Molecular Characterization of Three Novel Phospholipase A2 Proteins from the Venom of Atheris chlorechis, Atheris nitschei and Atheris squamigera

Abstract

Secretory phospholipase A₂ (sPLA₂) is known as a major component of snake venoms and displays higher-order catalytic hydrolysis functions as well as a wide range of pathological effects. Atheris is not a notoriously dangerous genus of snakes although there are some reports of fatal cases after envenomation due to the effects of coagulation disturbances and hemorrhaging. Molecular characterization of Atheris venom enzymes is incomplete and there are only a few reports in the literature. Here, we report, for the first time, the cloning and characterization of three novel cDNAs encoding phospholipase A₂ precursors (one each) from the venoms of the Western bush viper (Atheris chlorechis), the Great Lakes bush viper (Atheris nitschei) and the Variable bush viper (Atheris squamigera), using a "shotgun cloning" strategy. Open-reading frames of respective cloned cDNAs contained putative 16 residue signal peptides and mature proteins composed of 121 to 123 amino acid residues. Alignment of mature protein sequences revealed high degrees of structural conservation and identity with Group II venom PLA₂ proteins from other taxa within the Viperidae. Reverse-phase High Performance Liquid Chromatography (HPLC) profiles of these three snake venoms were obtained separately and chromatographic fractions were assessed for phospholipase activity using an egg yolk suspension assay. The molecular masses of mature proteins were all identified as approximately 14 kDa. Mass spectrometric analyses of the fractionated oligopeptides arising from tryptic digestion of intact venom proteins, was performed for further structural characterization.

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Article
Molecular Characterization of Three Novel Phospholipase
A2Proteins from the Venom of Atheris chlorechis,
Atheris nitschei and Atheris squamigera
He Wang 1,*, Xiaole Chen 2,*, Mei Zhou 3, Lei Wang 3, Tianbao Chen 3and Chris Shaw 3
1School of Integrative Medicine, Fujian University of Traditional Chinese Medicine, No.1 Qiu Yang Road,
Shangjie Town, Fuzhou 350122, Fujian, China
2School of Pharmacy, Fujian Medical University, No.1 Xueyuan Road, Shangjie Town, Fuzhou 350004,
Fujian, China
3Natural Drug Discovery Group, School of Pharmacy, Queen’s University Belfast, University Road,
Belfast BT7 1NN, UK; m.zhou@qub.ac.uk (M.Z.); l.wang@qub.ac.uk (L.W.); t.chen@qub.ac.uk (T.C.);
chris.shaw@qub.ac.uk (C.S.)
*Correspondence: hwang11@qub.ac.uk or wh4401@hotmail.com (H.W.); leochen5139@fjmu.edu.cn (X.C.);
Tel.: +86-591-2286-1151 (H.W.); +86-591-2286-2692 (X.C.)
Academic Editor: Bryan Grieg Fry
Received: 24 February 2016; Accepted: 20 May 2016; Published: 1 June 2016
Abstract:
Secretory phospholipase A
2
(sPLA
2
) is known as a major component of snake venoms
and displays higher-order catalytic hydrolysis functions as well as a wide range of pathological
effects. Atheris is not a notoriously dangerous genus of snakes although there are some reports of
fatal cases after envenomation due to the effects of coagulation disturbances and hemorrhaging.
Molecular characterization of Atheris venom enzymes is incomplete and there are only a few reports
in the literature. Here, we report, for the first time, the cloning and characterization of three
novel cDNAs encoding phospholipase A
2
precursors (one each) from the venoms of the Western
bush viper (Atheris chlorechis), the Great Lakes bush viper
(Atheris nitschei)
and the Variable bush
viper
(Atheris squamigera),
using a “shotgun cloning” strategy. Open-reading frames of respective
cloned cDNAs contained putative 16 residue signal peptides and mature proteins composed of
121 to 123 amino acid residues. Alignment of mature protein sequences revealed high degrees of
structural conservation and identity with Group II venom PLA
2
proteins from other taxa within
the Viperidae. Reverse-phase High Performance Liquid Chromatography (HPLC) profiles of these
three snake venoms were obtained separately and chromatographic fractions were assessed for
phospholipase activity using an egg yolk suspension assay. The molecular masses of mature
proteins were all identified as approximately 14 kDa. Mass spectrometric analyses of the fractionated
oligopeptides arising from tryptic digestion of intact venom proteins, was performed for further
structural characterization.
Keywords: snake venom; phospholipase A2; molecular cloning; Atheris; mass spectrometry
1. Introduction
As major components of snake venoms, proteins of the secretory phospholipase A
2
(sPLA
2
) family
have been structurally-defined, characterized and their catalytic mechanisms have been elucidated [
1
].
Traditionally, these snake venom enzymes have been divided into two main groups, Group I (GI)
and Group II (GII), used to distinguish between molecules based on their amino acid sequences,
disulfide-bridging patterns, unique functional loops and extension amino acids [
2
]. Phospholipase A
2
catalyzes the hydrolysis of the sn-2 fatty acyl bond of phospholipids in a calcium-dependent manner [
3
].
A broad range of physiological molecules, such as long-chain fatty acid phospholipids, short-chain fatty
Toxins 2016,8, 168; doi:10.3390/toxins8060168 www.mdpi.com/journal/toxins

Toxins 2016,8, 168 2 of 11
acid-containing oxidized phospholipids and platelet-activating factors, act as the natural substrates
of phospholipase A
2
[
4
]. The higher-order catalytic hydrolysis functions of these snake enzymes
play a central role in lipid metabolism of numerous cells and tissues, contributing to prey digestion.
In addition, much is known about the wide variety of pathological effects exhibited by the snake
venom phospholipase A
2
s including their neurotoxicity, myotoxicity, cytotoxicity, anticoagulant
effects, cardiotoxicity, hypotension induction, platelet aggregation/inhibition and anti-bacterial
activities [5–10], which in many cases, are fundamental to the toxicity of snake venoms [11,12].
Of the 400 species of snakes in Africa, bush vipers (Atheris, Viperidae) are not considered to
be highly-dangerous in terms of the numbers of documented bites and deaths. This may in part
be due to their arboreal behavior and their distribution in often inhospitable habitats [
13
]. A full
envenomation by Atheris is likely to be fatal, however, due to the injection of a large quantity of
toxic venom through their long and sharp fangs [
14
]. Envenomations involving Atheris vipers are
clinically-characterized by coagulation disturbances and hemorrhage, local pain, edema, bleeding
and a prolongation of coagulation time and non-clotting blood [
15
,
16
]. Some victims develop acute
renal failure and hypertension [
17
,
18
]. However, studies on characterization of venom enzymes to
explain these sequelae, are scarce and are incomplete [
19
]. In fact, even superficial reports on the
components of whole venoms are equally rare. Thus, hunting for and describing the toxic polypeptides
and/or proteins in Atheris venoms became a focus of our interest. Novel disintegrins had been isolated
and identified in a previous study [
20
], and in this work, cloning of precursor-encoding cDNAs
and characterization of the mature phospholipase A
2
proteins from Atheris chlorechis (A. chlorechis),
Atheris nitschei
(A. nitschei) and Atheris squamigera (A. squamigera) venoms, were carried out in an
attempt to explain the toxic effects of the venom of Atheris snakes in more depth.
2. Results
2.1. cDNAs Encoding PLA2Precursors and Bioinformatic Analyses
A single novel unique full-length phospholipase-encoding precursor was cloned from each of the
venom-derived libraries of A. chlorechis,A. nitschei and A. squamigera and each encoded a single copy
of PLA
2
(Figure 1). Here, the first genomic sequences for the three cDNAs encoding phospholipase
A
2
precursors from Atheris bush viper venoms, are reported and were named PLA
2
-A.C., PLA
2
-A.N.
and PLA
2
-A.S., respectively. These data have been deposited in the Genbank Nucleotide Sequence
Database under the accession codes, KP11968, KP119683 and KP119684, respectively. The open-reading
frame amino acid sequences of these proteins exhibited a similar structural pattern: all contained a
putative 16-residue signal peptide sequence and a mature protein sequence consisting of 123 residues
for A. nitschei, 121 residues for A. chlorechis and 122 residues for A. squamigera. All contained a
conserved Histidine/Aspartic acid dyad-motif and a cysteine-rich sequence that defines the PLA
2
hydrolytic function.
Comparison of these three precursors, established by deduction from cloned cDNA, is shown
in the alignment in Figure 2. They exhibit a remarkable degree of identity: the signal peptides differ
by only one residue, with the mature proteins showing high similarity especially in the features of
the catalytic network and the primary metal binding site. Except for PLA
2
-A.N., with two additional
cysteine residues, the positions of the 12 cysteines are homologous in the three precursors, meaning
that this uniform character of disulfide bridging pattern should lead to similar crystal structures of
those three PLA2s.

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(a)
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(b)
Figure 1. Cont.

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(c)
Figure1.NucleotideandtranslatedaminoacidsequencesofthethreePhospholipaseA2(PLA2)
precursors:(a)PLA2‐A.C.fromA.chlorechis;(b)PLA2‐A.N.fromA.nitschei;and(c)PLA2‐A.S.from
A.squamigera.Theputativesignalsequencesaredouble‐underlined,thematureproteinsare
single‐underlinedandstopcodonsareindicatedbyasterisks.Polyadenylationsitesareunderlined.
Comparisonofthesethreeprecursors,establishedbydeductionfromclonedcDNA,isshownin
thealignmentinFigure2.Theyexhibitaremarkabledegreeofidentity:thesignalpeptidesdifferby
onlyoneresidue,withthematureproteinsshowinghighsimilarityespeciallyinthefeaturesofthe
catalyticnetworkandtheprimarymetalbindingsite.ExceptforPLA2‐A.N.,withtwoadditional
cysteineresidues,thepositionsofthe12cysteinesarehomologousinthethreeprecursors,meaning
thatthisuniformcharacterofdisulfidebridgingpatternshouldleadtosimilarcrystalstructuresof
thosethreePLA2s.

Figure2.AlignmentofphospholipaseA2proteinsfromthevenomsofA.chlorechis,PLA2‐A.C.;
A.nitschei,PLA2‐A.N.;andA.squamigera,PLA2‐A.S.Identicalaminoacidresiduesareshadedgray.Putative
signalpeptidesaremarkedandpositionally‐conservedcysteinesinalloftheproteinsareoutlinedinred
Figure 1.
Nucleotide and translated amino acid sequences of the three Phospholipase A
2
(PLA
2
)
precursors: (
a
) PLA
2
-A.C. from A. chlorechis; (
b
) PLA
2
-A.N. from A. nitschei; and (
c
) PLA
2
-A.S.
from A. squamigera. The putative signal sequences are double-underlined, the mature proteins are
single-underlined and stop codons are indicated by asterisks. Polyadenylation sites are underlined.
Toxins2016,8,1684of11

(c)
Figure1.NucleotideandtranslatedaminoacidsequencesofthethreePhospholipaseA2(PLA2)
precursors:(a)PLA2‐A.C.fromA.chlorechis;(b)PLA2‐A.N.fromA.nitschei;and(c)PLA2‐A.S.from
A.squamigera.Theputativesignalsequencesaredouble‐underlined,thematureproteinsare
single‐underlinedandstopcodonsareindicatedbyasterisks.Polyadenylationsitesareunderlined.
Comparisonofthesethreeprecursors,establishedbydeductionfromclonedcDNA,isshownin
thealignmentinFigure2.Theyexhibitaremarkabledegreeofidentity:thesignalpeptidesdifferby
onlyoneresidue,withthematureproteinsshowinghighsimilarityespeciallyinthefeaturesofthe
catalyticnetworkandtheprimarymetalbindingsite.ExceptforPLA2‐A.N.,withtwoadditional
cysteineresidues,thepositionsofthe12cysteinesarehomologousinthethreeprecursors,meaning
thatthisuniformcharacterofdisulfidebridgingpatternshouldleadtosimilarcrystalstructuresof
thosethreePLA2s.

Figure2.AlignmentofphospholipaseA2proteinsfromthevenomsofA.chlorechis,PLA2‐A.C.;
A.nitschei,PLA2‐A.N.;andA.squamigera,PLA2‐A.S.Identicalaminoacidresiduesareshadedgray.Putative
signalpeptidesaremarkedandpositionally‐conservedcysteinesinalloftheproteinsareoutlinedinred
Figure 2.
Alignment of phospholipase A
2
proteins from the venoms of A. chlorechis, PLA
2
-A.C.;
A. nitschei
, PLA
2
-A.N.; and A. squamigera, PLA
2
-A.S. Identical amino acid residues are shaded gray.
Putative signal peptides are marked and positionally-conserved cysteines in all of the proteins are
outlined in red boxes, with the two additional cysteine residues outlined by blue boxes. The numbering
of amino acid residues is indicated above the first sequence, and gaps are shown as dashes.
The open-reading frames of the three PLA
2
proteins were employed in Basic Local Alignment
Search Tool (BLAST) searches using the National Center for Biotechnological Information (NCBI)
on-line portal. The results indicated that they had a relatively high identity with Group II

Toxins 2016,8, 168 5 of 11
phospholipase A
2
s from Viperidae snake venoms (Table 1, Figure 2). These studies revealed some very
interesting characteristics of the three novel enzymes: PLA
2
-A.C. and PLA
2
-A.S., which both possessed
12 cysteines, showed a disparate alignment map. PLA
2
-A.S. showed some unexpected identities with
PLA
2
-A.N. that has 14 cysteines, and they were both obviously most similar to the phospholipases A
2
classified as Group IIA—the majority cluster. However, PLA
2
-A.C. is nearly identical to phospholipase
A
2
AAR06850.1 from Bitis gabonica [
21
], which falls within the extraordinary Group IIB PLA
2
s that lack
one disulfide-bridge conserved in the Group IIA as mentioned in the work of Six et al. [2].
Table 1.
Comparison of the results of similarity searches by use of the Basic Local Alignment Search
Tool (BLAST) tool on the Phospholipase A
2
(PLA
2
) precursor sequences obtained from the venoms
of A. chlorechis (PLA
2
-A.C.), A. nitschei (PLA
2
-A.N.), and A. squamigera (PLA
2
-A.S.). The accession
numbers shown here are unique identifiers of the recorded protein sequences archived in Genbank.
Protein Accession Number Organism Identities Group
PLA2-A.C. AAR06850.1 Bitis gabonica 96% GIIB
PLA2-A.N. AAK49823.1 Echis coloratus 71% GIIA
ACQ57801.1 Macrovipera lebetina 71% GIIA
PLA2-A.S. AAK49822 Echis coloratus 73% GIIA
ACQ57801.1 Macrovipera lebetina 72% GIIA
2.2. Identification and Structural Analysis of PLA2Proteins
A large range of components was successfully resolved following reverse phase High Performance
Liquid Chromatography (HPLC) fractionation of the crude lyophilized venoms from A. chlorechis,
A. nitschei
and A. squamigera. Initial PLA
2
activity screening of eluted HPLC fractions was performed
separately by agarose/egg yolk suspension plates (Supplementary materials Figure S1, Table S1). A
number of fractions were identified as possessing phospholipase A
2
activity and were displayed as
isolated peaks in each chromatogram (Figure 3).
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boxes,withthetwoadditionalcysteineresiduesoutlinedbyblueboxes.Thenumberingofaminoacid
residuesisindicatedabovethefirstsequence,andgapsareshownasdashes.
Theopen‐readingframesofthethreePLA2proteinswereemployedinBasicLocalAlignment
SearchTool(BLAST)searchesusingtheNationalCenterforBiotechnologicalInformation(NCBI)
on‐lineportal.TheresultsindicatedthattheyhadarelativelyhighidentitywithGroupII
phospholipaseA2sfromViperidaesnakevenoms(Table1,Figure2).Thesestudiesrevealedsome
veryinterestingcharacteristicsofthethreenovelenzymes:PLA2‐A.C.andPLA2‐A.S.,whichboth
possessed12cysteines,showedadisparatealignmentmap.PLA2‐A.S.showedsomeunexpected
identitieswithPLA2‐A.N.thathas14cysteines,andtheywerebothobviouslymostsimilartothe
phospholipasesA2classifiedasGroupIIA—themajoritycluster.However,PLA2‐A.C.isnearly
identicaltophospholipaseA2AAR06850.1fromBitisgabonica[21],whichfallswithinthe
extraordinaryGroupIIBPLA2sthatlackonedisulfide‐bridgeconservedintheGroupIIAas
mentionedintheworkofSixetal.[2].
Table1.ComparisonoftheresultsofsimilaritysearchesbyuseoftheBasicLocalAlignmentSearch
Tool(BLAST)toolonthePhospholipaseA2(PLA2)precursorsequencesobtainedfromthevenomsof
A.chlorechis(PLA2‐A.C.),A.nitschei(PLA2‐A.N.),andA.squamigera(PLA2‐A.S.).Theaccessionnumbers
shownhereareuniqueidentifiersoftherecordedproteinsequencesarchivedinGenbank.
ProteinAccessionNumbe
r
Organism IdentitiesGroup
PLA2-A.C. AAR06850.1 Bitisgabonica96%GIIB
PLA2‐A.N.AAK49823.1Echiscoloratus71%GIIA
ACQ57801.1Macroviperalebetina71%GIIA
PLA2‐A.S.AAK49822Echiscoloratus73%GIIA
ACQ57801.1Macroviperalebetina72%GIIA
2.2.IdentificationandStructuralAnalysisofPLA2Proteins
AlargerangeofcomponentswassuccessfullyresolvedfollowingreversephaseHigh
PerformanceLiquidChromatography(HPLC)fractionationofthecrudelyophilizedvenomsfromA.
chlorechis,A.nitscheiandA.squamigera.InitialPLA2activityscreeningofelutedHPLCfractionswas
performedseparatelybyagarose/eggyolksuspensionplates(SupplementarymaterialsFigureS1,
TableS1).AnumberoffractionswereidentifiedaspossessingphospholipaseA2activityandwere
displayedasisolatedpeaksineachchromatogram(Figure3).

(a)
Absorbance [mA]
Tim e [mm :ss]
-5
354
713
1073
1432
00:00 48:00 96:00 144:00 192:00 240:00
Figure 3. Cont.

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Toxins2016,8,1686of11
(b)
(c)
Figure3.RegionofreversephaseHighPerformanceLiquidChromatography(HPLC)profilesof
venomsfrom(a)A.chlorechis;(b)A.nitschei;and(c)A.squamigera,indicating(arrow)elution
position/retentiontimesofPLA2‐A.C.,PLA2‐A.N.andPLA2‐A.S.,respectively.
ThemolecularmassesofeachproteinweredeterminedbyMatrix‐assistedlaser
desorption/ionization‐TimeofFlight(MALDI‐TOF)massspectrometryandsinglemajorionswith
m/zratiosaround14KDawereresolvedwhichareinaccordancewithdatafromthecDNA‐deduced
aminoacidsequences.TheprimarystructuresweresubsequentlyunequivocallyconfirmedbyLiquid
chromatography–tandemmassspectrometry(LC/MS/MS)fragmentationsequencing(Table2).

Absorbance [mA]
Time [mm:ss]
-102
202
507
811
1115
00:00 48:00 96:00 144:00 192:00 240:00
Absorban ce [mA]
Tim e [mm :ss]
-32
344
720
1096
1472
00:00 48:00 96:00 144:00 192:00 240:00
Figure 3.
Region of reverse phase High Performance Liquid Chromatography (HPLC) profiles
of venoms from (
a
)A. chlorechis; (
b
)A. nitschei; and (
c
)A. squamigera, indicating (arrow) elution
position/retention times of PLA2-A.C., PLA2-A.N. and PLA2-A.S., respectively.
The molecular masses of each protein were determined by Matrix-assisted laser
desorption/ionization-Time of Flight (MALDI-TOF) mass spectrometry and single major ions with
m/zratios around 14 KDa were resolved which are in accordance with data from the cDNA-deduced
amino acid sequences. The primary structures were subsequently unequivocally confirmed by Liquid
chromatography–tandem mass spectrometry (LC/MS/MS) fragmentation sequencing (Table 2).

Toxins 2016,8, 168 7 of 11
Table 2.
Assignment of the Liquid chromatography–tandem mass spectrometry (LC/MS/MS)
identified Atheris venom protein fragments (A. chlorechis,A. nitschei and A. squamigera) from fractions
shown in Figure 3.
Species Retention
Time (min)
Average Mass
Observation
Average Mass
Calculation MS/MS-Derived Sequence
A. chlorechis
103–104 13,960.5 Da 13,964 Da HLEQFGNMIDHVSGR
CCFVHDCCYGK
MGTYDTK
ELCECDR
VAAICFGNNR
NTYNSK
A. nitschei
106–109 13,975 Da 13,979 Da NLFQFGSMIK
NAIMNYSAYGCYCGWGGQGKPQDATDR
DKDPCK
VNTYNDNYR
WYPSK
A. squamigera
105–107 13,840 Da 13,841 Da NLFQFR
NMIHK
NAVMNYSAYGCYCGWGGQGKPQDATDR
113 13,847 Da NLFQFR CCFVHDCCYGR
ELCECDR
CQEESEQC
3. Discussion
Whole venoms from A. chlorechis,A. nitschei and A. squamigera were subjected to reverse
phase HPLC fractionation, followed by mass spectrometric analysis of each fraction, resulting in
consistent identification of PLA
2
proteins with the predicted masses of mature proteins deduced from
cDNA-encoded precursors. It can be seen from Figure 3that active PLA
2
fractions from A. squamigera
venom seems to contain more than one component. However, as shown in Table 2, the spectrum of the
peaks at 106 and 113 min differed by only six mass units from the theoretical molecular mass of 13841
Da. The discrepant masses of these two peaks probably could be accounted for by several amino-acid
substitutions in the sequences of related isoenzymes which often occur in surface exposed residues
which play a key role in recognition of target proteins [
22
]. Although the spectrum of A. squamigera
exhibited the probable existence of PLA
2
enzyme isoforms in the venom, the tiny mass difference of
the two peaks indicated a very low possibility. Therefore, we suppose it could just be due to slight
differences in hydrophobic properties or the isoelectric points of the two subunits constituting the
PLA
2
homodimer (related to the elutropic theory of reverse-phase chromatography); even external
conditions, such as room temperature, stability of ionization, etc., could also be responsible. However,
as a consequence of intrinsic molecular diversity, single snake venom can contain up to 16 discrete
PLA
2
homologs with phospholipase activity [
23
], we cannot affirm that the precursors obtained by
molecular cloning in our study represent the only PLA2proteins occurring in these snake venoms.
Comparative sequence analyses have revealed a high conservation of functionally important
domains and disulfide bonding patterns among the different groups of phospholipase A
2
s. This can
be seen clearly by our alignment of Groups I and II PLA2s from Genbank and PLA2-A.C., PLA2-A.N.
and PLA
2
-A.S., from our original experimental data, as shown in Figure 4. The sequence identity as
well as the BLAST data shown in Table 1proves our hypothesis on the relationship of PLA
2
-A.C.,
PLA
2
-A.N. and PLA
2
-A.S. PLA
2
-A.N. and PLA
2
-A.S. share the same N-terminal sequence NLFQ-,
show higher similarity in functional amino-acid sequence and both belong to Group IIA. In contrast,
PLA
2
-A.C. has a different N-terminal amino acid sequence, HLEQ-, to the previous two PLA
2
s and
was identical to AAR 06850.1—the PLA
2
from Gaboon viper venom [
21
], which was identified as a
Group IIB PLA
2
. Structural comparisons provide a basis for speculation as to the novel phospholipase
A
2
protein metal binding sites, disulfide-bond assignments and residues participating in the catalytic
site. These three novel phospholipase A
2
s share, in common with the other PLA
2
s, the Ca
2+
binding
loop, active site, and pivotal amino-acid residues such as the His 48/Asp 49 core and calcium-binding
assistants, Tyr 28, Gly 30 and Gly 32 (shown in Figure 4). Many of the similar pre-determinations of
former newly-discovered PLA
2
s, derived from such homologies, have been confirmed by enzyme
activity assays and by X-ray crystallographic analysis. It is thus justifiable to consider the relationships

Toxins 2016,8, 168 8 of 11
between primary structures and enzyme properties of these three PLA
2
s and the effects of their key
structural frameworks as well as the location and types of residues involved in activity or toxicity.
Toxins2016,8,1688of11
assistants,Tyr28,Gly30andGly32(showninFigure4).Manyofthesimilarpre‐determinationsof
formernewly‐discoveredPLA2s,derivedfromsuchhomologies,havebeenconfirmedbyenzyme
activityassaysandbyX‐raycrystallographicanalysis.Itisthusjustifiabletoconsiderthe
relationshipsbetweenprimarystructuresandenzymepropertiesofthesethreePLA2sandtheeffects
oftheirkeystructuralframeworksaswellasthelocationandtypesofresiduesinvolvedin
activityortoxicity.


Figure4.AlignmentsofPLA2‐A.C.,PLA2‐A.N.,andPLA2‐A.S.structuresandtheselectedPLA2sfrom
theGenbankdatabase.Identicalresiduesareshadedwithblackandconservedresidueswithgray.
Thefunctionalregionsareunderlinedandthehighly‐conservedactivecenterisexhibitedusingan
arrow.Thenumberofaminoacidsisillustratedabovethefirstsequence,andtheaccessionnumbers
ofeachproteinattheNationalCenterforBiotechnologyInformation(NCBI)Genbankismarkedat
thefrontofthearray.TheN‐terminalsequencesofPLA2‐A.N.andPLA2‐A.S.areindicatedbyared
box;PLA2‐A.C.andthesimilarPLA2AAR06850.1areindicatedbyabluebox.
LittleisknownaboutthetoxicityofenzymesfromvenomsoftheAtherisgenus,althoughthe
coagulationdisturbances,hemorrhaging,acuterenalfailureandhypertensionfollowingsnakebite,
canallbefatal.Ourgrouphascharacterizedthedisintegrinproteinsinapreviousstudy[20]and
thesecouldexplaintheplateletaggregationinhibitionandbloodclottingblockingtoxicityofthe
Atherisvenoms.Itwouldthusbeinterestingtopredictpharmacologicaleffectsandlookforthe
Figure 4.
Alignments of PLA
2
-A.C., PLA
2
-A.N., and PLA
2
-A.S. structures and the selected PLA
2
s from
the Genbank database. Identical residues are shaded with black and conserved residues with gray.
The functional regions are underlined and the highly-conserved active center is exhibited using an
arrow. The number of amino acids is illustrated above the first sequence, and the accession numbers of
each protein at the National Center for Biotechnology Information (NCBI) Genbank is marked at the
front of the array. The N-terminal sequences of PLA
2
-A.N. and PLA
2
-A.S. are indicated by a red box;
PLA2-A.C. and the similar PLA2AAR06850.1 are indicated by a blue box.
Little is known about the toxicity of enzymes from venoms of the Atheris genus, although the
coagulation disturbances, hemorrhaging, acute renal failure and hypertension following snakebite,
can all be fatal. Our group has characterized the disintegrin proteins in a previous study [
20
] and these
could explain the platelet aggregation inhibition and blood clotting blocking toxicity of the Atheris
venoms. It would thus be interesting to predict pharmacological effects and look for the experimental
evidence for the involvement of these novel phospholipase A
2
s
.
As direct experimental proof is not

Toxins 2016,8, 168 9 of 11
yet available, it is not possible to predict the properties of these proteins at the moment, and thus the
question remains open.
4. Experimental
4.1. Materials
Lyophilized venoms from the Western bush viper (A. chlorechis), the Great Lakes bush viper
(A. nitschei)
and the Variable bush viper (A. squamigera), were obtained from a commercial source
(Latoxan, Valence, France).
4.2. Molecular Cloning of the Phospholipase A2Precursor-Encoding cDNAs
Five milligrams of each venom sample were separately dissolved in 1 mL of lysis/binding
buffer (Dynal Biotech, Wirral, UK); the polyadenylated mRNA was extracted by oligo-dT Dynabeads
(Dynal Biotech, Wirral, UK) and was isolated as described by the manufacturer. A SMART
TM
Rapid
Amplification of cDNA ends (RACE) cDNA Amplification kit (BD Clontech, Basingstoke, UK) was
then employed. Both 5
'
-and 3
'
cDNA ends were synthesized using SMART (Switching Mechanism
At 5' end of RNA Transcription) cDNA synthesis technology. The reaction was performed by using a
nested universal primer (NUP) and a sense primer (S1: 5
'
-ATGAGGACTCTCTGGATAGTGGCCG-3
'
)
that was designed to be complementary to a highly-conserved domain of the 5
'
-untranslated region
of previously-characterized phospholipase A
2
cDNAs from related snake species(Accession Nos.:
GU012263, AY430405, DQ288157, AM114013 and DQ295886). The DNA fragments of expected size
were purified and inserted into the pGEM-T vector system (Promega, Southampton, UK). Plasmid
DNAs selected after transformation by Escherichia coli (E. coli) cells, were amplified and then purified by
use of a Rapid HiYield
™
Gel/PCR DNA Extraction Kit (RBC Bioscience, Duren, Germany). Sequences
of the products were obtained use of an automated 3730 capillary DNA sequencer (Applied Biosystems,
Wamington, UK).
4.3. Chromatographic Fractionation and Activity Determination
Fractionation of components of all three snake venoms was achieved by gradient reverse-phase
HPLC (Cecil CE 4200 Adept, Cambridge, UK), using a Phenomenex C
5
(300 A, 250
ˆ
10 mm) column.
Five milligram samples from each venom were separately reconstituted in 0.05% (v/v) trifluoroacetic
acid (TFA)/water, injected onto the column and eluted with a linear gradient formed from 0.05/99.5
(v/v) TFA/water to 0.05/19.95/80.0 (v/v/v) TFA/water/acetonitrile, by increasing the percentage of
the latter buffer gradually over 240 min at a flow rate of 1 mL/min. The fractions were collected at
minute intervals and samples (200
µ
L) were removed from each fraction in triplicate, concentrated and
stored at 4 ˝C prior to functional studies.
The determination of phospholipase A
2
(PLA
2
) activity in fractions was achieved by use of
agarose/egg yolk suspension plates containing phosphatidylcholine as substrate. A 20% portion of
HPLC fractions from each of the snake venoms were concentrated and reconstituted in 0.1% Albumin
from bovine serum/phosphate buffer saline (BSA/PBS). The plates were prepared with 4% egg
yolk emulsion (Oxoid, Basingstoke, UK) in agarose solution in the presence of calcium, punched
with 8 wells per plate
and each well was loaded with 10
µ
L sample. The diameters of cleared egg
yolk zones after the 20 h incubation (37
˝
C) were measured to locate the fractions that possessed
PLA2activity.
4.4. Identification and Structural Investigations
Reverse phase High Performance Liquid Chromatography (RP-HPLC) fractions possessing PLA
2
activity were subjected to matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry
(MALDI–TOF MS) on a linear time-of-flight Voyager DE mass spectrometer (Perseptive Biosystems,
Warrington, UK) in positive detection mode using
α
-cyano-4-hydroxycinnamic acid as the matrix.

Toxins 2016,8, 168 10 of 11
Internal mass calibration of the instrument with known standards established the accuracy of mass
determination as
˘
0.1%. Those fractions with masses of approximately 14 kDa coincident with those
deduced from cloned cDNAs were digested using trypsin and the resultant tryptic peptides were
subjected to primary structural analysis by use of an LCQ Fleet electrospray ion-trap mass spectrometer
(Thermo, Cheshire, UK).
4.5. Ethical Statement
Venoms used in this study were obtained non-invasively from captive snakes.
Supplementary Materials:
The following are available online at www.mdpi.com/2072-6651/8/6/168/s1,
Figure S1:
Dose response curve of diameter of PLA
2
clearance zones against log
10
enzyme concentration of
the standard PLA
2
from honey bee venom. Linear regression analysis was employed, and the regression equation
(Y= 0.7039X
´
0.34) was obtained to analyse the equivalent PLA
2
content of sample venoms. The clearance zones
of samples in the agarose/egg yolk suspension plate assay were used to estimate content of PLA
2
s in sample
venom fractions. The results are summarised in Table S1. Table S1: Clearance zones and corresponding estimated
quantities of PLA2in sample venoms from A. chlorechis,A. nitschei and A. squamigera.
Acknowledgments:
This project was supported by the National Science Foundation, China (Grant No. 81402842)
and the Natural Science Foundation of Fujian Province, China (Grant No. 2015J05162).
Author Contributions:
He Wang, Xiaole Chen and Mei Zhou conceived and designed the experiments; He
Wang performed the experiments. He Wang and Chris Shaw analyzed the data. Tianbao Chen and Lei Wang
contributed reagents/materials/analysis tools. He Wang wrote the manuscript. Chris Shaw and Xiaole Chen
revised the manuscript.
Conflicts of Interest: The authors declare that they have no conflicts of interest.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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