
340
will affect and govern the overall venom activity. The high PLA activity may result from an
341
efficient folding of its structure (Aoki et al., 2007; Richmond and Smith, 2011; Rungsa et al.,
342
2016b). Via sequence homology and structural modelling, the VesT1.02 N-terminal domain
343
was conserved whereas the C-terminal domain was completely missing. However, the first 24
344
amino acid residues in the N-terminal domain were absent. The β5, β9 and lid loop, essential
345
in the catalysis and the substrate selectivity of triglycerides or phospholipids, were conserved.
346
In the open conformation, the hydrophobic chains of lipids can interact with the clustered
347
hydrophobic residues of the β9 loop and lid domain, whereas the β5 loop is involved in the
348
formation of the oxyanion holes (Carrière et al., 1998).
TE D
M AN U
SC
339
Previous studies have focused on the open forms of the human pancreatic lipase. It is
350
suggested that the β5 loop may adopt a flexible conformation to accommodate the
351
phospholipid molecule. Moreover, replacement of the glutamine residue by a threonine in the
352
β5 loops was predicted to eliminate the salt bridge that stabilises the interaction between the
353
β5 loop and the core of the protein. Therefore, the presence of different residues at this
354
position may cause a different conformation (Carrière et al., 1998; Sukprasert et al., 2013;
355
Withers-Martinez et al., 1996). The α5 helix of VesT1.02 is 7 residues longer than that in
356
HPL and thus may adopt a different loop conformation. The Gln95 allows orientation
357
towards phosphatidylcholine and possibly helps to stabilize substrate binding (Hou et al.,
358
2016; Withers-Martinez et al., 1996). The β9 loop near the active site and shortened lid loop
359
contributes to an increase of the active site accessibility, suggesting that VesT1.01 is only
360
exhibiting PLA activity (Aoki et al., 2007; Carrière et al., 1998; Withers-Martinez et al.,
361
1996).
AC C
EP
349
ACCEPTED MANUSCRIPT 362
In summary, V. tropica crude venom clearly shows high toxicity and allergenicity
363
(Rungsa et al., 2016a, b; Sukprasert et al., 2013). The three isoforms of PLAs in this study
364
were shown to be the major toxin components in the venom. The computational modelling
365
and structure features provide a potential explanation for the high thermo-stability and
366
activity.
368
RI PT
367
5. Acknowledgements
This work was financially supported by the Post-Doctoral Training Program from (1)
370
Research and Technology Transfer Affairs, Khon Kaen University (KKU) and Graduate
371
School, KKU, Thailand (Grant no. 583334), (2) KKU Research Fund, fiscal years
372
2011−2014, (3) Thailand Research Fund (TRF)−Khon Kaen University (KKU) joint funded
373
TRF Basic Research Grant (TRF-BRG), years 2015-2017 (BRG5780014).
SC
369
M AN U
374 375
References
376
Abe, T., Sugita, M., Fujikura, T., Hiyoshi, J., Akasu, M., 2000. Giant hornet (Vespa
377
mandarinia) venomous phospholipases: The purification, characterization and
378
inhibitory properties by biscoclaurine alkaloids. Toxicon 38, 1803−1816. An, S., Chen, L., Wei, J.F., Yang, X., Ma, D., Xu, X., Xu, X., He, S., Lu, J., Lai, R., 2012.
380
Purification and characterization of two new allergens from the venom of Vespa
381
magnifica. PloS one 7, e31920.
TE D
379
Aoki, J., Inoue, A., Makide, K., Saiki, N., Arai, H., 2007. Structure and function of
383
extracellular phospholipase A1 belonging to the pancreatic lipase gene family.
384
Biochimie 89, 197−204.
386 387
Arnold, K., Bordoli, L., Kopp, J., Schwede, T., 2006. The SWISS-MODEL workspace: a
AC C
385
EP
382
web-based environment for protein structure homology modelling. Bioinformatics (Oxford, England) 22, 195−201.
388
Bordoli, L., Kiefer, F., Arnold, K., Benkert, P., Battey, J., Schwede, T., 2009. Protein
389
structure homology modeling using SWISS-MODEL workspace. Nat. Protoc. 4,
390
1−13.
391
Borodina, I., Jensen, B.M., Wagner, T., Hachem, M.A., Søndergaard, I., Poulsen, L.K., 2011.
392
Expression of enzymatically inactive wasp venom phospholipase A1 in Pichia
393
pastoris. PloS one 6, 21267.
ACCEPTED MANUSCRIPT 394
Carrière, F., Withers-Martinez, C., van Tilbeurgh, H., Roussel, A., Cambillau, C., Verger, R.,
395
1998. Structural basis for the substrate selectivity of pancreatic lipases and some
396
related proteins. Biochim. Biophys. Acta 1376, 417−432. Caruso, B., Bonadonna, P., Bovo, C., Melloni, N., Lombardo, C., Senna, G., Lippi, G., 2016.
398
Wasp venom allergy screening with recombinant allergen testing. Diagnostic
399
performance of rPol d 5 and rVes v 5 for differentiating sensitization to Vespula and
400
Polistes subspecies. Clin. Chim. Acta 453, 170−173.
RI PT
397
Chou, C.C., Hou, M.H., 2008. Crystallization and preliminary X-ray diffraction analysis of
402
phospholipase A1 isolated from hornet (Vespa basalis) venom. Acta Crystallogr.
403
Sect. F Struct. Biol. Cryst. Commun. 64, 1118−1120.
405 406 407
Chrisope, G.L., Fox, C.W., Marshall, R.T., 1976. Lecithin agar for detection of microbial phospholipases. Appl. Environ. Microbiol. 31, 784−786.
M AN U
404
SC
401
Das, R.N., Mukherjee, K., 2008. Asian wasp envenomation and acute renal failure: a report of two cases. Mcgill J. Med. 11, 25−28.
408
Dias, N.B., de Souza, B.M., Gomes, P.C., Brigatte, P., Palma, M.S., 2015. Peptidome
409
profiling of venom from the social wasp Polybia paulista. Toxicon 107, Part B,
410
290−303.
Diego-García, E., Peigneur, S., Debaveye, S., Gheldof, E., Tytgat, J., Caliskan, F., 2013.
412
Novel potassium channel blocker venom peptides from Mesobuthus gibbosus
413
(Scorpiones: Buthidae). Toxicon 61, 72−82.
TE D
411
dos Santos, L.D., da Silva Menegasso, A.R., dos Santos Pinto, J.R., Santos, K.S., Castro,
415
F.M., Kalil, J.E., Palma, M.S., 2011. Proteomic characterization of the multiple
416
forms of the PLAs from the venom of the social wasp Polybia paulista. Proteomics
417
11, 1403-1412.
419
AC C
418
EP
414
Golden, D.B.K., 2007. Insect sting anaphylaxis. Immunol. Allergy Clin. North Am. 27, 261−72.
420
Henriksen, A., King, T.P., Mirza, O., Monsalve, R.I., Meno, K., Ipsen, H., Larsen, J.N.,
421
Gajhede, M., Spangfort, M.D., 2001. Major venom allergen of yellow jackets, Ves v
422
5: structural characterization of a pathogenesis-related protein superfamily. Proteins
423
45, 438−448.
424
Higashijima, T., Uzu, S., Nakajima, T., Ross, E.M., 1988. Mastoparan, a peptide toxin from
425
wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G
426
proteins). J. Biol. Chem. 263, 6491−6494.
ACCEPTED MANUSCRIPT 427
Ho, C. L., Chen, W. C., Lin, Y.-L., 1998. Structures and biological activities of new wasp
428
venom peptides isolated from the black-bellied hornet (Vespa basalis) venom.
429
Toxicon 36, 609−617. Ho, C. L., Ko, J. L., 1988. Purification and characterization of a lethal protein with
431
phospholipase A1 activity from the hornet (Vespa basalis) venom. Biochim.
432
Biophys. Acta 963, 414−422.
RI PT
430
433
Ho, C. L., Lin, Y. L., Li, S. F., 1999. Three toxins with phospholipase activity isolated from
434
the yellow-legged hornet (Vespa verutina) venom. Toxicon 37, 1015-1024.
435
Hou, M. H., Chuang, C. Y., Ko, T. P., Hu, N. J., Chou, C. C., Shih, Y. P., Ho, C. L., Wang,
437
A. H. J., 2016. Crystal structure of vespid phospholipase A1 reveals insights into the
438
mechanism for cause of membrane dysfunction. Insect Biochem. Mol. Biol. 68,
439
79−88.
M AN U
SC
436
440
Incamnoi, P., Patramanon, R., Thammasirirak, S., Chaveerach, A., Uawonggul, N.,
441
Sukprasert, S., Rungsa, P., Daduang, J., Daduang, S., 2013. Heteromtoxin (HmTx),
442
a novel heterodimeric phospholipase A2 from Heterometrus laoticus scorpion
443
venom. Toxicon 61, 62−71.
Justo Jacomini, D.L., Campos Pereira, F.D., Aparecido dos Santos Pinto, J.R., dos Santos,
445
L.D., da Silva Neto, A.J., Giratto, D.T., Palma, M.S., de Lima Zollner, R., Brochetto
446
Braga, M.R., 2013. Hyaluronidase from the venom of the social wasp Polybia
447
paulista (Hymenoptera, Vespidae): Cloning, structural modeling, purification, and
448
immunological analysis. Toxicon 64, 70−80.
EP
TE D
444
Justo Jacomini, D.L., Gomes Moreira, S.M., Campos Pereira, F.D., Zollner, R.d.L., Brochetto
450
Braga, M.R., 2014. Reactivity of IgE to the allergen hyaluronidase from Polybia
451
AC C
449
paulista (Hymenoptera, Vespidae) venom. Toxicon 82, 104−111.
452
Kularatne, K., Kannangare, T., Jayasena, A., Jayasekera, A., Waduge, R., Weerakoon, K.,
453
Kularatne, S.A., 2014. Fatal acute pulmonary oedema and acute renal failure
454 455
following multiple wasp/hornet (Vespa affinis) stings in Sri Lanka: two case reports. J. Med. Case Rep. 8, 188.
456
Kularatne, S.A., Gawarammana, I.B., de Silva, P.H., 2003. Severe multi-organ dysfunction
457
following multiple wasp (Vespa affinis) stings. Ceylon Med. J. 48, 146−147.
ACCEPTED MANUSCRIPT 458
Liu, Z., Chen, S., Zhou, Y., Xie, C., Zhu, B., Zhu, H., Liu, S., Wang, W., Chen, H., Ji, Y.,
459
2015. Deciphering the Venomic Transcriptome of Killer-Wasp Vespa velutina. Sci.
460
Rep. 5, 9454. Lu, G., Kochoumian, L., King, T.P., 1995. Sequence identity and antigenic cross-reactivity of
462
white face hornet venom allergen, also a hyaluronidase, with other proteins. J. Biol.
463
Chem. 270, 4457−4465.
RI PT
461
464
Nakajima, T., Yasuhara, T., Uzu, S., Wakamatsu, K., Miyazawa, T., Fukuda, K., Tsukamoto,
465
Y., 1985. Wasp venom peptides; wasp kinins, new cytotrophic peptide families and
466
their physico-chemical properties. Peptides 6, Suppl. 3, 425−430.
Niu, C., Zhu, L., Xu, X., Li, Q., 2016. Rational design of disulfide bonds increases
468
thermostability of a Mesophilic 1,3-1,4-β- Glucanase from Bacillus terquilensis.
469
PLoS ONE 11(4): e0154036.
SC
467
Peigneur, S., Van Der Haegen, A., Möller, C., Waelkens, E., Diego-García, E., Marí, F.,
471
Naudé, R., Tytgat, J., 2013. Unraveling the peptidome of the South African cone
472
snails Conus pictus and Conus natalis. Peptides 41, 8−16.
M AN U
470
Perez-Riverol, A., Campos Pereira, F.D., Musacchio Lasa, A., Romani Fernandes G. L.,
474
Santos-Pinto, J.R., Justo-Jacomini, D.L., Oliveira de Azevedo, G., Bazon M. L.,
475
Palma, M.S., Zollner, R.L., Brochetto-Braga, M.R., 2016. Molecular cloning,
476
expression and IgE-immunoreactivity of phospholipase A1, a major allergen from
477
Polybia paulista (Hymenoptera: Vespidae) venom. Toxicon 124:44-52.
478
Richmond, G.S., Smith, T.K., 2011. Phospholipases A(1). Int. J. Mol. Sci. 12, 588−612.
479
Rosenberg, P., Ishay, J., Gitter, S., 1977. Phospholipases A and B activities of the oriental
482
EP
481
hornet (Vespa Orientalis) venom and venom apparatus. Toxicon 15, 141−155. Roussel, A., Yang, Y., Ferrato, F., Verger, R., Cambillau, C., Lowe, M., 1998. Structure and
AC C
480
TE D
473
activity of rat pancreatic lipase-related protein 2. J. Biol. Chem. 273, 32121−32128.
483
Rungsa, P., Incamnoi, P., Sukprasert, S., Uawonggul, N., Klaynongsruang, S., Daduang, J.,
484
Patramanon, R., Roytrakul, S., Daduang, S., 2016a. Cloning, structural modelling
485 486
and characterization of VesT2s, a wasp venom hyaluronidase (HAase) from Vespa tropica. J. Venom. Anim. Toxins Incl. Trop. Dis. 22, 28.
487
Rungsa, P., Incamnoi, P., Sukprasert, S., Uawonggul, N., Klaynongsruang, S., Daduang, J.,
488
Patramanon, R., Roytrakul, S., Daduang, S., 2016b. Comparative proteomic analysis
489
of two wasps venom, Vespa tropica and Vespa affinis. Toxicon 119, 159−167.
ACCEPTED MANUSCRIPT 490
Santos, L.D., Santos, K.S., de Souza, B.M., Arcuri, H.A., Cunha-Neto, E., Castro, F.M.,
491
Kalil,
J.E.,
Palma,
M.S.,
2007.
Purification,
sequencing
and
structural
492
characterization of the phospholipase A1 from the venom of the social wasp Polybia
493
paulista (Hymenoptera, Vespidae). Toxicon 50, 923−937. Santos, L.D., Silva Menegasso A. R., Santos Pinto, J.R., Santos, K.S., Castro, F.M., Palma,
495
M.S., 2011. Proteomic characterization of the multiple forms of the PLAs from the
496
venom of the social wasp Polybia paulista. Proteomics 11, 1403–1412.
497 498
RI PT
494
Schmidt, J.O., Yamane, S., Matsuura, M., Starr, C.K., 1986. Hornet venoms: Lethalities and lethal capacities. Toxicon 24, 950−954.
Seismann, H., Blank, S., Braren, I., Greunke, K., Cifuentes, L., Grunwald, T., Bredehorst, R.,
500
Ollert, M., Spillner, E., 2010. Dissecting cross-reactivity in hymenoptera venom
501
allergy by circumvention of α-1,3-core fucosylation. Mol. Immunol. 47, 799−808.
SC
499
Soldatova, L., Kochoumian, L., King, T.P., 1993. Sequence similarity of a hornet (D.
503
maculata) venom allergen phospholipase A1 with mammalian lipases. FEBS Lett
504
320, 145-149.
M AN U
502
Sookrung, N., Wong-Din-Dam, S., Tungtrongchitr, A., Reamtong, O., Indrawattana, N.,
506
Sakolvaree, Y., Visitsunthorn, N., Manuyakorn, W., Chaicumpa, W., 2014.
507
Proteome and allergenome of Asian wasp, Vespa affinis, venom and IgE reactivity
508
of the venom components. J. Proteome Res. 13, 1336–1344.
TE D
505
Srisong, H., Daduang, S., Lopata, A.L., 2016. Current advances in ant venom proteins
510
causing hypersensitivity reactions in the Asia-Pacific region. Mol. Immunol. 69,
511
24−32.
EP
509
Sukprasert, S., Rungsa, P., Uawonggul, N., Incamnoi, P., Thammasirirak, S., Daduang, J.,
513
Daduang, S., 2013. Purification and structural characterisation of phospholipase A1
514 515
AC C
512
(Vespapase, Ves a 1) from Thai banded tiger wasp (Vespa affinis) venom. Toxicon 61, 151−164.
516
Uawonggul, N., Thammasirirak, S., Chaveerach, A., Arkaravichien, T., Bunyatratchata, W.,
517
Ruangjirachuporn, W., Jearranaiprepame, P., Nakamura, T., Matsuda, M.,
518
Kobayashi, M., Hattori, S., Daduang, S., 2007. Purification and characterization of
519
Heteroscorpine-1 (HS-1) toxin from Heterometrus laoticus scorpion venom.
520
Toxicon 49, 19−29.
ACCEPTED MANUSCRIPT 521
Withers-Martinez, C., Carrière, F., Verger, R., Bourgeois, D., Cambillau, C., 1996. A
522
pancreatic lipase with a phospholipase A1 activity: crystal structure of a chimeric
523
pancreatic lipase-related protein 2 from guinea pig. Structure 4, 1363−1374.
524
Yang, H., Xu, X., Ma, D., Zhang, K., Lai, R., 2008. A phosholipase A1 platelet activator
526 527
from the wasp venom of Vespa magnifica (Smith). Toxicon 51, 289−296. Yang, X., Wang, Y., Lee, W. H., Zhang, Y., 2013. Antimicrobial peptides from the venom
RI PT
525
gland of the social wasp Vespa tropica. Toxicon 74, 151−157.
Yoon, K.A., Kim, K., Nguyen, P., Seo, J.B., Park, Y.H., Kim, K.G., Seo, H.Y., Koh, Y.H.,
529
Lee, S.H., 2015. Comparative functional venomics of social hornets Vespa crabro
530
and Vespa analis. J. Asia-Pacific Entomol. 18, 815−823.
SC
528
AC C
EP
TE D
M AN U
531
ACCEPTED MANUSCRIPT Figure Legends
533
Fig. 1. Purification profile using HPLC-C18 column. Soluble Vespa tropica venom was
534
loaded on to a C18 reverse phase HPLC column. A linear gradient of 0% solvent A (0.1%
535
trifluoroacetic acid (TFA) in water), and 80% solvent B (0.085% TFA in acetonitrile) were
536
run for 100 min at a flow rate of 1 mL/min. The absorbance was monitored at 214 and 280
537
nm. The purification revealed 21 fractions. Only P16 and P17 exhibited the phospholipase
538
activity on lecithin agar plates.
539
Fig. 2. Phospholipase activity assay on lecithin agar. Vespa tropica crude venom (Venom),
540
phospholipase A1 from Thermomyces lanuginosus (Control), the purified fractions (P16 and
541
P17) (VesT1.01s and VesT1.02, respectively) from RP-HPLC were added in agarose wells
542
containing 1.2% lecithin. After 24 h incubation at 37 °C, the plates were inspected for clear
543
zones of activity.
544
Fig. 3. An electrophoresis profile of the purified fraction containing the Vespa tropica
545
phospholipase. Purified fractions P16 (VesT1.01s) and P17 (VesT1.02) were resolved by
546
13% SDS-PAGE under reducing conditions with Coomassie blue R-250. Lane M was the
547
molecular weight marker in kDa.
548
Fig. 4. A 2D-PAGE profile of the P16 purified fraction. First dimension was isoelectric
549
focusing (pH 3–10, non-linear gradient). Second dimension was 13% SDS-PAGE. The gel
550
was stained with Coomassie brilliant blue G-250 dye solution. Molecular weight marker in
551
kDa is shown on the right panel and the pH gradient is indicated at the top panel.
552
Fig. 5. The specific PLA1 activity assay. The VesT1s (Vespa tropica PLA1s) were assayed
553
using fluorometric substrate (Invitrogen, USA). Five U/mL lecitase was used as positive
554
control. The others substrates were used at a concentration of five µg/mL. For the thermal
555
stability test, VesT1s were exposed to 100 ˚C for 5 mins and then kept at 4 ˚C.
556
Fig. 6. The completed nucleotide sequence and deduced amino acid sequence of Vespa
557
tropica phospholipase (VesT1.02). (A) The complete sequence of VesT1.02. The 3′ UTRs
558
are indicated by small letters. The first 14 amino acid residues obtained from the Edman
559
degradation were show in the red box. Sequences from LC-MS/MS analysis were shown in
560
the blue boxes. The catalytic triad (Ser, Asp and His) is bolded and italicized. The stop codon
561
is indicated with “stop”. The cysteine residues were labelled with triangle marks (). (B)
562
Disulfide linkages of Ves a 1. The 12 cysteine residues that form the following disulfide
563
bridges Cys87/294, Cys176/245, Cys181/266, Cys219/228, Cys240/246 and Cys267/269 are
564
linked by solid lines.
AC C
EP
TE D
M AN U
SC
RI PT
532
ACCEPTED MANUSCRIPT Fig. 7. The homology modelling of Vespa tropica phospholipase A1 (VesT1.02) (A)
566
Secondary structure topology diagram of VesT1.02 by using PDBsum (Laskowski, 2009).
567
(B) Structure analysis of VesT1.02 using homologies modelling (C) Alignments of structural
568
dominants of VesT1.02 with other PLA, focusing only β5, β9 loops and lid domain by
569
ClustalW program (HLP: human pancreatic lipase, rPLRP2: rat pancreatic lipase related
570
protein 2, Dol m1: Dolichovespula maculata PLA1, VesT 1: Vespa tropica PLA1 ).
571
Fig. 8. Sequence alignment of the deduced amino acid sequence of Vespa tropica
572
phospholipase with the other phospholipases. VesT1.02 was aligned with the known lipases
573
and phospholipases: human pancreatic lipase (LIPP_Human), guinea pig phospholipase
574
(LIPR2_CAVPO), human hepatic lipase (PLA1A_Human) and human lipoprotein lipase
575
(LPL_Human). Some allergens from vespid venoms, defined as PLA1, were included in this
576
alignment. They were Ves g 1 (Vespula germanica), Ves v 1 (Vespula vulgaris), Dol m 1.01
577
(Dolichovespula maculata), Pol a 1 (Polistes anularis), Pol d 1 (Polistes dominulus), Pol p 1
578
(Polybia polista) and Ves a 1 (Vespa affinis).
M AN U
SC
RI PT
565
579 580 581
EP AC C
583
TE D
582
ACCEPTED MANUSCRIPT Table 1 Primer used for the amplification of phospholipase gene Reverse Primer
F1 GATTC(C/T)T(A/T/G/C)CCAAT(A/T/C)CCTTAC
R1 AGCAGGATCAAGCCCAATAA
F2 CATGGTTTTACTTCAACTGC
R2 GCAAACGCACTCATTTCTTG
F3 CCCTTGGAACTGTCGATTTC
R3 TGCATTTAATCCAACGCAAA
F4 CGGTTGCGGTCTTCCTATTA
R4 TGCATTTAATCCAACGCAAA
F5 GGCGCACATATTTCAGGTTT
R5 GGCGCACATATTTCAGGTTT
RI PT
Forward Primers
SC
1
AC C
EP
TE D
M AN U
2
ACCEPTED MANUSCRIPT
Table 2. Comparison of the N-terminal sequences of several venom phospholipase. N-terminal sequence
Molecular mass
Reference
Vespa tropica (VesT1.02)
FLPIPYSDDTVKMIa
33 .99
In this study
Vespa affinis
FNPCPYSDDTVKMIITLRENKKHDFb
33.44/33.47
(Sukprasert et al., 2013)
Vespa verutina
FNPCPYSDDTVKMIILTRENKKHDFa
34.982
(Ho et al., 1999)
33.961
(de Souza et al., 2009)
TE D
M AN U
Amino acid sequences from the automated Edman degradation. Deduced amino acid sequences from the cDNA template.
EP
b
AC C
a
SC
Polybia paulista LIPECPFNEYDILFFVYTRQQRDa
RI PT
Source
ACCEPTED MANUSCRIPT
Table 3. Proteomic identification of the Phospholipase in V. tropica; the purified phospholipase were excised from the gels (SDS-PAGE and 2D-PAGE).
VesT1.01a Vespa crabro
Accession Protein Theorical code score MW/pI P0CH87.1 435 34104/8.91
K.VQELGLGK.Y K.KVQELGLGK.Y K.YNVPMANIR.L R.LVGNYIATVTK.M K.TGSFYVPVESK.A R.NECVCVGLNAK.T K.HECCLIGVPKSK.N -.FNPCPYSDDTVK.M R.LIGHSLGAHISGFAGK.K K.KVQELGLGK.Y K.YNVPMANIR.L R.LVGNYIATVTK.M K.TGSFYVPVESK.A R.NECVCVGLNAK.T K.HECCLIGVPKSK.N -.FNPCPYSDDTVK.M R.LIGHSLGAHISGFAGK.K K.KVQELGLGK.Y K.YNVPMANIR.L R.LVGNYIATVTK.M K.TGSFYVPVESK.A R.NECVCVGLNAK.T K.HECCLIGVPKSK.N -.FNPCPYSDDTVK.M
P0CH87.1 284
VesT1.02
P0CH87.1 259
34104/8.91
AC C
EP
TE D
M AN U
VesT1.01b Vespa crabro
Vespa crabro
Peptides sequences
RI PT
Protein
SC
Spots
34104/8.91
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Highlights: •
The Vespa tropica phospholipase A1 (VesT1s), one of major allergens is found as three isoforms in the venom.
•
The VesT1s are non-glycoproteins.
•
The shortened lid and shortened β9 loop, playing important roles in substrate
The VesT1s have been shown to be highly thermally stable. The insertion of a Pro
EP
TE D
M AN U
SC
residue might be involved in this thermo-stability.
AC C
•
RI PT
selectivity, cause this enzyme to only exhibit PLA activity.
S0041-0101(18)30127-2
DOI:
10.1016/j.toxicon.2018.03.015
Reference:
TOXCON 5849
To appear in:
Toxicon
Received Date: 8 January 2018 Revised Date:
16 March 2018
Accepted Date: 28 March 2018
Please cite this article as: Rungsa, P., Peigneur, S., Daduang, S., Tytgat, J., Purification and biochemical characterization of VesT1s, a novel phospholipase A1 isoform isolated from the venom of the greater banded wasp Vespa tropica, Toxicon (2018), doi: 10.1016/j.toxicon.2018.03.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Purification and biochemical characterization of VesT1s, a novel phospholipase A1
2
isoform isolated from the venom of the greater banded wasp Vespa tropica
3
Prapenpuksiri Rungsa1,2, Steve Peigneur3, Sakda Daduang1,2*, Jan Tytgat3*
4 5
1
6
Khon Kaen University, Khon Kaen 40002, Thailand
7
2
8
Kaen University, Khon Kaen 40002, Thailand
9
3
Division of Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon
Herestraat 49, 3000 Leuven, Belgium
11
*corresponding author:
[email protected] (J. Tytgat)
*These authors contributed equally.
14
Abstract
M AN U
[email protected] (S. Daduang)
12
15
SC
Toxicology & Pharmacology, University of Leuven (KU Leuven), O&N 2, PO Box 992,
10
13
RI PT
Protein and Proteomics Research Center for Commercial and Industrial Purposes (ProCCI),
Vespa tropica, a social wasp locally found in Thailand is responsible for many out off
17
the record accidental stings due to close encounters with human activities and because of the
18
animal’s highly potent venom. Phospholipase (PLA) is one of the major proteins commonly
19
found in insect venom. In this work, V. tropica phospholipase was successfully isolated,
20
purified and characterized. Three isoforms PLAs have been purified using reversed phase
21
HPLC, and are named VesT1s (VesT1.01a, VesT1.01b and VesT1.02). They are not
22
glycoproteins. VesT1.01s has a molecular weight of 33.72 kDa while for VesT1.02 a mass of
23
34 kDa was found.
EP
TE D
16
The deduced sequence of the mature VesT1.02 protein is composed of 301 amino acid
25
residues (1005 bp), including the catalytic triad (Ser-His-Asp), which is similar to other wasp
26
venom PLAs. The 12 cysteine residues found are conserved among venom PLA1. They form
27
six disulfide bonds, and therefore have no free sulfhydryl groups. Based on homology
28
modelling, VesT1.02 belongs to the α/β hydrolase fold family. Its structure is composed of
29
10 β-sheets and 11 α-helixes, characterized by a β-strand/εSer/α-helix structural motif, which
30
contains the Gly-X-Ser-X-Gly consensus sequence. The shortened lid and shortened β9 loop,
31
which play important roles in substrate selectivity, cause this enzyme to only exhibit PLA
32
activity. Moreover, these PLAs have been shown to be highly thermally stable after heating at
AC C
24
ACCEPTED MANUSCRIPT 33
100 °C for 5 mins. We propose that an inserted Pro residue might be involved in this high
34
thermo-stability.
35
37
Highlights: •
The Vespa tropica phospholipase A1 (VesT1s), one of major allergens is found as three isoforms in the venom.
38
RI PT
36
39
•
The VesT1s are non-glycoproteins.
40
•
The shortened lid and shortened β9 loop, playing important roles in substrate selectivity, cause this enzyme to only exhibit PLA activity.
41
43
•
The VesT1s have been shown to be highly thermally stable. The insertion of a Pro
SC
42
residue might be involved in this thermo-stability.
M AN U
44 45
Key words: greater banded wasp, hornet, wasp, molecular modelling, phospholipase A1,
46
venom, vespid, Vespa tropica
47 48
1. Introduction
Arthropod venom such as bees, wasps, hornets and ants are responsible for causing
50
several allergic reactions in approximately 1% of children and 3% of adults (Golden, 2007;
51
Srisong et al., 2016). The increasing number of insect stings is causing serious problem in
52
tropical regions. Arthropod venoms contain several pharmacologically active molecules, such
53
as proteins and peptides, which are used by the host as venom in order to repel predators, for
54
self-defense, or for protection of their nest (Dias et al., 2015). These stings cause pain,
55
edema, local tissue damage, hemolysis, circulatory failure and immunogenic reactions
56
usually leading to anaphylactic shock (Hou et al., 2016; Yang et al., 2008).
EP
AC C
57
TE D
49
Previous studies have shown important allergic toxins in vespid venom such as
58
hyaluronidase, phospholipase and antigen 5 (An et al., 2012; Caruso et al., 2016; Rungsa et
59
al., 2016b; Sukprasert et al., 2013). Vespid hyaluronidase (Hyase), with a molecular mass of
60
45 kDa, is a glycosylated enzyme that hydrolyses hyaluronic acid (HA), one of the primary
61
components of the extracellular matrix of vertebrates and mainly acts as a “spreading factor”
62
to enhance venom distribution after stinging (Justo Jacomini et al., 2013; Rungsa et al.,
63
2016a). Antigen 5 (Ag 5), a 23 kDa protein, is a non-glycosylated protein identified as an
64
allergic protein that strongly induces an acute hypersensitivity response in human after being
65
stung (Henriksen et al., 2001; Lu et al., 1995). Phospholipase A (PLA1or PLA2), with a
ACCEPTED MANUSCRIPT molecular mass of 10-34 kDa, is a glycosylated or non-glycosylated protein, known as an
67
allergic toxin which primarily is responsible for IgE-mediated allergic reactions (Borodina et
68
al., 2011; dos Santos et al., 2011; Seismann et al., 2010; Sukprasert et al., 2013). PLA1 is an
69
enzyme that hydrolyzes the sn-1 fatty acids from phospholipids resulting in the formation of
70
2-acyl-lysophospholipids. PLA1 is believed to be able to disrupt the phospholipids packing of
71
several kinds of biological membranes, causing severe haemolysis which leads to cardiac
72
dysfunction and hereby is responsible for lethality in animals and human (Chou and Hou,
73
2008; Santos et al., 2007; Yang et al., 2008).
RI PT
66
Eighteen species of wasps belonging to the order of Hymenoptera and genera
75
Vespidae are commonly found in Thailand, especially the Thai banded wasps, Vespa affinis
76
and Vespa tropica. These two wasps are mostly distributed in the forests and are generally
77
considered harmful to humans, but many out off the record stinging accidents in Thailand do
78
occur. Notwithstanding this, in the Asia pacific region several cases of intoxication have been
79
reported after stings by V. affinis. Stings can result in serious symptomology including
80
haemolysis, hepatic dysfunction, oligoanuria and azotaemia (Das and Mukherjee, 2008;
81
Kularatne et al., 2014; Kularatne et al., 2003). V. tropica specimens are bigger than V. affinis
82
with bodies having a length of 3-4 cm. Stinging of these wasps generally induces severe pain,
83
local tissue damage and occasionally results in death in large vertebrates and sometimes
84
humans (Yang et al., 2013). Wasp venom is highly potent and also lethal in insects (PD50 of
85
crude venom approximately 3 µg/g body weight in crickets) and mice (LD50 of approximately
86
2.8 mg/kg) (Rungsa et al., 2016b; Schmidt et al., 1986). A higher phospholipase protein
87
activity is seen for V. tropica venom compared to V. affinis. This study describes the
88
purification, identification, biochemically characterization and homology modelling analysis
89
of PLA from V. tropica venom.
M AN U
TE D
EP
AC C
90
SC
74
91
2. Materials and Methods
92
2.1 Venom collection
93
About 5,000 specimens of V. tropica were collected from Nakorn Pranom Province,
94
Thailand. Fresh venom was collected according to our previously reported method (Rungsa et
95
al., 2016b).
96
2.2 Partial purification of phospholipase
97
The freeze-dried venom (10 mg total venom proteins) was solubilized in 0.1%
98
trifluoroacetic acid (TFA) and fractionated using high performance liquid chromatography
99
(HPLC) on a C18 reversed-phase column (dimensions 4.6 × 250 mm, 5 µm Vydac 218 MS
ACCEPTED MANUSCRIPT C18). The column was equilibrated with two solvents, 0.1% trifluoroacetic acid (TFA) in
101
water (solution A) and 0.085% TFA in CH3CN (solution B). The elution was performed on a
102
linear gradient of solution B from 0% to 80%, with a flow rate of 1 mL/min for 100 min
103
using a Liquid Chromatography system (Gilson, USA). The absorbance was monitored at 214
104
and 280 nm. Individual fractions were collected manually and freeze dried under vacuum
105
(Diego-García et al., 2013).
106
2.3 N-terminal sequence determination
RI PT
100
The N-terminal amino acid sequence of V. tropica PLA was determined by automated
108
Edman degradation using a Shimadzu PPSQ-30 protein sequencer (Peigneur et al., 2013).
109
The obtained sequence was compared with other protein sequences by using BLAST
110
(http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi).
111
2.4 Polyacrylamide gel electrophoresis (PAGE)
SC
107
A one-dimensional polyacrylamide gel electrophoresis was performed following a
113
standard method using 13% (w/v) separating gel and 4% (w/v) stacking gels. The two-
114
dimension polyacrylamide gel electrophoresis (2D-PAGE) was run according to the methods
115
described elsewhere (Incamnoi et al., 2013). The purified protein was separated using 7 cm
116
immobilized dry trips (pH3-11 NL, GE Healthcare). For the first-dimension, the strip was
117
rehydrated for 12 h, after which the isoelectric was focused for a total 9250 voltage-hours
118
using the EttanTM IPGphor system (GE Healthcare Bio-Sciences, Sweden). For the second-
119
dimension a 13% (w/v) separating gel was used, with staining in Coomassie blue G-250.
120
2.5 Protein identification using liquid chromatography
TE D
M AN U
112
The protein bands or spots were excised from the PAGE gel and digested with 20 ng
122
trypsin (Promega, USA) as described previously (Rungsa et al., 2016b). The digested
123
peptides were separated by Nano scale LC separation of tryptic peptides, performed with a
124
NanoAcquity system (Waters Corp., MA) equipped with a Symmetry C18 Trap column and a
125
BEH130 C18 analytical reversed phase column (Waters Corp., MA). All samples were
126
analyzed in triplicate. Analysis of tryptic peptides was performed using a SYNAPT™ HDMS
127
mass spectrometer (Waters Corp., UK). The peptide sequences were submitted to a database
128
search using a local MASCOT sever with the following search parameters: a specified trypsin
129
enzymatic cleavage with one possible missed cleavage, +/−0.6 Da mass tolerances for MS
130
and MS/MS, a peptide tolerance of 1.2 Da, 1+, 2+, 3+ ions, methionine oxidation variable
131
modification, carbamidomethyl (C) fixed modification, monoisotopic mass, and 20 numbers
132
of responses (Uawonggul et al., 2007).
133
2.6 Agar Phospholipase activity
AC C
EP
121
ACCEPTED MANUSCRIPT 134
PLA activity was measured using a lecithin agar assay as described previously
135
(Incamnoi et al., 2013). Briefly, crude venom or purified samples were loaded to 3 mm/wells
136
in an agarose gel containing 1.2% lecithin with 10 mM CaCl2. After 24 h of incubation at 37
137
°C, the plates were observed for the presence of a clear zone to appear in the medium.
138
2.7 Phospholipase A1 activity and thermal stability assay The PLA1 activity was carried out using the EnzCheck® Phospholipase A1 Assay kit
140
(Invitrogen, USA) according to the manufacturer’s protocol. The Leacitase and buffer were
141
used as the positive and negative control. The thermal stability of these enzymes was
142
determined by dilution in 50 mM Tris–HCl (pH 8.0), heated at 100 ˚C for 5 min and kept on
143
ice for 5 min. After cooling down, the samples were tested for PLA1 activity.
144
2.8 Pro-Q® Emerald 300 glycoprotein gel stain
SC
RI PT
139
After separation of the protein by SDS-electrophoresis, the gel was stabilized by a
146
fixing solution and incubated at room temperature for 30 min. These steps were repeated to
147
ensure that SDS was fully removed from the gel. The carbohydrates were oxidized in an
148
oxidizing solution and then washed again prior to staining. The gels were stained with a Pro-
149
Q® Emerald 300 staining solution for 120 min (protected from light) and washed before
150
scanning. The gels were scanned using 300 nm UV transilluminator in Gel Doc™ 2000
151
system (Bio-Rad, USA).
152
2.9 SYPRO® Ruby protein gel stain
TE D
M AN U
145
The gels were immersed in a 10% methanol and 7% acetic acid solution for fixation
154
and they were then stained overnight in SYPRO® Ruby (protected from light) for maximum
155
signal strength. The gels were washed to reduce the fluorescence background and increase
156
sensitivity, and they were scanned using a UV imaging system (optimal with UV
157
transillumination at 300 nm) in a Gel Doc™ 2000 system (Bio-Rad, USA).
158
2.10 Preparation of total RNA and cDNA synthesis
AC C
159
EP
153
Total RNA was extracted from the sting apparatus using the TRIzol® reagent
160
(Invitrogen, USA). RT-PCR was applied according to a protocol of the SMARTer cDNA
161
Library Construction Kit (Clontech Lab Bio, USA). RACE was performed using the In-
162
fusion RACE system of Rapid amplification of cDNA end (Invitrogen, USA) as described in
163
the instruction manual. The gene specific primers and degenerated primers were designed
164
based on the conserved region of vespid phospholipase and the V. tropica N-terminal
165
sequence. The PCR of the cDNA was used to obtain the 3’-end using the specific primers
ACCEPTED MANUSCRIPT 166
(Table 1). The RACE PCR products were cloned into the pGEM® -T easy vector (Promega,
167
USA) for sequencing.
168
2.11 Sequencing analysis and homology modelling The amino acid sequence obtained from the nucleotide sequence was analysed using
170
the basic local alignment search tool BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and
171
compared with other PLA using the ClustalW program (http://www2.ebi.ac.uk/clustalw/).
172
Homology modelling was performed through the SWISS-MODEL program using the
173
automated protein homology modelling template at the ExPASY (Switzerland) and a
174
template search with the Alignment Mode program from the Protein Data Base (PDB;
175
http://swissmodel.expasy.org/) (Arnold et al., 2006; Bordoli et al., 2009). The model was
176
previewed
177
(http://www.expasy.org/spdbv) and Chimera software (https://www.cgl.ucsf.edu/chimera/).
178
To compute pI/MW of V. tropica PLA used tool of ExPASy Bioinformatics tool
179
(http://web.expasy.org/compute_pi/).
180
(http://clavius.bc.edu/clotelab/DiANNA/) was used to predict the formation of disulfide
181
bonds.
analyzed
using
182
Swiss-Pdb
Viewer
Deep
M AN U
and
SC
RI PT
169
The
3. Results
184
3.1 The partial purification and characterization
1.1
v4
web
software
server
TE D
183
DiANNA
View
The soluble venom was fractionated by reverse-phase HPLC and revealed 21
186
fractions as shown in Fig. 1. All fractions were screened for lecithin agar phospholipase
187
activity. The purified fractions P16 and P17 exhibited phospholipase activity on lecithin agar
188
after incubation at 37 ˚C for 24 hr (Fig. 2). Since the peptides were isolated from V. tropica
189
venom, they were designated “VesT 1”. The P16 and P17 were called VesT1.01 and
190
VesT1.02, respectively.
AC C
191
EP
185
Prior to purification, crude venom had been assayed for PLA activity using
192
EnzCheck® Phospholipase A1 Assay kit. The crude venom exhibited 5.54 U/µg protein of
193
specific activity. After purification, fractions P16 and P17 had been checked for their specific
194
activity. They were 185.6 and 214.6 U/µg, respectively. Then, the phospholipase fractions
195
(VesT1.01 and VesT1.02) were subjected to a 13% SDS-PAGE to evaluate the purified
196
products. The VesT1.02 fraction showed a single band under reducing condition, which was
197
approximately 34 kDa (Fig. 3). The peptide sequencing of this fraction, using a Beckman LF
198
300 Protein Sequencer (Palo Alto, CA, USA) revealed the first 14 amino acids of the N-
ACCEPTED MANUSCRIPT 199
terminal sequence (FLPIPYSDMTVKMI). This N-terminal sequence was used to search for
200
homologous
201
(http://www.ncbi.nlh.gov/BLAST/). The homology search indicated that VesT1.02 shares
202
homology with other PLA1 in vespid venom (Table 2).
sequences
in
the
protein
search
engine
tool
BLAST
The VesT1.01 fraction showed two bands under reducing conditions and
204
corresponded to 2 spots at same molecular mass of approximately 33.71 kDa with a slightly
205
different pI, 8.80 and 8.82 respectively (Fig. 3 and Fig. 4). The different and purified bands or
206
spots were trypsinized and subsequently identified using a LC-MS/MS. The peptides were
207
identified by the Protein MASCOT Search Engine using the NCBI Protein Database (Table
208
3). The P16 spots (named VesT1.01a and VesT1.01b) and the band of P17 (VesT1.02)
209
contained peptides which are similar to the PLA in Vespa carbo (P0CH87.1).
210
3.2 Phospholipase activity and the thermal stability assay.
SC
RI PT
203
The quantitative analysis of PLA1 activity was determined using a fluorometric PLA1
212
specific substrate. Five µg/mL of crude venom exhibited an activity of 11.0844 U/mL, as
213
compared with 5 U/mL activity of lecitase (Fig. 5). The results indicate that VesT1.01s (9.28
214
U/mL) and VesT1.02 (10.73 U/ mL) are PLA1s which cleave the sn-1 position. The thermo-
215
stability of V. tropica PLA and VesT1s were investigated. Both the venom and VesT1.01s
216
showed to contain PLA1 activity after exposure at 100 ˚C for 5 min (3.705 U/ mL).
217
Interestingly, VesT1.02 had no PLA1 activity (Fig. 5).
218
3.3 Glycoprotein detection
TE D
M AN U
211
VesT1.01s and VesT1.02 were investigated to detect possible carbohydrates
220
attachments, using the fluorescence probe (Pro-Q Emerald 300). The PLA fractions were
221
separated by SDS-PAGE. The fluorescent substance is assumed to react with the glycoprotein
222
after being oxidized to aldehydes. The VesT1.01s and VesT1.02 did not exhibit any
223
carbohydrates attachment. This gel was post-stained with SYPRO® Ruby solution to evoke
224
total protein (data not shown). The obtained result suggest VesT1s (VesT1.01s and
225
VesT1.02) enzymes to be non-glycoproteins.
226
3.4 cDNA cloning and sequencing analysis
AC C
EP
219
227
The mature sequence encoding VesT1.02 was determined using RT-PCR and 3′ rapid
228
amplification of cDNA ends (3′RACE). The VesT1.02 gene is composed of an open reading
229
frame of 1005 bp and 287 bp of 3′ untranslated region (3′ UTR) (Fig. 6A). VesT1.02 mature
230
protein contains 301 amino acid residues (1005 bp) including stops codon. The sequence of
231
VesT1.02 is rich in the amino acids Lys, Ile, Leu and Gly, with a theoretical pI of 8.80 and a
ACCEPTED MANUSCRIPT 232
predicted molecular mass of 33,243.3 Da. The VesT1.02 amino acid sequence deduced from
233
the nucleotide sequence obtained from RT-PCR was identical to the N-terminal sequence of
234
VesT1.02 (red box, Fig. 6A) and corroborated with the results obtained by LC-MS/MS (blue
235
boxes, Fig. 6A). An amino acid sequence similarity search revealed that the toxin shares similarities
237
with wasp venom PLAs: VesT1.02 contains the conserved catalytic triad (Ser137, Asp165
238
and His230). This sequence is also conserved in members of the pancreatic lipase family
239
(Fig. 8) (Aoki et al., 2007). As expected for wasp venom PLA1, the six disulfide bonds were
240
conserved. These six disulfide bonds are responsible for the structure stabilization of the
241
VesT1.02 protein (red arrows, Fig. 6A). The disulfide bonds are formed as follow Cys87/294,
242
Cys176/245, Cys181/266, Cys219/228, Cys240/246 and Cys267/269 (Fig. 6B). No free
243
sulfhydryl group were detected.
244
3.5 Structure homology modelling and molecular phylogeny analysis
M AN U
SC
RI PT
236
The 3D-structure homology modelling of V. tropica PLA1 (VesT1.02) was created
246
using the SWISS-MODEL program. The X-ray crystal structure of human pancreatic lipase
247
(1n8s.1.A) was used as template for the computational homology modelling (Fig.7B). The
248
VesT1.02 protein presents 29.82 % sequence identity with human pancreatic lipase.
249
Crystallography studies of human pancreatic lipase shows that each lipase is composed of
250
two domains, an N-terminal domain and a C-terminal domain (Roussel et al., 1998). The
251
phospholipase A1 from V. tropica venom (VesT1.02) was identified as a member of the
252
pancreatic lipase family, which shares 19.06 % sequence homology with the N-terminal
253
catalytic domain of human pancreatic lipase. Based on this model, VesT1.02 represents a
254
structure containing 10 β-sheets and 11 α-helixes (Fig. 7A). Comparative analysis of the
255
modelled structures among the sequence of VesT1.02 and human pancreatic lipase, clearly
256
showed that VesT1.02 lacks 24 amino acid residues at the N terminal domain (Fig. 7 and 8).
257
The region containing the α5 helix and its preceding loop (α5 loop) is 10 amino acids longer
258
in VesT1.02 compared to the human pancreatic lipase (1n8s.1.A). However, the C-terminal
259
domain of VesT1.02, which is required for colipase binding is completely missing. The
260
shorter lid domain and β9 loop allows PLA to be more selective for phospholipids (Fig. 7C).
261
The VesT1.02 model indicated the presence of highly conserved N-terminal domains that are
262
essential for catalytic PLA activity (Fig. 8) (Aoki et al., 2007).
263
4. Discussion
AC C
EP
TE D
245
ACCEPTED MANUSCRIPT In general, vespid venom are composed of mainly three bioactive type of compounds:
265
(1) high molecular weight proteins, enzymes and allergens such as phospholipase,
266
hyaluronidase and antigen5 (Abe et al., 2000; Ho and Ko, 1988; Justo Jacomini et al., 2014;
267
Rosenberg et al., 1977; Rungsa et al., 2016a, b; Sukprasert et al., 2013); (2) biologically
268
active amines, including histamine; and (3) small peptides and bioactive molecules, such as
269
mastoparan, kinins and chemolytic peptides (Higashijima et al., 1988; Nakajima et al., 1985;
270
Yang et al., 2013), which, upon envenomation, cause severe pain, local damage, allergic
271
reaction and even death in a very low concentrations. This suggest that these components are
272
potentially interesting for the development of novel pharmaceutical compounds. One such
273
category of components with a potential pharmaceutical application are phospholipases. The
274
wasp venom PLA and PLB are major allergens and they also act as toxins. They are found in
275
the venom of several species from the Asian-Pacific region, Europe and America. The PLA is
276
reported to be one of the most abundant proteins in V.tropica and V.affinis venom (Rungsa et
277
al., 2016b; Sookrung et al., 2014; Sukprasert et al., 2013). PLA1 were classified to be a major
278
allergen of the V.affinis venom. It reacted with IgE in all wasp sera when tested in allergic
279
pateints, indicating 100% allergenic properties in human (Sookrung et al., 2014). Also in the
280
Brazilian social wasp, Polybia paulista, PLA was identified to be the major allergen after
281
being detected in the allergenic response of sensitized patients (Pereez-Riverol et al., 2016).
282
The PLA hydrolyses sn-1 and/or sn-2 acyl group(s) of phospholipids, specifically of
283
membrane phospholipids. This results in membrane damage, severe hemolysis including
284
diverse pharmacological effects like hemorrhage, edema, neurotoxicity, cardiotoxicity,
285
myotoxicity, necrosis, anticoagulant and hypotension (Santos et al., 2007). On the basis here
286
of, PLAs are identified as a lethal factor of the venom (dos Santos et al., 2011; Ho et al.,
287
1998; Ho and Ko, 1988; Liu et al., 2015; Yoon et al., 2015). V. tropica venom is highly
288
potent against several animals. Its venom contains a higher PLA activity but apparently lower
289
protein proportion than PLA of V. affinis (Rungsa et al., 2016b; Schmidt et al., 1986). The
290
purification and characterization reveals at least three isoform of PLAs in V. tropica venom,
291
named VesT1.01a, VesT1.01b and VesT1.02. The PLA activity of VesT1.01s and VesT1.02
292
were determined using the lecithin agar phospholipase activity. The result showed cloudy
293
haloes of degradation of lecithin on the plates. The action of phospholipase results in the
294
formation of a calcium complex with free fatty acids released from phospholipids present in
295
soybean. These complexes will cause cloudy haloes (Chrisope et al., 1976; Incamnoi et al.,
296
2013; Sukprasert et al., 2013). The specific PLA1 activities were investigated using the
AC C
EP
TE D
M AN U
SC
RI PT
264
ACCEPTED MANUSCRIPT 297
fluorometric PLA1 substrate. The activity is comparable with previously described activities
298
(Rungsa et al., 2016b; Sukprasert et al., 2013). VesT1.01a and VesT1.01b are iso-enzymes with the same molecular mass (33.72
300
kDa) and a closely related pI of 8.80 and 8.82. This result was similar to other wasp PLA1s
301
such as those found in the venom of V. affinis (Ves a 1.01; 33.4415 kDa and Ves a 1.02;
302
33.4744 kDa) or V. verutina (vertoxin 2a; 33.360 kDa and 2b; 33.374 kDa) (Ho et al., 1999;
303
Sukprasert et al., 2013). The spots from 2D-PAGE were digested and the proteins were
304
identified using LC-MS/MS. It was shown that several peptide sequences are closely related.
305
VesT1.02 is a PLA1 with a molecular mass of approximately 34 kDa, which corresponds to
306
the experimental mass of PLA from V. tropica venom (Rungsa et al., 2016b). Comparison of
307
the first 14 amino acid residues, obtained by Edman degradation, of VesT1.02 with known
308
vespid phospholipase A1 yielded 79 % identity with proteins of the venoms of V. velutina
309
and V. affinis (Ho et al., 1999; Sukprasert et al., 2013). For VesT1.02 PLA, the presence of a
310
phenylalanine at the first position of N-terminus is quite common in many wasp venom PLAs
311
(Hou et al., 2016). The peptides form LC-MS/MS analysis were similar to many wasp venom
312
PLA1 and corresponded to the VesT1.02 sequences. The six disulphide bonds of VesT1.02
313
are conserved (Chou and Hou, 2008; Ho and Ko, 1988; Soldatova et al., 1993; Sukprasert et
314
al., 2013). The lack of a cysteine at position 4, normally conserved among vespid PLA, may
315
cause less tendency of miss folding and a higher stability of the protein structure. The lower
316
activity of PLA1 from the venom of V. tropica might be due to the free cysteine residue
317
which allows miss-paring of cysteines leading to the unstable conformation of the substrate
318
binding site and catalytic domain.
EP
TE D
M AN U
SC
RI PT
299
Although the PLA from these 2 species (V. tropica and V. affinis) showed high
320
homology (91.69%) in primary structure, the activity of the crude venoms was surprisingly
321
different. A three-fold higher potency was observed for V. tropica venom compared to the V.
322
affinis venom. This three-fold difference in venom potency of V. tropica may follow from the
323
higher hyaluronidase activity in his venom since this hyaluronidase activity might synergize
324
the action of other venom toxins.
AC C
319
325
Sukprasert et al. (2013) reported that V. affinis crude venom showed thermos-stability
326
after heating at 100 °C for 5 min. However, after purification, V. affinis PLA completely lost
327
activity. In this study, V. tropica crude venom and VesT1.01s showed thermo-stability after
328
heating at 100 °C for 5 min. This thermo-stability results from the insertion of proline
329
residues at the mini-lid domain and might protect the enzyme from the high temperature or
ACCEPTED MANUSCRIPT 330
possibly plays an important role in the enzyme-substrate stabilization. These proline residues
331
improved the flexibility of the structure resulting in the high thermo-stability. Furthermore,
332
the absence of possible mismatching in disulfide bond formation may be an additional effect
333
in strengthening the structure stability after heating (Niu et al., 2016). It was suggested that carbohydrate attachment contributes to the biological activity
335
and immunogenicity (Rungsa et al., 2016a). Santos (2011) investigated glycosylation and
336
identified 4 out of 7 isoforms of PLA in Polybia paulista with several having carbohydrates
337
attached to the structure. The VesT1s do not contain (a) carbohydrate(s), similar to what was
338
reported previously (Santos et al., 2007; Sukprasert et al., 2013).
RI PT
334
Since the PLA is normally one of the major venom components, the activity of PLA
340
will affect and govern the overall venom activity. The high PLA activity may result from an
341
efficient folding of its structure (Aoki et al., 2007; Richmond and Smith, 2011; Rungsa et al.,
342
2016b). Via sequence homology and structural modelling, the VesT1.02 N-terminal domain
343
was conserved whereas the C-terminal domain was completely missing. However, the first 24
344
amino acid residues in the N-terminal domain were absent. The β5, β9 and lid loop, essential
345
in the catalysis and the substrate selectivity of triglycerides or phospholipids, were conserved.
346
In the open conformation, the hydrophobic chains of lipids can interact with the clustered
347
hydrophobic residues of the β9 loop and lid domain, whereas the β5 loop is involved in the
348
formation of the oxyanion holes (Carrière et al., 1998).
TE D
M AN U
SC
339
Previous studies have focused on the open forms of the human pancreatic lipase. It is
350
suggested that the β5 loop may adopt a flexible conformation to accommodate the
351
phospholipid molecule. Moreover, replacement of the glutamine residue by a threonine in the
352
β5 loops was predicted to eliminate the salt bridge that stabilises the interaction between the
353
β5 loop and the core of the protein. Therefore, the presence of different residues at this
354
position may cause a different conformation (Carrière et al., 1998; Sukprasert et al., 2013;
355
Withers-Martinez et al., 1996). The α5 helix of VesT1.02 is 7 residues longer than that in
356
HPL and thus may adopt a different loop conformation. The Gln95 allows orientation
357
towards phosphatidylcholine and possibly helps to stabilize substrate binding (Hou et al.,
358
2016; Withers-Martinez et al., 1996). The β9 loop near the active site and shortened lid loop
359
contributes to an increase of the active site accessibility, suggesting that VesT1.01 is only
360
exhibiting PLA activity (Aoki et al., 2007; Carrière et al., 1998; Withers-Martinez et al.,
361
1996).
AC C
EP
349
ACCEPTED MANUSCRIPT 362
In summary, V. tropica crude venom clearly shows high toxicity and allergenicity
363
(Rungsa et al., 2016a, b; Sukprasert et al., 2013). The three isoforms of PLAs in this study
364
were shown to be the major toxin components in the venom. The computational modelling
365
and structure features provide a potential explanation for the high thermo-stability and
366
activity.
368
RI PT
367
5. Acknowledgements
This work was financially supported by the Post-Doctoral Training Program from (1)
370
Research and Technology Transfer Affairs, Khon Kaen University (KKU) and Graduate
371
School, KKU, Thailand (Grant no. 583334), (2) KKU Research Fund, fiscal years
372
2011−2014, (3) Thailand Research Fund (TRF)−Khon Kaen University (KKU) joint funded
373
TRF Basic Research Grant (TRF-BRG), years 2015-2017 (BRG5780014).
SC
369
M AN U
374 375
References
376
Abe, T., Sugita, M., Fujikura, T., Hiyoshi, J., Akasu, M., 2000. Giant hornet (Vespa
377
mandarinia) venomous phospholipases: The purification, characterization and
378
inhibitory properties by biscoclaurine alkaloids. Toxicon 38, 1803−1816. An, S., Chen, L., Wei, J.F., Yang, X., Ma, D., Xu, X., Xu, X., He, S., Lu, J., Lai, R., 2012.
380
Purification and characterization of two new allergens from the venom of Vespa
381
magnifica. PloS one 7, e31920.
TE D
379
Aoki, J., Inoue, A., Makide, K., Saiki, N., Arai, H., 2007. Structure and function of
383
extracellular phospholipase A1 belonging to the pancreatic lipase gene family.
384
Biochimie 89, 197−204.
386 387
Arnold, K., Bordoli, L., Kopp, J., Schwede, T., 2006. The SWISS-MODEL workspace: a
AC C
385
EP
382
web-based environment for protein structure homology modelling. Bioinformatics (Oxford, England) 22, 195−201.
388
Bordoli, L., Kiefer, F., Arnold, K., Benkert, P., Battey, J., Schwede, T., 2009. Protein
389
structure homology modeling using SWISS-MODEL workspace. Nat. Protoc. 4,
390
1−13.
391
Borodina, I., Jensen, B.M., Wagner, T., Hachem, M.A., Søndergaard, I., Poulsen, L.K., 2011.
392
Expression of enzymatically inactive wasp venom phospholipase A1 in Pichia
393
pastoris. PloS one 6, 21267.
ACCEPTED MANUSCRIPT 394
Carrière, F., Withers-Martinez, C., van Tilbeurgh, H., Roussel, A., Cambillau, C., Verger, R.,
395
1998. Structural basis for the substrate selectivity of pancreatic lipases and some
396
related proteins. Biochim. Biophys. Acta 1376, 417−432. Caruso, B., Bonadonna, P., Bovo, C., Melloni, N., Lombardo, C., Senna, G., Lippi, G., 2016.
398
Wasp venom allergy screening with recombinant allergen testing. Diagnostic
399
performance of rPol d 5 and rVes v 5 for differentiating sensitization to Vespula and
400
Polistes subspecies. Clin. Chim. Acta 453, 170−173.
RI PT
397
Chou, C.C., Hou, M.H., 2008. Crystallization and preliminary X-ray diffraction analysis of
402
phospholipase A1 isolated from hornet (Vespa basalis) venom. Acta Crystallogr.
403
Sect. F Struct. Biol. Cryst. Commun. 64, 1118−1120.
405 406 407
Chrisope, G.L., Fox, C.W., Marshall, R.T., 1976. Lecithin agar for detection of microbial phospholipases. Appl. Environ. Microbiol. 31, 784−786.
M AN U
404
SC
401
Das, R.N., Mukherjee, K., 2008. Asian wasp envenomation and acute renal failure: a report of two cases. Mcgill J. Med. 11, 25−28.
408
Dias, N.B., de Souza, B.M., Gomes, P.C., Brigatte, P., Palma, M.S., 2015. Peptidome
409
profiling of venom from the social wasp Polybia paulista. Toxicon 107, Part B,
410
290−303.
Diego-García, E., Peigneur, S., Debaveye, S., Gheldof, E., Tytgat, J., Caliskan, F., 2013.
412
Novel potassium channel blocker venom peptides from Mesobuthus gibbosus
413
(Scorpiones: Buthidae). Toxicon 61, 72−82.
TE D
411
dos Santos, L.D., da Silva Menegasso, A.R., dos Santos Pinto, J.R., Santos, K.S., Castro,
415
F.M., Kalil, J.E., Palma, M.S., 2011. Proteomic characterization of the multiple
416
forms of the PLAs from the venom of the social wasp Polybia paulista. Proteomics
417
11, 1403-1412.
419
AC C
418
EP
414
Golden, D.B.K., 2007. Insect sting anaphylaxis. Immunol. Allergy Clin. North Am. 27, 261−72.
420
Henriksen, A., King, T.P., Mirza, O., Monsalve, R.I., Meno, K., Ipsen, H., Larsen, J.N.,
421
Gajhede, M., Spangfort, M.D., 2001. Major venom allergen of yellow jackets, Ves v
422
5: structural characterization of a pathogenesis-related protein superfamily. Proteins
423
45, 438−448.
424
Higashijima, T., Uzu, S., Nakajima, T., Ross, E.M., 1988. Mastoparan, a peptide toxin from
425
wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G
426
proteins). J. Biol. Chem. 263, 6491−6494.
ACCEPTED MANUSCRIPT 427
Ho, C. L., Chen, W. C., Lin, Y.-L., 1998. Structures and biological activities of new wasp
428
venom peptides isolated from the black-bellied hornet (Vespa basalis) venom.
429
Toxicon 36, 609−617. Ho, C. L., Ko, J. L., 1988. Purification and characterization of a lethal protein with
431
phospholipase A1 activity from the hornet (Vespa basalis) venom. Biochim.
432
Biophys. Acta 963, 414−422.
RI PT
430
433
Ho, C. L., Lin, Y. L., Li, S. F., 1999. Three toxins with phospholipase activity isolated from
434
the yellow-legged hornet (Vespa verutina) venom. Toxicon 37, 1015-1024.
435
Hou, M. H., Chuang, C. Y., Ko, T. P., Hu, N. J., Chou, C. C., Shih, Y. P., Ho, C. L., Wang,
437
A. H. J., 2016. Crystal structure of vespid phospholipase A1 reveals insights into the
438
mechanism for cause of membrane dysfunction. Insect Biochem. Mol. Biol. 68,
439
79−88.
M AN U
SC
436
440
Incamnoi, P., Patramanon, R., Thammasirirak, S., Chaveerach, A., Uawonggul, N.,
441
Sukprasert, S., Rungsa, P., Daduang, J., Daduang, S., 2013. Heteromtoxin (HmTx),
442
a novel heterodimeric phospholipase A2 from Heterometrus laoticus scorpion
443
venom. Toxicon 61, 62−71.
Justo Jacomini, D.L., Campos Pereira, F.D., Aparecido dos Santos Pinto, J.R., dos Santos,
445
L.D., da Silva Neto, A.J., Giratto, D.T., Palma, M.S., de Lima Zollner, R., Brochetto
446
Braga, M.R., 2013. Hyaluronidase from the venom of the social wasp Polybia
447
paulista (Hymenoptera, Vespidae): Cloning, structural modeling, purification, and
448
immunological analysis. Toxicon 64, 70−80.
EP
TE D
444
Justo Jacomini, D.L., Gomes Moreira, S.M., Campos Pereira, F.D., Zollner, R.d.L., Brochetto
450
Braga, M.R., 2014. Reactivity of IgE to the allergen hyaluronidase from Polybia
451
AC C
449
paulista (Hymenoptera, Vespidae) venom. Toxicon 82, 104−111.
452
Kularatne, K., Kannangare, T., Jayasena, A., Jayasekera, A., Waduge, R., Weerakoon, K.,
453
Kularatne, S.A., 2014. Fatal acute pulmonary oedema and acute renal failure
454 455
following multiple wasp/hornet (Vespa affinis) stings in Sri Lanka: two case reports. J. Med. Case Rep. 8, 188.
456
Kularatne, S.A., Gawarammana, I.B., de Silva, P.H., 2003. Severe multi-organ dysfunction
457
following multiple wasp (Vespa affinis) stings. Ceylon Med. J. 48, 146−147.
ACCEPTED MANUSCRIPT 458
Liu, Z., Chen, S., Zhou, Y., Xie, C., Zhu, B., Zhu, H., Liu, S., Wang, W., Chen, H., Ji, Y.,
459
2015. Deciphering the Venomic Transcriptome of Killer-Wasp Vespa velutina. Sci.
460
Rep. 5, 9454. Lu, G., Kochoumian, L., King, T.P., 1995. Sequence identity and antigenic cross-reactivity of
462
white face hornet venom allergen, also a hyaluronidase, with other proteins. J. Biol.
463
Chem. 270, 4457−4465.
RI PT
461
464
Nakajima, T., Yasuhara, T., Uzu, S., Wakamatsu, K., Miyazawa, T., Fukuda, K., Tsukamoto,
465
Y., 1985. Wasp venom peptides; wasp kinins, new cytotrophic peptide families and
466
their physico-chemical properties. Peptides 6, Suppl. 3, 425−430.
Niu, C., Zhu, L., Xu, X., Li, Q., 2016. Rational design of disulfide bonds increases
468
thermostability of a Mesophilic 1,3-1,4-β- Glucanase from Bacillus terquilensis.
469
PLoS ONE 11(4): e0154036.
SC
467
Peigneur, S., Van Der Haegen, A., Möller, C., Waelkens, E., Diego-García, E., Marí, F.,
471
Naudé, R., Tytgat, J., 2013. Unraveling the peptidome of the South African cone
472
snails Conus pictus and Conus natalis. Peptides 41, 8−16.
M AN U
470
Perez-Riverol, A., Campos Pereira, F.D., Musacchio Lasa, A., Romani Fernandes G. L.,
474
Santos-Pinto, J.R., Justo-Jacomini, D.L., Oliveira de Azevedo, G., Bazon M. L.,
475
Palma, M.S., Zollner, R.L., Brochetto-Braga, M.R., 2016. Molecular cloning,
476
expression and IgE-immunoreactivity of phospholipase A1, a major allergen from
477
Polybia paulista (Hymenoptera: Vespidae) venom. Toxicon 124:44-52.
478
Richmond, G.S., Smith, T.K., 2011. Phospholipases A(1). Int. J. Mol. Sci. 12, 588−612.
479
Rosenberg, P., Ishay, J., Gitter, S., 1977. Phospholipases A and B activities of the oriental
482
EP
481
hornet (Vespa Orientalis) venom and venom apparatus. Toxicon 15, 141−155. Roussel, A., Yang, Y., Ferrato, F., Verger, R., Cambillau, C., Lowe, M., 1998. Structure and
AC C
480
TE D
473
activity of rat pancreatic lipase-related protein 2. J. Biol. Chem. 273, 32121−32128.
483
Rungsa, P., Incamnoi, P., Sukprasert, S., Uawonggul, N., Klaynongsruang, S., Daduang, J.,
484
Patramanon, R., Roytrakul, S., Daduang, S., 2016a. Cloning, structural modelling
485 486
and characterization of VesT2s, a wasp venom hyaluronidase (HAase) from Vespa tropica. J. Venom. Anim. Toxins Incl. Trop. Dis. 22, 28.
487
Rungsa, P., Incamnoi, P., Sukprasert, S., Uawonggul, N., Klaynongsruang, S., Daduang, J.,
488
Patramanon, R., Roytrakul, S., Daduang, S., 2016b. Comparative proteomic analysis
489
of two wasps venom, Vespa tropica and Vespa affinis. Toxicon 119, 159−167.
ACCEPTED MANUSCRIPT 490
Santos, L.D., Santos, K.S., de Souza, B.M., Arcuri, H.A., Cunha-Neto, E., Castro, F.M.,
491
Kalil,
J.E.,
Palma,
M.S.,
2007.
Purification,
sequencing
and
structural
492
characterization of the phospholipase A1 from the venom of the social wasp Polybia
493
paulista (Hymenoptera, Vespidae). Toxicon 50, 923−937. Santos, L.D., Silva Menegasso A. R., Santos Pinto, J.R., Santos, K.S., Castro, F.M., Palma,
495
M.S., 2011. Proteomic characterization of the multiple forms of the PLAs from the
496
venom of the social wasp Polybia paulista. Proteomics 11, 1403–1412.
497 498
RI PT
494
Schmidt, J.O., Yamane, S., Matsuura, M., Starr, C.K., 1986. Hornet venoms: Lethalities and lethal capacities. Toxicon 24, 950−954.
Seismann, H., Blank, S., Braren, I., Greunke, K., Cifuentes, L., Grunwald, T., Bredehorst, R.,
500
Ollert, M., Spillner, E., 2010. Dissecting cross-reactivity in hymenoptera venom
501
allergy by circumvention of α-1,3-core fucosylation. Mol. Immunol. 47, 799−808.
SC
499
Soldatova, L., Kochoumian, L., King, T.P., 1993. Sequence similarity of a hornet (D.
503
maculata) venom allergen phospholipase A1 with mammalian lipases. FEBS Lett
504
320, 145-149.
M AN U
502
Sookrung, N., Wong-Din-Dam, S., Tungtrongchitr, A., Reamtong, O., Indrawattana, N.,
506
Sakolvaree, Y., Visitsunthorn, N., Manuyakorn, W., Chaicumpa, W., 2014.
507
Proteome and allergenome of Asian wasp, Vespa affinis, venom and IgE reactivity
508
of the venom components. J. Proteome Res. 13, 1336–1344.
TE D
505
Srisong, H., Daduang, S., Lopata, A.L., 2016. Current advances in ant venom proteins
510
causing hypersensitivity reactions in the Asia-Pacific region. Mol. Immunol. 69,
511
24−32.
EP
509
Sukprasert, S., Rungsa, P., Uawonggul, N., Incamnoi, P., Thammasirirak, S., Daduang, J.,
513
Daduang, S., 2013. Purification and structural characterisation of phospholipase A1
514 515
AC C
512
(Vespapase, Ves a 1) from Thai banded tiger wasp (Vespa affinis) venom. Toxicon 61, 151−164.
516
Uawonggul, N., Thammasirirak, S., Chaveerach, A., Arkaravichien, T., Bunyatratchata, W.,
517
Ruangjirachuporn, W., Jearranaiprepame, P., Nakamura, T., Matsuda, M.,
518
Kobayashi, M., Hattori, S., Daduang, S., 2007. Purification and characterization of
519
Heteroscorpine-1 (HS-1) toxin from Heterometrus laoticus scorpion venom.
520
Toxicon 49, 19−29.
ACCEPTED MANUSCRIPT 521
Withers-Martinez, C., Carrière, F., Verger, R., Bourgeois, D., Cambillau, C., 1996. A
522
pancreatic lipase with a phospholipase A1 activity: crystal structure of a chimeric
523
pancreatic lipase-related protein 2 from guinea pig. Structure 4, 1363−1374.
524
Yang, H., Xu, X., Ma, D., Zhang, K., Lai, R., 2008. A phosholipase A1 platelet activator
526 527
from the wasp venom of Vespa magnifica (Smith). Toxicon 51, 289−296. Yang, X., Wang, Y., Lee, W. H., Zhang, Y., 2013. Antimicrobial peptides from the venom
RI PT
525
gland of the social wasp Vespa tropica. Toxicon 74, 151−157.
Yoon, K.A., Kim, K., Nguyen, P., Seo, J.B., Park, Y.H., Kim, K.G., Seo, H.Y., Koh, Y.H.,
529
Lee, S.H., 2015. Comparative functional venomics of social hornets Vespa crabro
530
and Vespa analis. J. Asia-Pacific Entomol. 18, 815−823.
SC
528
AC C
EP
TE D
M AN U
531
ACCEPTED MANUSCRIPT Figure Legends
533
Fig. 1. Purification profile using HPLC-C18 column. Soluble Vespa tropica venom was
534
loaded on to a C18 reverse phase HPLC column. A linear gradient of 0% solvent A (0.1%
535
trifluoroacetic acid (TFA) in water), and 80% solvent B (0.085% TFA in acetonitrile) were
536
run for 100 min at a flow rate of 1 mL/min. The absorbance was monitored at 214 and 280
537
nm. The purification revealed 21 fractions. Only P16 and P17 exhibited the phospholipase
538
activity on lecithin agar plates.
539
Fig. 2. Phospholipase activity assay on lecithin agar. Vespa tropica crude venom (Venom),
540
phospholipase A1 from Thermomyces lanuginosus (Control), the purified fractions (P16 and
541
P17) (VesT1.01s and VesT1.02, respectively) from RP-HPLC were added in agarose wells
542
containing 1.2% lecithin. After 24 h incubation at 37 °C, the plates were inspected for clear
543
zones of activity.
544
Fig. 3. An electrophoresis profile of the purified fraction containing the Vespa tropica
545
phospholipase. Purified fractions P16 (VesT1.01s) and P17 (VesT1.02) were resolved by
546
13% SDS-PAGE under reducing conditions with Coomassie blue R-250. Lane M was the
547
molecular weight marker in kDa.
548
Fig. 4. A 2D-PAGE profile of the P16 purified fraction. First dimension was isoelectric
549
focusing (pH 3–10, non-linear gradient). Second dimension was 13% SDS-PAGE. The gel
550
was stained with Coomassie brilliant blue G-250 dye solution. Molecular weight marker in
551
kDa is shown on the right panel and the pH gradient is indicated at the top panel.
552
Fig. 5. The specific PLA1 activity assay. The VesT1s (Vespa tropica PLA1s) were assayed
553
using fluorometric substrate (Invitrogen, USA). Five U/mL lecitase was used as positive
554
control. The others substrates were used at a concentration of five µg/mL. For the thermal
555
stability test, VesT1s were exposed to 100 ˚C for 5 mins and then kept at 4 ˚C.
556
Fig. 6. The completed nucleotide sequence and deduced amino acid sequence of Vespa
557
tropica phospholipase (VesT1.02). (A) The complete sequence of VesT1.02. The 3′ UTRs
558
are indicated by small letters. The first 14 amino acid residues obtained from the Edman
559
degradation were show in the red box. Sequences from LC-MS/MS analysis were shown in
560
the blue boxes. The catalytic triad (Ser, Asp and His) is bolded and italicized. The stop codon
561
is indicated with “stop”. The cysteine residues were labelled with triangle marks (). (B)
562
Disulfide linkages of Ves a 1. The 12 cysteine residues that form the following disulfide
563
bridges Cys87/294, Cys176/245, Cys181/266, Cys219/228, Cys240/246 and Cys267/269 are
564
linked by solid lines.
AC C
EP
TE D
M AN U
SC
RI PT
532
ACCEPTED MANUSCRIPT Fig. 7. The homology modelling of Vespa tropica phospholipase A1 (VesT1.02) (A)
566
Secondary structure topology diagram of VesT1.02 by using PDBsum (Laskowski, 2009).
567
(B) Structure analysis of VesT1.02 using homologies modelling (C) Alignments of structural
568
dominants of VesT1.02 with other PLA, focusing only β5, β9 loops and lid domain by
569
ClustalW program (HLP: human pancreatic lipase, rPLRP2: rat pancreatic lipase related
570
protein 2, Dol m1: Dolichovespula maculata PLA1, VesT 1: Vespa tropica PLA1 ).
571
Fig. 8. Sequence alignment of the deduced amino acid sequence of Vespa tropica
572
phospholipase with the other phospholipases. VesT1.02 was aligned with the known lipases
573
and phospholipases: human pancreatic lipase (LIPP_Human), guinea pig phospholipase
574
(LIPR2_CAVPO), human hepatic lipase (PLA1A_Human) and human lipoprotein lipase
575
(LPL_Human). Some allergens from vespid venoms, defined as PLA1, were included in this
576
alignment. They were Ves g 1 (Vespula germanica), Ves v 1 (Vespula vulgaris), Dol m 1.01
577
(Dolichovespula maculata), Pol a 1 (Polistes anularis), Pol d 1 (Polistes dominulus), Pol p 1
578
(Polybia polista) and Ves a 1 (Vespa affinis).
M AN U
SC
RI PT
565
579 580 581
EP AC C
583
TE D
582
ACCEPTED MANUSCRIPT Table 1 Primer used for the amplification of phospholipase gene Reverse Primer
F1 GATTC(C/T)T(A/T/G/C)CCAAT(A/T/C)CCTTAC
R1 AGCAGGATCAAGCCCAATAA
F2 CATGGTTTTACTTCAACTGC
R2 GCAAACGCACTCATTTCTTG
F3 CCCTTGGAACTGTCGATTTC
R3 TGCATTTAATCCAACGCAAA
F4 CGGTTGCGGTCTTCCTATTA
R4 TGCATTTAATCCAACGCAAA
F5 GGCGCACATATTTCAGGTTT
R5 GGCGCACATATTTCAGGTTT
RI PT
Forward Primers
SC
1
AC C
EP
TE D
M AN U
2
ACCEPTED MANUSCRIPT
Table 2. Comparison of the N-terminal sequences of several venom phospholipase. N-terminal sequence
Molecular mass
Reference
Vespa tropica (VesT1.02)
FLPIPYSDDTVKMIa
33 .99
In this study
Vespa affinis
FNPCPYSDDTVKMIITLRENKKHDFb
33.44/33.47
(Sukprasert et al., 2013)
Vespa verutina
FNPCPYSDDTVKMIILTRENKKHDFa
34.982
(Ho et al., 1999)
33.961
(de Souza et al., 2009)
TE D
M AN U
Amino acid sequences from the automated Edman degradation. Deduced amino acid sequences from the cDNA template.
EP
b
AC C
a
SC
Polybia paulista LIPECPFNEYDILFFVYTRQQRDa
RI PT
Source
ACCEPTED MANUSCRIPT
Table 3. Proteomic identification of the Phospholipase in V. tropica; the purified phospholipase were excised from the gels (SDS-PAGE and 2D-PAGE).
VesT1.01a Vespa crabro
Accession Protein Theorical code score MW/pI P0CH87.1 435 34104/8.91
K.VQELGLGK.Y K.KVQELGLGK.Y K.YNVPMANIR.L R.LVGNYIATVTK.M K.TGSFYVPVESK.A R.NECVCVGLNAK.T K.HECCLIGVPKSK.N -.FNPCPYSDDTVK.M R.LIGHSLGAHISGFAGK.K K.KVQELGLGK.Y K.YNVPMANIR.L R.LVGNYIATVTK.M K.TGSFYVPVESK.A R.NECVCVGLNAK.T K.HECCLIGVPKSK.N -.FNPCPYSDDTVK.M R.LIGHSLGAHISGFAGK.K K.KVQELGLGK.Y K.YNVPMANIR.L R.LVGNYIATVTK.M K.TGSFYVPVESK.A R.NECVCVGLNAK.T K.HECCLIGVPKSK.N -.FNPCPYSDDTVK.M
P0CH87.1 284
VesT1.02
P0CH87.1 259
34104/8.91
AC C
EP
TE D
M AN U
VesT1.01b Vespa crabro
Vespa crabro
Peptides sequences
RI PT
Protein
SC
Spots
34104/8.91
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Highlights: •
The Vespa tropica phospholipase A1 (VesT1s), one of major allergens is found as three isoforms in the venom.
•
The VesT1s are non-glycoproteins.
•
The shortened lid and shortened β9 loop, playing important roles in substrate
The VesT1s have been shown to be highly thermally stable. The insertion of a Pro
EP
TE D
M AN U
SC
residue might be involved in this thermo-stability.
AC C
•
RI PT
selectivity, cause this enzyme to only exhibit PLA activity.