toxins
Article
Comparative Venomics of the Vipera ammodytes
transcaucasiana and Vipera ammodytes montandoni
from Turkey Provides Insights into Kinship
Benjamin-Florian Hempel 1ID , Maik Damm 1, Bayram Göçmen 2ID , Mert Karis 2ID ,
Mehmet Anıl Oguz 2, Ayse Nalbantsoy 3,* and Roderich D. Süssmuth 1,*
1Department of Chemistry, Technische Universität Berlin, 10623 Berlin, Germany;
2Department of Biology, Ege University, 35100 Izmir, Turkey; [email protected] (B.G.);
3Department of Bioengineering, Ege University, 35100 Izmir, Turkey
*Correspondence: [email protected] (A.N.); [email protected] (R.D.S.)
Tel.: +49-30-314-24205 (R.D.S.)
Received: 22 September 2017; Accepted: 28 December 2017; Published: 1 January 2018
Abstract:
The Nose-horned Viper (Vipera ammodytes) is one of the most widespread and venomous
snakes in Europe, which causes high frequent snakebite accidents. The first comprehensive venom
characterization of the regional endemic Transcaucasian Nose-horned Viper (Vipera ammodytes
transcaucasiana) and the Transdanubian Sand Viper (Vipera ammodytes montandoni) is reported
employing a combination of intact mass profiling and bottom-up proteomics. The bottom-up analysis
of both subspecies identified the major snake protein families of viper venoms. Furthermore, intact
mass profiling revealed the presence of two tripeptidic metalloprotease inhibitors and their precursors.
While previous reports applied multivariate analysis techniques to clarify the taxonomic status of the
subspecies, an accurate classification of Vipera ammodytes transcaucasiana is still part of the ongoing
research. The comparative analysis of the viper venoms on the proteome level reveals a close
relationship between the Vipera ammodytes subspecies, which could be considered to clarify the
classification of the Transcaucasian Nose-horned Viper. However, the slightly different ratio of
some venom components could be indicating interspecific variations of the two studied subspecies
or intraspecies alternations based on small sample size. Additionally, we performed a bioactivity
screening with the crude venoms against several human cancerous and non-cancerous cell lines,
which showed interesting results against a human breast adenocarcinoma epithelial cell line. Several
fractions of Vipera a. transcaucasiana demonstrated a strong cytotoxic effect on triple negative MDA
MB 231 breast cancer cells.
Keywords:
Viperidae;Vipera ammodytes; Transcaucasian Nose-horned Viper; Transdanubian Sand
Viper; snake venomics; intact mass profiling; tripeptide metalloprotease inhibitor; cytotoxicity
Key Contribution:
The detailed characterization and comparison of the venom proteome Vipera
ammodytes transcaucasiana and Vipera ammodytes montandoni showed an impressive matching of
the venom composition, which could help to overcome the question of the taxonomic status of
Vipera ammodytes transcaucasiana. Additional bioactivity screenings against several cancerous and
non-cancerous cell lines showed promising results against breast cancer cells.
1. Introduction
Venom research has an ongoing significance for various disciplines and applications ranging
from drug development, pharmacology for rational antivenom production even to the cosmetics
Toxins 2018,10, 23; doi:10.3390/toxins10010023 www.mdpi.com/journal/toxins
Toxins 2018,10, 23 2 of 24
industry [
1
–
3
]. After the discovery of the first venom-derived therapeutic, Captopril, in 1975, which
was developed from the Brazilian pit viper (Bothrops jararaca), in the following years, investigations
of several other venomous snakes revealed further venom-based drugs with different medical
applications [
4
,
5
]. Nevertheless, there is still an uncountable number of venomous animals in diverse
habitats to be suspected, of which only a small part of venom proteomes have been characterized,
and as a consequence the potential for new applications of these venoms and individual components
thereof still have to be explored [3,6,7].
The advances in “-omics” technologies allowed for the characterization of an increasing number
of animal venoms and plays a pivotal role for the development of new potential drugs against
several human diseases. The increase in sensitivity and the development of soft ionization methods,
e.g., electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI), for mass
spectrometry and next-generation high-throughput sequencing dramatically enhanced the analysis of
venoms [
3
,
8
–
10
]. Nowadays, there exist different well-established protocols to characterize the venom
in its entirety. The so-called bottom-up strategies can be divided in gel-based approaches and liquid
chromatographic (LC)-based approaches. A combination of both strategies, termed “snake venomics”,
uses both separation techniques successively followed by an in-gel digestion of excised protein bands
and a mass spectrometric measurement by tandem mass analysis [
11
–
13
]. Recently described methods
for the proteomic analysis of snake venoms include the “intact mass profiling”, which directly separate
the components out of the crude venom without any previous fractionation. The power of this tool has
already been demonstrated at the example of whole snake venom proteomes [
14
,
15
]. The intact mass
analysis of native proteins compared to chemically reduced proteins allows for a classification, based
on the number of existing intra- and intermolecular disulfide bonds, and represents an important
characteristic of different viper-venom protein families [
16
–
18
]. However, the method is critical for
proteins of higher molecular masses, e.g., snake venom metalloproteases, because the high resolution
of accurate isotopic masses becomes challenging [
15
]. The subsequent step to the intact mass profiling
is the MS/MS analysis by collision-induced dissociation (CID) of intact molecular masses, termed
“top-down venomics”. The major advantage of this approach is a high-throughput analysis of venoms
without the necessity of further pre-MS separation steps. A decisive drawback is the application
mainly limited to peptides and small-sized proteins (>15 kDa), which are the main constituents of
Elapidae (e.g., three-finger toxins, Kunitz-type inhibitor, etc.) [
19
–
21
]. In contrast, Viperidae venoms
contain higher molecular mass components that makes them less suitable for the top-down approach
as Elapidae venoms [
14
,
15
]. Finally, the mass spectrometry-based absolute intact mass quantification
by isotope dilution is a further cutting-edge approach, which could replace the semi-quantitative
densitometric determination [17,22,23].
The combination of several workflows allows for an encompassing characterization of different
kinds of venoms. In particular, the venom of vipers is a promising source of new substances and
therapeutics, due to their different venom compositions [
1
,
4
]. They are distributed in a wide range all
over the world, and are especially located around the Mediterranean Sea [
24
]. A great variety of habitats
and zones of subtropical climate along the north coast side of Turkey provides suitable places to shelter
for many species that belong to the Viperidae family [
25
,
26
]. Important major protein families found in
analyzed viperid venoms are snake venom metalloproteases (svMP), snake venom serine proteases
(svSP), hyaluronidases, 5
0
-nucleotidase, phospholipases A
2
(PLA
2
), disintegrins, C-type lectin like
proteins (CTL), cysteine-rich secretory proteins (CRISP), natriuretic peptides, bradykinin-potentiating
peptides (BPP), nerve growth factors (NGF), snake venom vascular endothelial growth factors (VEGF-F)
and Kunitz-type protease inhibitors [27,28].
Our ongoing studies on snake venoms focus on the venom characterization of unrecorded Vipera in
the Turkish area and initial cytotoxicity screenings against cancerous as well as non-cancerous cell lines
of potent bioactive peptides and proteins. From this point of view, we aimed to screen viper venoms
from different regions of Turkey. For this purpose, the regional endemic Transcaucasian Nose-horned
Viper (Vipera ammodytes transcaucasiana) from central Anatolia and the V. a. montandoni from Northwest
Toxins 2018,10, 23 3 of 24
of Turkey (Turkish Thrace) were chosen for a comparative venom investigation. The Nose-horned Viper
(Vipera ammodytes) is one of the most venomous snakes in Europe and common pathophysiological
conditions range from local tissue damage, hemorrhage, pain, paralysis up to necrosis and in some cases
even death [29,30]. After the description of Vipera ammodytes ammodytes (Linnaeus, 1758), five further
subspecies have been described: V. a. meridionalis [
31
], V. a. montandoni [
32
], V. a. transcaucasiana [
33
],
V. a. ruffoi [
34
] and V. a. gregorwallneri [
35
]. V. a. transcaucasiana is considered a separate species by some
authors [
36
]. Heckes et al. (2005) and Tomovic (2006) accepted only four valid taxa for V. ammodytes
(V. a. ammodytes,V. a. meridionalis,V. a. montandoni and V. a. transcaucasiana) with an extensive
investigation on the species [
37
,
38
]. Phylogenetic and phylogeographic studies, using mtDNA gene
sequences obtained from cytochrome b(cyt b), 16S rRNA and the noncoding control region, supported
the validation of subspecies status of V. a. ammodytes,V. a. meridionalis and V. a. montandoni, but in turn
V. a. ruffoi and V. a. gregorwallneri were only accepted as synonyms to the nominotypic subspecies,
V. a. ammodytes. In addition, the taxonomic status of V. a. transcaucasiana was tentatively classified as
subspecies due to a low sample size [39].
The occurrence of Vipera ammodytes distributes around the Mediterranean Sea and reaches from
the Alps over to Turkey, Georgia, Azerbaijan and Iran. The Transcaucasian Nose-horned Viper (Vipera
ammodytes transcaucasiana (Vat)) shows a distribution in the Northeast of Turkey and sections of
Georgia along the Black Sea coast and some inland provinces in Turkey (see Figure 1, red) [
37
,
40
].
The Transdanubian Sand Viper (Vipera ammodytes montandoni (Vam)) is spread from Turkish Thrace,
Bulgaria to Romania and shares its distribution area in parts with all three other subspecies (see Figure 1,
blue) [
37
]. Beside those previously mentioned, there exist two further subspecies whose venoms were
already characterized: The Western Sand Viper (Vipera ammodytes ammodytes) can be found from the
Alps over Croatia to the borders of Macedonia (see Figure 1, yellow) and the Eastern Sand Viper (Vipera
ammodytes meridionalis), only endemic in Greece and several Hellenic islands (see Figure 1, green) [
41
].
All subspecies of Vipera ammodytes can be found from sea level up to 2000 m a.s.l. in many kinds of
suitable habitats (forests, meadows, arid regions, rocky areas, and even sandy coastal parts), thus
there is no special habitat selectivity. The Nose-horned viper (Vipera ammodytes) is one of the most and
venomous species in Europe and therefore of significance for public health [41,42].
Previous investigations on the neutralization of lethality by several antisera against Vipera
ammodytes subspecies revealed low paraspecific neutralization potency [
43
,
44
]. Therefore, the
elucidation of the undescribed venom proteome is significant for public health and could help to
bypass the lack of sufficient venom neutralization. Here, we give deeper insight into the composition of
the venom proteome and peptidome of the two Nose-Horned vipers by bottom-up venomics and an
intact mass profiling of the crude venoms. The detailed characterization and comparison of the venom
proteomes with other subspecies showed a remarkable matching of the venom components, which
could be an additional helpful tool to overcome the controversial question of the taxonomic status of
Vipera ammodytes transcaucasiana in connection with the phylogentic analysis [39].
Toxins 2018,10, 23 4 of 24
Toxins2018,10,23 3of23
fromNorthwestofTurkey(TurkishThrace)werechosenforacomparativevenominvestigation.The
Nose‐hornedViper(Viperaammodytes)isoneofthemostvenomoussnakesinEuropeandcommon
pathophysiologicalconditionsrangefromlocaltissuedamage,hemorrhage,pain,paralysisupto
necrosisandinsomecasesevendeath[29,30].AfterthedescriptionofViperaammodytesammodytes
(Linnaeus,1758),fivefurthersubspecieshavebeendescribed:V.a.meridionalis[31],V.a.montandoni
[32],V.a.transcaucasiana[33],V.a.ruffoi[34]andV.a.gregorwallneri[35].V.a.transcaucasianais
consideredaseparatespeciesbysomeauthors[36].Heckesetal.(2005)andTomovic(2006)accepted
onlyfourvalidtaxaforV.ammodytes(V.a.ammodytes,V.a.meridionalis,V.a.montandoniandV.a.
transcaucasiana)withanextensiveinvestigationonthespecies[37,38].Phylogeneticand
phylogeographicstudies,usingmtDNAgenesequencesobtainedfromcytochromeb(cytb),16S
rRNAandthenoncodingcontrolregion,supportedthevalidationofsubspeciesstatusofV.a.
ammodytes,V.a.meridionalisandV.a.montandoni,butinturnV.a.ruffoiandV.a.gregorwallneriwere
onlyacceptedassynonymstothenominotypicsubspecies,V.a.ammodytes.Inaddition,thetaxonomic
statusofV.a.transcaucasianawastentativelyclassifiedassubspeciesduetoalowsamplesize[39].
TheoccurrenceofViperaammodytesdistributesaroundtheMediterraneanSeaandreachesfrom
theAlpsovertoTurkey,Georgia,AzerbaijanandIran.TheTranscaucasianNose‐hornedViper
(Viperaammodytestranscaucasiana(Vat))showsadistributionintheNortheastofTurkeyandsections
ofGeorgiaalongtheBlackSeacoastandsomeinlandprovincesinTurkey(seeFigure1,red)[37,40].
TheTransdanubianSandViper(Viperaammodytesmontandoni(Vam))isspreadfromTurkishThrace,
BulgariatoRomaniaandsharesitsdistributionareainpartswithallthreeothersubspecies(see
Figure1,blue)[37].Besidethosepreviouslymentioned,thereexisttwofurthersubspecieswhose
venomswerealreadycharacterized:TheWesternSandViper(Viperaammodytesammodytes)canbe
foundfromtheAlpsoverCroatiatothebordersofMacedonia(seeFigure1,yellow)andtheEastern
SandViper(Viperaammodytesmeridionalis),onlyendemicinGreeceandseveralHellenicislands(see
Figure1,green)[41].AllsubspeciesofViperaammodytescanbefoundfromsealevelupto2000m
a.s.l.inmanykindsofsuitablehabitats(forests,meadows,aridregions,rockyareas,andevensandy
coastalparts),thusthereisnospecialhabitatselectivity.
TheNose‐hornedviper(Viperaammodytes)
isoneofthemostandvenomousspeciesinEuropeandthereforeofsignificanceforpublichealth
[41,42].
Figure1.GeographicaldistributionofsubspeciesfromViperaammodytes.Thedistributionareasofthe
fourViperaammodytessubspeciesarehighlightedincolor:V.a.ammodytes(yellow),V.a.montandoni
(blue),V.a.meridionalis(green)andV.a.transcaucasiana(red).Overlappingdistributionareasare
Figure 1.
Geographical distribution of subspecies from Vipera ammodytes. The distribution areas of the
four Vipera ammodytes subspecies are highlighted in color: V. a. ammodytes (yellow), V. a. montandoni
(blue), V. a. meridionalis (green) and V. a. transcaucasiana (red). Overlapping distribution areas
are highlighted by shaded colors. The locations for catches of V. a. montandoni (star, blue) and
V. a. transcaucasiana (star, red) are marked and exemplary snake habitats are shown.
2. Results and Discussion
2.1. The Venom Proteome
2.1.1. Intact Mass Profiling
First, we applied an intact venom molecular mass profiling to obtain an overview of molecular
masses from all venom components, including low abundant and low molecular mass compounds.
Therefore, the crude venom as well as the RP-HPLC separated peptide fractions were used. The initial
profiling of Vipera ammodytes transcaucasiana (Vat) revealed 117 molecular masses for different venom
components (see Figure 2a, Tables 1and S1): 55 (<1 kDa), 11 (1–3 kDa) and 28 (3–9 kDa), which
represents the peptide part of the venom in total with 13.49%. Higher molecular masses from 10 to
28 kDa were detected 23 times in the following composition: 1 (10 kDa), 14 (12–16 kDa), 2 (21 kDa) and
6 (25–28 kDa). The venom of Vipera ammodytes montandoni (Vam) showed a comparable distribution
pattern with 115 different venom components (see Figure 2b, Tables 1and S2): 47 (<1 kDa), 26 (1–3 kDa)
and 19 (3–8 kDa), which corresponds to a slightly higher peptide content of 17.49%. We also found
25 components with molecular masses between 13 and 34 kDa: 10 (13–16 kDa), 2 (21 kDa), 7 (24–27 kDa)
and 6 (32 kDa).
Toxins 2018,10, 23 5 of 24
Toxins2018,10,23 5of23
Figure2.IntactmolecularmassprofilesofV.a.transcaucasiana(Vat)andV.a.montandoni.(Vam).The
totalioncounts(TIC)ofcrudevenomsfrom:(a)Vat;and(b)VamweremeasuredbyHPLC‐ESI‐MS.
ThepeaknomenclatureisbasedonthechromatogramfractionsandisshowninFigure3.The
identifiedmolecularmassesofintactproteinsandpeptidesarelistedforVatinTableS1andVamin
TableS2.
Figure 2.
Intact molecular mass profiles of V. a. transcaucasiana (Vat) and V. a. montandoni. (Vam). The
total ion counts (TIC) of crude venoms from: (
a
) Vat; and (
b
) Vam were measured by HPLC-ESI-MS.
The peak nomenclature is based on the chromatogram fractions and is shown in Figure 3. The identified
molecular masses of intact proteins and peptides are listed for Vat in Table S1 and Vam in Table S2.
Toxins2018,10,23 6of23
Figure3.ChromatogramsofV.a.transcaucasiana(Vat)andV.a.montandoni.(Vam)venomsseparated
bysemi‐preparativereversed‐phaseHPLC.Venomseparationof:(a)Vat;and(b)Vamwasperformed
byaSupelcoDiscoveryBIOwidePoreC18‐3RP‐HPLCcolumnandUVabsorbancemeasuredat214
nm.
Furthermore,theinitialmassprofilingenabledustoidentifytwopeptidesasmembersofthe
tripeptidemetalloproteaseinhibitor(svMP‐i)family.Theproteaseinhibitorsservetoavoiding
damagesduringstorageofthevenomintheglandtissueaswellastopreventautoproteolysisofthe
venom.Protectiveeffectshavebeendescribedforthewell‐knownendogenouspyroglutamic
tripeptidemetalloproteaseinhibitors,e.g.,thesmalltripeptidespEQWandpENW[45].Theirstrong
inhibitoryeffectagainstsvMPfromdifferentviperswasintensivelystudied[46–48].Inourstudies,
thevenomsofV.a.transcaucasianaandV.a.montandoniexhibittwomolecularmasseswithm/z444.23
andm/z430.17(seeFigure4andTablesS1andS2).Whilethemolecularmassofm/z444.23
correspondstoawellseparatedpeptidesignalfoundinthevenomsofVat(seeFigure2;Peak4/6)
andVam(seeFigure2;Peak3,4/5),theintensityofthesignalatm/z430.17forVat(seeFigure2;Peak
7)andVam(seeFigure2;Peak6)islessprominent.
Figure 3. Cont.
Toxins 2018,10, 23 6 of 24
Toxins2018,10,23 6of23
Figure3.ChromatogramsofV.a.transcaucasiana(Vat)andV.a.montandoni.(Vam)venomsseparated
bysemi‐preparativereversed‐phaseHPLC.Venomseparationof:(a)Vat;and(b)Vamwasperformed
byaSupelcoDiscoveryBIOwidePoreC18‐3RP‐HPLCcolumnandUVabsorbancemeasuredat214
nm.
Furthermore,theinitialmassprofilingenabledustoidentifytwopeptidesasmembersofthe
tripeptidemetalloproteaseinhibitor(svMP‐i)family.Theproteaseinhibitorsservetoavoiding
damagesduringstorageofthevenomintheglandtissueaswellastopreventautoproteolysisofthe
venom.Protectiveeffectshavebeendescribedforthewell‐knownendogenouspyroglutamic
tripeptidemetalloproteaseinhibitors,e.g.,thesmalltripeptidespEQWandpENW[45].Theirstrong
inhibitoryeffectagainstsvMPfromdifferentviperswasintensivelystudied[46–48].Inourstudies,
thevenomsofV.a.transcaucasianaandV.a.montandoniexhibittwomolecularmasseswithm/z444.23
andm/z430.17(seeFigure4andTablesS1andS2).Whilethemolecularmassofm/z444.23
correspondstoawellseparatedpeptidesignalfoundinthevenomsofVat(seeFigure2;Peak4/6)
andVam(seeFigure2;Peak3,4/5),theintensityofthesignalatm/z430.17forVat(seeFigure2;Peak
7)andVam(seeFigure2;Peak6)islessprominent.
Figure 3.
Chromatograms of V. a. transcaucasiana (Vat) and V. a. montandoni. (Vam) venoms separated
by semi-preparative reversed-phase HPLC. Venom separation of: (
a
) Vat; and (
b
) Vam was performed
by a Supelco Discovery BIO wide Pore C18-3 RP-HPLC column and UV absorbance measured at
214 nm.
Furthermore, the initial mass profiling enabled us to identify two peptides as members of
the tripeptide metalloprotease inhibitor (svMP-i) family. The protease inhibitors serve to avoiding
damages during storage of the venom in the gland tissue as well as to prevent autoproteolysis of
the venom. Protective effects have been described for the well-known endogenous pyroglutamic
tripeptide metalloprotease inhibitors, e.g., the small tripeptides pEQW and pENW [
45
]. Their strong
inhibitory effect against svMP from different vipers was intensively studied [
46
–
48
]. In our studies, the
venoms of V. a. transcaucasiana and V. a. montandoni exhibit two molecular masses with m/z444.23 and
m/z430.17 (see Figure 4and Tables S1 and S2). While the molecular mass of m/z444.23 corresponds
to a well separated peptide signal found in the venoms of Vat (see Figure 2; Peak 4/6) and Vam (see
Figure 2; Peak 3, 4/5), the intensity of the signal at m/z430.17 for Vat (see Figure 2; Peak 7) and Vam
(see Figure 2; Peak 6) is less prominent.
Accordingly, the measured mass of m/z430.17 matches to the predicted monoisotopic mass of the
pyroglutamic tripeptide pENW, while the measured m/z444.23 matches to the predicted monoisotopic
mass of the pyroglutamic tripeptide pEKW. The identities of the pyroglutamic tripeptides pEKW and
pENW were ultimately confirmed by ESI-MS/MS experiments (see Figure 4). In addition, comparison
with spectra for the pEKW inhibitor from the literature coincides with our experimentally observed
fragmentation pattern [
49
]. As an additional evidence for tripeptide metalloprotease inhibitors in the
venom of both vipers, the presence of several Gly- and Pro-rich protein fragments of the endogenous
precursors were detected in the venoms of V. a. transcaucasiana as well as V. a. montandoni [
50
].
The peptide PEGPPLMEPHE, a previously described tripeptide precursor fragment [
51
] from the
related snake V. a. ammodytes, was detected in both investigated venoms (see Figures S1 and S2 and
Tables S1 and S2, Peak 9 and 8). Furthermore, several other precursor peptide fragments of this type
were detected in the venoms of Vat (GGGGGGW, PPQMPGPKVPP) and Vam (DNEPPKKVPPN)
(see Figures S3–S6 and Tables S1 and S2). Taken together, the metalloprotease inhibitors and their
precursor peptides form the major share of the peptidome of Vat (28.80%) as well as of Vam (45.93%),
with the tripeptide pEKW as the main component. Even if details on secretion and processing of the
tripeptides are still unknown, a formation of the pyroglutamic inhibitors is assumed to happen during
the exocytosis. A processing in the gland lumen itself is rather unlikely based on missing glutaminyl
cyclases [49].
Toxins 2018,10, 23 7 of 24
Table 1.
Venom proteins and peptides identified from Vipera ammodytes transcaucasiana and Vipera ammodytes montandoni. Venomic components were assigned by crude
venom intact mass profiling and bottom-up approach. Peak numbers and retention time (RT) based on RP-HPLC (see Figure 3) and TIC (see Figure 2) annotation.
SDS-PAGE and intact mass profile analysis provided the molecular weight. Most abundant masses are asterisked (*). For peptide fractions, only the most abundant
masses were noted. Detailed lists of all exhibit masses are mentioned in Tables S1 and S2.
V. a. transcaucasiana V. a. montandoni
RT
(min)
Fraction
No. Protein Species SDS PAGE
Mav (kDa) Most Abundant IMP (m/z)RT
(min)
Fraction
No. Protein Species SDS PAGE
Mav (kDa)
Most Abundant
IMP (m/z)
2.14 1 unknown - 571.25 * - - - - -
3.45 2 unknown - 484.20 * 3.46 1 unknown - 484.21 *
- - - - - 6.45 2 svMP-i, unknown - 1234.65 *
12.86 3 unknown - 600.32 * - - - - -
15.48 4 svMP-i, svMP
unknown - 547.22 * 15.43 3 svMP-i, unknown - 444.22 *
19.47 5 svMP, unknown - 814.35 * 19.19 4 svMP-i, unknown - 444.23 *
21.08 6 svMP-i, unknown - 444.22 * 20.48 5 svMP-i, unknown - 444.22 *
24.49 7 svMP-i, unknown - 430.17 * 24.52 6 svMP-i, unknown - 430.17 *
27.85 8 unknown - 569.28 * 26.99 7 unknown - 1072.60 *
- - - - - 31.95 8 svMP-i, unknown - 3930.96 *
33.07 9 svMP-i, unknown - 5775.64 * 33.02 9 unknown - 3761.72 *
33.86 10 unknown - 4176.85 * 33.91 10 unknown - 3796.72 *
34.95 11 BPP, unknown - 3769.75 * 35.20 11 unknown - 3796.73 *
36.97 12 unknown - 1143.64 * 36.44 12 unknown - 1144.62 *
- - - - - 39.50 13 unknown - 1314.73 *
40.43 13 unknown - 1143.64 * 40.75 14 unknown - 1144.62 *
42.44 14 svMP-i, unknown - 1143.64 * 42.94 15 unknown - -
46.90 15 VEGF-F 15 * - 47.16 16 VEGF-F 14 * 1159.59 *
- - - - - 48.69 17 VEGF-F 14 * 1159.59 *
54.79 16 VEGF-F, svMP,
unknown 15, 27 *, 50 10,676.97, 21,311.88 * 55.03 18 unknown 14, 25 * 21,199.58 *, 21,298.85
- - - - - 58.69 19 unknown - 13,553.82 *, 13,590.75
Toxins 2018,10, 23 8 of 24
Table 1. Cont.
V. a. transcaucasiana V. a. montandoni
RT
(min)
Fraction
No. Protein Species SDS PAGE
Mav (kDa) Most Abundant IMP (m/z)RT
(min)
Fraction
No. Protein Species SDS PAGE
Mav (kDa)
Most Abundant
IMP (m/z)
61.54 17 PLA2, unknown 13 *, 25, 40 13,553.83 *, 13,590.76, 13,814.21,
13,842.19, 13,911.15 62.75 20 PLA213 * 13,553.82 *, 13,890.28,
13,988.22
63.85 18 PLA213 *, 25, 40 13,918.28 *, 14,016.22 - - - - -
66.42 19 PLA2, CRISP 13, 25 *, 50 12,346.55, 24,653.41 *, 24,752.38,
24,848.30 66.68 21 PLA2, CRISP 13.23 * 24,654.40 *, 24,750.41
- - - - 69.08 22 CRISP 23 * 24,547.04 *
71.28 20 PLA2, svMP, CRISP 13, 22.5 *, 27.5,
60 13,624.69, 13,625.73, 13,676.78,
24,515.96 * 71.38 23 PLA213, 21 * 13,624.69
- - - - - 74.21 24 PLA213 *, 21 13,676.81 *
- - - - - 76.93 25
PLA
2
, svSP, unknown
15, 23, 37 * -
78.24 21 svSP 35 * - - - - - -
78.90 22 unknown 35 * - - - - - -
79.43 23 svSP 35 *, 85 - 79.74 26 svSP, unknown 15, 37 *, 60,
>200 32,026.88 *, 32,899.08
80.23 24 svSP 35 *, 85 - 80.39 27 svSP 37 * 32,686.16 *, 35,124.93
80.95 25 svSP 35 *, 85 - 81.17 28 CRISP, svSP,
unknown 15, 25, 37 * 32,686.34 *, 33,342.02
81.59 26 svSP 40 *, 100 - - - - - -
84.01 27 svMP 70 * - - - - - -
85.34 28 svSP 35 * - 84.65 29 svMP, trypsin-like,
unknown 15, 25, 37, 60 * 24,547.96, 27,654.98
87.17 29 CTL, svSP, PDE 13 *, 35, 65 16,108.34 *, 16,208.30 87.19 30 CTL, svSP, svMP 13 *, 30, 37, 55 13,890.25 *
- - - - - 88.14 31 svSP, unknown 30, 37 *, 55 -
- - - - - 89.12 32 svSP, unknown 30 *, 37 -
92.85 30 CTL, LAAO 13, 20, 40, 60 * - 93.23 33 CTL, svSP, LAAO 11, 20, 30, 37,
50, 55 * -
Toxins 2018,10, 23 9 of 24
Table 1. Cont.
V. a. transcaucasiana V. a. montandoni
RT
(min)
Fraction
No. Protein Species SDS PAGE
Mav (kDa) Most Abundant IMP (m/z)RT
(min)
Fraction
No. Protein Species SDS PAGE
Mav (kDa)
Most Abundant
IMP (m/z)
93.88 31 CTL, LAAO 20, 60 * - 94.64 34 LAAO 30, 37, 55 * -
101.76 32 CTL, DI, svMP,
unknown 20, 35, 60 * - - - - - -
103.42 33
svSP, svMP, unknown
20, 35, 60 * - 103.23 35 CTL, svSP, LAAO 11, 20, 30, 37,
50 * -
104.82 34 svMP, LAAO, svSP 20, 35, 65 * - 104.83 36 aminopeptidase,
svMP, LAAO,
unknown 30, 50 *, 70 -
105.70 35 svMP 65 * - 106.47 37 svMP, LAAO 30, 50 * -
108.84 36 svSP, LAAO, svMP 35, 60 * - 108.82 38 unknown 30, 37, 50 * -
Toxins 2018,10, 23 10 of 24
Toxins2018,10,23 9of23
Figure4.CID‐MS/MSspectraoftwosnakevenommetalloproteinase(svMP)inhibitortripeptides.
Thetwosnakevenommetalloproteinaseinhibitortripeptides:(a)pEKW;and(b)pENWwere
identifiedbyintactmassprofilinginthevenomsofVatandVam(hereVat).TheHPLC‐ESI‐MS1
spectraofselectedparentionsareshownattheleftcorner.Standardfragmentationionswere
indicatedattheionmasspeaksandaminoacidrelatedionsbyasteriskedsinglelettercode.
Accordingly,themeasuredmassofm/z430.17matchestothepredictedmonoisotopicmassof
thepyroglutamictripeptidepENW,whilethemeasuredm/z444.23matchestothepredicted
monoisotopicmassofthepyroglutamictripeptidepEKW.Theidentitiesofthepyroglutamic
tripeptidespEKWandpENWwereultimatelyconfirmedbyESI‐MS/MSexperiments(seeFigure4).
Inaddition,comparisonwithspectraforthepEKWinhibitorfromtheliteraturecoincideswithour
experimentallyobservedfragmentationpattern[49].Asanadditionalevidencefortripeptide
metalloproteaseinhibitorsinthevenomofbothvipers,thepresenceofseveralGly‐andPro‐rich
proteinfragmentsoftheendogenousprecursorsweredetectedinthevenomsofV.a.transcaucasiana
aswellasV.a.montandoni[50].ThepeptidePEGPPLMEPHE,apreviouslydescribedtripeptide
precursorfragment[51]fromtherelatedsnakeV.a.ammodytes,wasdetectedinbothinvestigated
venoms(seeFiguresS1andS2andTablesS1andS2,Peak9and8).Furthermore,severalother
precursorpeptidefragmentsofthistypeweredetectedinthevenomsofVat(GGGGGGW,
PPQMPGPKVPP)andVam(DNEPPKKVPPN)(seeFiguresS3–S6andTablesS1andS2).Taken
together,themetalloproteaseinhibitorsandtheirprecursorpeptidesformthemajorshareofthe
peptidomeofVat(28.80%)aswellasofVam(45.93%),withthetripeptidepEKWasthemain
component.Evenifdetailsonsecretionandprocessingofthetripeptidesarestillunknown,a
formationofthepyroglutamicinhibitorsisassumedtohappenduringtheexocytosis.Aprocessing
intheglandlumenitselfisratherunlikelybasedonmissingglutaminylcyclases[49].
Adrawbackoftheintactmassprofilingisthathighmolecularmassesbecomedifficultto
properlydetect.Areasonisthatthehigherchargedstatesofmolecularionscauseahighpeakdensity
whichincreasinglychallengesthelimitsinresolutionoftheorbitrapmassanalyzer.Hence,the
Figure 4.
CID-MS/MS spectra of two snake venom metalloproteinase (svMP) inhibitor tripeptides.
The two snake venom metalloproteinase inhibitor tripeptides: (
a
) pEKW; and (
b
) pENW were identified
by intact mass profiling in the venoms of Vat and Vam (here Vat). The HPLC-ESI-MS1 spectra of selected
parent ions are shown at the left corner. Standard fragmentation ions were indicated at the ion mass
peaks and amino acid related ions by asterisked single letter code.
A drawback of the intact mass profiling is that high molecular masses become difficult to properly
detect. A reason is that the higher charged states of molecular ions cause a high peak density
which increasingly challenges the limits in resolution of the orbitrap mass analyzer. Hence, the
incomplete characterization of the venoms discriminating higher masses requires the complementary
bottom-up approach.
2.1.2. Bottom-Up Venomics
The bottom-up analysis by the combined approach, termed snake venomics, was performed with
lyophilized snake venoms of Vat and Vam. Subsequent to fractionation by reversed phase-HPLC
(see Figure 3), the protein containing fractions were size-separated by SDS-PAGE (see Figure 5).
The prominent bands were excised followed by tryptic in-gel digestion and de novo sequencing via
MS/MS. The quantitative venom composition was calculated based on the RP-HPLC peak integration
and in case of co-eluting components, the ratio of optical intensities and densities from SDS-PAGE was
deduced [17,22,23].
Toxins 2018,10, 23 11 of 24
Toxins2018,10,23 10of23
incompletecharacterizationofthevenomsdiscriminatinghighermassesrequiresthecomplementary
bottom‐upapproach.
2.1.2.Bottom‐UpVenomics
Thebottom‐upanalysisbythecombinedapproach,termedsnakevenomics,wasperformed
withlyophilizedsnakevenomsofVatandVam.Subsequenttofractionationbyreversedphase‐
HPLC(seeFigure3),theproteincontainingfractionsweresize‐separatedbySDS‐PAGE(seeFigure
5).Theprominentbandswereexcisedfollowedbytrypticin‐geldigestionanddenovosequencing
viaMS/MS.ThequantitativevenomcompositionwascalculatedbasedontheRP‐HPLCpeak
integrationandincaseofco‐elutingcomponents,theratioofopticalintensitiesanddensitiesfrom
SDS‐PAGEwasdeduced[17,22,23].
Figure5.VenomfractionanalysisofV.a.transcaucasianaandV.a.montandonibySDS‐PAGE.TheRP‐
HPLCfractions(indicatedabovethelanearebasedonFigure3)ofthe(a)Vatand(b)Vamvenoms
wereanalyzedbySDS‐PAGEunderreducingconditions.Alphabeticallymarkedbandsperlinewere
excisedforsubsequenttrypticin‐geldigestion.
AconcludinganalysisoftheVatandVamvenomsrenderedthefollowingresults(seeFigure6):
themostabundanttoxinfamilyoftheVatvenomisrepresentedbysnakevenomphospholipasesA2
(svPLA2,44.96%)followedbyvascularendothelialgrowthfactors(VEGF‐F,9.81%),snakevenom
serineproteases(svSP,9.47%),snakevenommetalloproteases(svMP,8.76%),L‐aminoacidoxidases
(LAAO,6.41%),cysteine‐richsecretoryproteins(CRISP,3.41%)andC‐typelectinlikeproteins(CTL,
2.99%).Theremainingconstituents,suchasadisintegrinandaphosphodiesterase,weresummarized
asotherproteins(0.07%)andunannotatedproteins(0.60%)(seeFigure6a),respectively.Similarly,
thevenomofVamiscomposedofsnakevenomphospholipasesA2(svPLA2,52.44%)asmainpart
followedbyvascularendothelialgrowthfactors(VEGF‐F,10.69%),snakevenomserineproteases
(svSP,5.48%),L‐aminoacidoxidases(LAAO,4.83%),cysteine‐richsecretoryproteins(CRISP,3.81%),
snakevenommetalloproteases(svMP,1.79%),C‐typelectinlikeproteins(CTL,0.26%),several
trypsin‐likeproteinsandoneaminopeptidase(otherproteins,0.11%)andnon‐characterizedproteins
(3.11%)(seeFigure6b).
Figure 5.
Venom fraction analysis of V. a. transcaucasiana and V. a. montandoni by SDS-PAGE.
The RP-HPLC fractions (indicated above the lane are based on Figure 3) of the (
a
) Vat and (
b
) Vam
venoms were analyzed by SDS-PAGE under reducing conditions. Alphabetically marked bands per
line were excised for subsequent tryptic in-gel digestion.
A concluding analysis of the Vat and Vam venoms rendered the following results (see Figure 6):
the most abundant toxin family of the Vat venom is represented by snake venom phospholipases A
2
(svPLA
2
, 44.96%) followed by vascular endothelial growth factors (VEGF-F, 9.81%), snake venom
serine proteases (svSP, 9.47%), snake venom metalloproteases (svMP, 8.76%), L-amino acid oxidases
(LAAO, 6.41%), cysteine-rich secretory proteins (CRISP, 3.41%) and C-type lectin like proteins (CTL,
2.99%). The remaining constituents, such as a disintegrin and a phosphodiesterase, were summarized
as other proteins (0.07%) and unannotated proteins (0.60%) (see Figure 6a), respectively. Similarly, the
venom of Vam is composed of snake venom phospholipases A
2
(svPLA
2
, 52.44%) as main part followed
by vascular endothelial growth factors (VEGF-F, 10.69%), snake venom serine proteases (svSP, 5.48%),
L-amino acid oxidases (LAAO, 4.83%), cysteine-rich secretory proteins (CRISP, 3.81%), snake venom
metalloproteases (svMP, 1.79%), C-type lectin like proteins (CTL, 0.26%), several trypsin-like proteins
and one aminopeptidase (other proteins, 0.11%) and non-characterized proteins (3.11%) (see Figure 6b).
In total, 118 fragments with 84 different sequences for Vat (see Tables 1and S1) and 87 fragments
to 66 sequences for Vam (see Tables 1and S2) could be assigned by bottom-up analysis with a
subsequent de novo sequencing. The assignment revealed, e.g., for the phospholipase A
2
protein
family, homologs of both chains from the heterodimeric Vaspin (acidic, Uniprot-ID: CAE47105.1;
basic, Uniprot-ID: CAE47300.1), one of Vipoxin (B chain, Uniprot-ID: 1AOK_B) and the Ammodytin.
This monomeric PLA
2
was identified in the case of Vat as the neutral Ammodytin I2(A) variant
(Uniprot-ID: CAE47197.1) and in the venom of Vam by two isoforms of the acidic Ammodytin I1(A)
(Uniprot-ID: CAE47141.1) and I1(C) (Uniprot-ID: CAE47172.1). The main snake venom serine proteases
(svSP) could be identified as homologs of Nikobin (Uniprot-ID: E5AJX2.1) and the Cadam10-svSP11
(Uniprot-ID: JAV48393.1). The identity of several bottom-up determined proteins, e.g., Vipoxin or
Ammodytin, could be additionally assigned by intact mass profiling through comparison to their
average protein family masses.
Toxins 2018,10, 23 12 of 24
Toxins2018,10,23 11of23
a b
Figure6.Semi‐quantitativevenomcompositionofV.a.transcaucasianaandV.a.montandoni.The
relativeoccurrenceofdifferenttoxinfamiliesof:(a)Vatand(b)Vamarerepresentedbypiecharts.
IdentificationofphospholipasesA2(PLA
2
,blue),vascularendothelialgrowthfactors(VEGF‐F,red),
snakevenomserineproteases(svSP,green),snakevenommetalloproteases(svMP,violet),
L
‐amino
acidoxidases(LAAO,lightblue),cysteinerichsecretoryproteins(CRISP,orange),C‐typelectinlike
proteins(CTL,darkblue),otherproteins(other,darkred),unknownproteins(n/a,darkgreen)and
peptides(lightred).Groupsofdifferentpeptidesizesaresummarizedinanadditionalpiechartas
percentagesofthetotalpeptidecontentandclusteredto<1kDa(dullpurple),1–3kDa(dullbrown)
and3–9kDa(dullgreen).
Intotal,118fragmentswith84differentsequencesforVat(seeTables1andS1)and87fragments
to66sequencesforVam(seeTables1andS2)couldbeassignedbybottom‐upanalysiswitha
subsequentdenovosequencing.Theassignmentrevealed,e.g.,forthephospholipaseA
2
protein
family,homologsofbothchainsfromtheheterodimericVaspin(acidic,Uniprot‐ID:CAE47105.1;
basic,Uniprot‐ID:CAE47300.1),oneofVipoxin(Bchain,Uniprot‐ID:1AOK_B)andtheAmmodytin.
ThismonomericPLA
2
wasidentifiedinthecaseofVatastheneutralAmmodytinI2(A)variant
(Uniprot‐ID:CAE47197.1)andinthevenomofVambytwoisoformsoftheacidicAmmodytinI1(A)
(Uniprot‐ID:CAE47141.1)andI1(C)(Uniprot‐ID:CAE47172.1).Themainsnakevenomserine
proteases(svSP)couldbeidentifiedashomologsofNikobin(Uniprot‐ID:E5AJX2.1)andthe
Cadam10‐svSP11(Uniprot‐ID:JAV48393.1).Theidentityofseveralbottom‐updeterminedproteins,
e.g.,VipoxinorAmmodytin,couldbeadditionallyassignedbyintactmassprofilingthrough
comparisontotheiraverageproteinfamilymasses.
2.2.ComparativeVenomicsofViperaammodytes
Correlationsbetweenvenomcompositionandrelationshipfromsnakesbelongingtothesame
generahavebeenshownintheliterature[52,53].Nevertheless,manyexamplesforinterspecies
variationsinthecompositionofsnakevenomsarealsodescribed[54–57].Variationsinthevenom
compositioncanbeassociatedtodifferentdiets,regionalseparationofpopulations,sexorage[58–
62].Likewise,Tashimaetal.[63]reportedsignificantvariationsinthevenomcompositionofclose‐
relatedpitvipers,butcouldidentifytaxonomymarkers,whichcanbeemployedforanunambiguous
differentiation.Todate,thecomparisonofvenomcompositionsfromrelatedspeciesinthecontextof
taxonomicclassificationisstillacontroversialdebate[57].Inthefollowing,wecomparethevenom
compositionofthetworelatedNose‐Hornedvipers,VatandVam,atthelevelofsubspeciesthatmay
aidinrecognizingofacloserelationship,whichcouldhelptoapotentialtaxonomicassessment.
Theclosephylogeneticrelationshipofthetwostudiedsnakeswaspreviouslyimpliedby
Ursenbacheretal.[39]usingmolecularphylogeography.Ourstudyattemptstounderpintheimplied
taxonomicstatusofVatbycomparativeanalysisatthevenomiclevel,underscoredbythecomparison
oftheUVchromatograms(seeFigure3)aswellastheTICs(seeFigure2)ofthevenoms.Thecurve
progressionandpeakdistributioninthechromatogramsshowasuperimposablearrangement
containingthesametoxinfamilies,whichwasconfirmedbySDS‐PAGEfollowedbysimilardenovo
sequencingasdescribedabove(seeTable1andTablesS1andS2).
Figure 6.
Semi-quantitative venom composition of V. a. transcaucasiana and V. a. montandoni.
The relative occurrence of different toxin families of: (
a
) Vat and (
b
) Vam are represented by pie
charts. Identification of phospholipases A2 (PLA
2
, blue), vascular endothelial growth factors (VEGF-F,
red), snake venom serine proteases (svSP, green), snake venom metalloproteases (svMP, violet), L-amino
acid oxidases (LAAO, light blue), cysteine rich secretory proteins (CRISP, orange), C-type lectin like
proteins (CTL, dark blue), other proteins (other, dark red), unknown proteins (n/a, dark green) and
peptides (light red). Groups of different peptide sizes are summarized in an additional pie chart as
percentages of the total peptide content and clustered to <1 kDa (dull purple), 1–3 kDa (dull brown)
and 3–9 kDa (dull green).
2.2. Comparative Venomics of Vipera ammodytes
Correlations between venom composition and relationship from snakes belonging to the same
genera have been shown in the literature [
52
,
53
]. Nevertheless, many examples for interspecies
variations in the composition of snake venoms are also described [
54
–
57
]. Variations in the venom
composition can be associated to different diets, regional separation of populations, sex or age [
58
–
62
].
Likewise, Tashima et al. [
63
] reported significant variations in the venom composition of close-related
pitvipers, but could identify taxonomy markers, which can be employed for an unambiguous
differentiation. To date, the comparison of venom compositions from related species in the context of
taxonomic classification is still a controversial debate [
57
]. In the following, we compare the venom
composition of the two related Nose-Horned vipers, Vat and Vam, at the level of subspecies that may
aid in recognizing of a close relationship, which could help to a potential taxonomic assessment.
The close phylogenetic relationship of the two studied snakes was previously implied by
Ursenbacher et al. [
39
] using molecular phylogeography. Our study attempts to underpin the
implied taxonomic status of Vat by comparative analysis at the venomic level, underscored by
the comparison of the UV chromatograms (see Figure 3) as well as the TICs (see Figure 2) of the
venoms. The curve progression and peak distribution in the chromatograms show a superimposable
arrangement containing the same toxin families, which was confirmed by SDS-PAGE followed by
similar de novo sequencing as described above (see Tables 1, S1 and S2).
A closer look at the intact mass profiling exhibits the svMP-i pEKW and pENW as most abundant
peptide part in both venoms, but, with ca. 7.0% and 1.0%, they are more prominent in Vam then in
Vat (3.1% and 0.4%). On the other hand, the major peptide of fraction 11 (Vat m/z3796.75 and Vam
m/z3796.73) with 0.9% to 0.4% is twice as abundant in Vat, as m/z1143.64 (Vat) and m/z1144.43
(Vam) as major Vat peptide in fractions 12, 13 and 14. In total, the snakes have eight close related
peptide masses. This shows that the lower molecular masses are in the main contents similar, but differ
strongly between the studied venoms in the lower abundant peptides and mostly in the abundance of
3–9 kDa masses. Additionally, the intact mass profiling exemplarily revealed six proteins from the
venoms of Vat and Vam that are either in part or fully identical between these two vipers, as well as
matched database entries for other species members (see Figure 7). Two molecular masses ~24 kDa
were observed each in peak 19 of Vat (24,652.41 Da and 24,751.38 Da) and peak 21 of Vam (24,653.40 Da
Toxins 2018,10, 23 13 of 24
and 24,749.41 Da), which were both determined by de novo sequencing as CRISPs. The remaining
four molecular masses ~13 kDa belong to the PLA
2
family and were found in the strong peaks 17
and 20 of Vat (13,552.83 Da, 13,589.76 Da, 13,623.69 Da and 13,675.78 Da), and in peaks 19, 23 and 24
(13,552.82 Da, 13,589.75 Da, 13,623.69 Da and 13,675.81 Da) of Vam (Figures S6–S13), respectively.
Toxins2018,10,23 12of23
AcloserlookattheintactmassprofilingexhibitsthesvMP‐ipEKWandpENWasmost
abundantpeptidepartinbothvenoms,but,withca.7.0%and1.0%,theyaremoreprominentinVam
theninVat(3.1%and0.4%).Ontheotherhand,themajorpeptideoffraction11(Vatm/z3796.75and
Vamm/z3796.73)with0.9%to0.4%istwiceasabundantinVat,asm/z1143.64(Vat)andm/z1144.43
(Vam)asmajorVatpeptideinfractions12,13and14.Intotal,thesnakeshaveeightcloserelated
peptidemasses.Thisshowsthatthelowermolecularmassesareinthemaincontentssimilar,but
differstronglybetweenthestudiedvenomsinthelowerabundantpeptidesandmostlyinthe
abundanceof3–9kDamasses.Additionally,theintactmassprofilingexemplarilyrevealedsix
proteinsfromthevenomsofVatandVamthatareeitherinpartorfullyidenticalbetweenthesetwo
vipers,aswellasmatcheddatabaseentriesforotherspeciesmembers(seeFigure7).Twomolecular
masses~24kDawereobservedeachinpeak19ofVat(24,652.41Daand24,751.38Da)andpeak21of
Vam(24,653.40Daand24,749.41Da),whichwerebothdeterminedbydenovosequencingasCRISPs.
Theremainingfourmolecularmasses~13kDabelongtothePLA
2
familyandwerefoundinthe
strongpeaks17and20ofVat(13,552.83Da,13,589.76Da,13,623.69Daand13,675.78Da),andin
peaks19,23and24(13,552.82Da,13,589.75Da,13,623.69Daand13,675.81Da)ofVam(FiguresS6–
S13),respectively.
Figure7.ComparativevenomanalysisofV.a.transcaucasianaandV.a.montandoni.Intactmass
profilingofcrudevenomsfromVat(red)andVam(blue)showsseveralidenticalmasses[M+H]
+
of
differenttoxinfamilies:(a)VipoxinAchaininpeak20(Vat)andpeak23(Vam);and(b)fromtopto
bottom:twophospholipaseA
2
(PLA
2
,~13–14kDa)inpeaks17and20(Vat)andinpeaks19and24
(Vam),aswellasoneCRISP(~25kDa)inpeak19(Vat)andpeak21(Vat).
Thepreviouslymentionedmolecularmassof13,623.69DaidentifiedfromVat(peak20)and
Vam(peak23)wasassignedtothecloselyrelatedacidicphospholipaseA
2
homologVipoxinAchain
(Uniprot‐ID:P04084,includingoxidizedcysteines)M
av
=13,625.04DaoftheV.a.meridionalis[64].
Untilnow,VipoxinwasknownasoneofthemostabundantcomponentsfromthetwootherVipera
ammodytessubspecies[41].Inbothtestedvenoms,thesefractionswerealsothemostabundantpeaks,
whichunderscoretheimportanceofthistoxininthegeneralvenomcomposition.Additionally,by
denovosequencing,weidentifiedfragmentsoftheVipoxinBchaininvenomsofVat(peak18)and
Vam(peak20).TheintactmassprofilingfurthershowsonlyforVat(peak18)amolecularmassof
13,813.21DathatcorrelatestotheaveragemassofthebasicphospholipaseA
2
VipoxinBchain
(Uniprot‐ID:P14420,includingoxidizedcysteines)M
av
=13,813.77DaoftheV.a.meridionalis.
Figure 7.
Comparative venom analysis of V. a. transcaucasiana and V. a. montandoni. Intact mass
profiling of crude venoms from Vat (red) and Vam (blue) shows several identical masses [M + H]
+
of
different toxin families: (
a
) Vipoxin A chain in peak 20 (Vat) and peak 23 (Vam); and (
b
) from top to
bottom: two phospholipase A
2
(PLA
2
, ~13–14 kDa) in peaks 17 and 20 (Vat) and in peaks 19 and 24
(Vam), as well as one CRISP (~25 kDa) in peak 19 (Vat) and peak 21 (Vat).
The previously mentioned molecular mass of 13,623.69 Da identified from Vat (peak 20) and
Vam (peak 23) was assigned to the closely related acidic phospholipase A2homolog Vipoxin A chain
(Uniprot-ID: P04084, including oxidized cysteines) M
av
= 13,625.04 Da of the V. a. meridionalis [
64
].
Until now, Vipoxin was known as one of the most abundant components from the two other Vipera
ammodytes subspecies [
41
]. In both tested venoms, these fractions were also the most abundant peaks,
which underscore the importance of this toxin in the general venom composition. Additionally, by
de novo sequencing, we identified fragments of the Vipoxin B chain in venoms of Vat (peak 18)
and Vam (peak 20). The intact mass profiling further shows only for Vat (peak 18) a molecular
mass of 13,813.21 Da that correlates to the average mass of the basic phospholipase A
2
Vipoxin B
chain (Uniprot-ID: P14420, including oxidized cysteines) M
av
= 13,813.77 Da of the V. a. meridionalis.
Furthermore, the measured molecular mass of 13,552.8 Da in both venoms mass-correlates with
the neutral phospholipase A
2
Ammodytin I2 (Uniprot-ID: P34180, including oxidized cysteines)
M
av
= 13,553.30 Da of V. a. ammodytes [
65
]. The presence of the same metalloprotease inhibitors and
identical fragments of the precursor in both snake venoms, mentioned before, are further indicators of
a close kinship.
Considering the study by Georgieva et al. [
41
] on the venom proteomes of V. a. ammodytes and
V. a. meridionalis, we could compare our datasets in a wider context of the venoms of these subspecies.
Georgieva et al. showed that several PLA
2
s constitute an important part of the venoms, which we
also determined in high quantities, e.g., Vammin A, several Ammodytin variants and Vipoxin B [
41
].
Toxins 2018,10, 23 14 of 24
In addition, from venoms of Vat and Vam, we could also identify several venom families shared
between the other V. ammodytes subspecies members, such as PLA
2
, LAAO, growth factors as well as
serine- and metalloproteinases. All these findings, which are solely based on the comparative venom
analysis of four snakes of the same species, could be an indicator for a closer relationship and help to
make a classification of the four snakes of Vipera ammodytes as subspecies comprehensible.
On the other hand, a view to other related Vipera species that are sharing the geographical
habitat with the Vipera ammodytes species in parts show remarkable differences in the venom
composition. For example, the wide distributed Vipera berus berus, which was recently compared to
the
V. a. ammodytes
, exhibit svSP (31%) as the main toxin family followed by svMP (19%). In contrast,
the most abundant families of the Vat and Vam play a subordinate role in the V. b. berus venom with
10% for PLA
2
or were not even detected in the case of the VEGF-F [
51
]. Furthermore, the venom
composition of the close related Vipera species Vipera anatolica is focused on the presence of the toxin
families svMP (42%) and CRISP (16%) with a similar occurrence of PLA
2
and VEGF-F to V. b. berus [
14
].
Even if there are many similarities in the comparative analysis of the Vat and Vam venoms, some
small differences remain, especially in the peptide content and in the protease pattern as well as in
the LAAOs, which could be considered as parameters to distinguish between subspecies. However,
venom compositions are susceptible to variation due to the influence of various factors (e.g., age,
diet, sex and geographic origin) and could be more likely attributed to the occurrence of intraspecific
variations [
58
–
62
]. The venom variations influenced by diet or habitat are not suspected in the cases
of V. a. transcaucasiana and V. a. montandoni, whose analyzed specimens share the same geographical
origin, but due to the small pooled sample size of each subspecific population, complete variation
compensation cannot be excluded. Additionally, the Vat mass profile shows two dominant peaks (peak
17 and 18), which includes PLA
2
s (13,813.21 Da and 13,917.24 Da) that could not be detected in the Vam
venom profile. In contrast, the Vam venom contains two abundant peaks (peaks 20 and 22) containing
a PLA
2
(13,889.25 Da) and a CRISP (24,546.04 Da) as a major difference that are missing in the Vat
profile (see Figures 2and 4). These small differences, even between subspecies, play a crucial role in the
development of effective antidotes and the understanding of reduced effects of polyvalent antivenoms.
For example, a study of V. a. ammodytes antidotes has shown strong reduced neutralization potency
against V. a. montandoni [44].
2.3. Cytotoxicity Screening
Snake venoms constitute complex mixtures of enzymes, peptides and proteins with a high toxicity
potential, which can selectively and specifically act on various cellular targets by modulating the
physiological function. This turns snake venoms into an attractive source for potential anticancer
agents [
66
]. As part of our ongoing studies on Turkish snake venoms, the potency against various
human cancer cells, and the cytotoxicities of the crude venom for V. a. transcaucasiana and
V. a. montandoni
were tested on a panel of cancer cell lines together with non-cancerous cell lines
in a MTT assay. For V. a. transcaucasiana and V. a. montandoni crude venoms the MTT assay resulted as
IC50 values of 1.34–22.75 µg/mL and 0.06–50.00 µg/mL (see Table 2), respectively.
Toxins 2018,10, 23 15 of 24
Table 2.
IC
50
values of Vipera ammodytes transcaucasiana and Vipera ammodytes montandoni venoms
against various human cell lines. The half maximal inhibitory concentration (IC
50
in
µ
g/mL) for the
venom of Vat and Vam were determined after 48 h exposure. Parthenolide was used as reference
compound. Noncancerous human cells: HEK293 (human embryonic kidney). Cancerous human cells:
U87MG (epithelial-like glioblastoma-astrocytoma); SHSY5Y (neuroblastoma); MDA-MB-231 (breast
epithelial adenocarcinoma); A549 (lung adenocarcinoma); MPanc-96 (pancreas adenocarcinoma);
MCF-7 (epithelial breast adenocarcinoma); CaCo-2 (epithelial colorectal adenocarcinoma); 253-JBV
(bladder carcinoma); HeLa (epithelial cervical carcinoma); PC-3 (prostate adenocarcinoma). A minus
(−) mentioned not tested cell lines and error in mean ±SD.
Cell Line V. ammodytes transcaucasiana
IC50 (µg/mL)
V. ammodytes montandoni
IC50 (µg/mL)
Parthenolide IC50
(µg/mL)
HEK293 1.34 ±0.72 3.55 ±0.61 1.23 ±0.24
U87MG 6.02 ±1.38 1.02 ±0.20 3.33 ±0.59
SHSY5Y - 0.06 ±0.01 0.15 ±0.01
MDA-MB-231 1.84 ±0.76 2.36 ±0.20 4.80 ±1.10
MCF-7 21.75 ±2.45 >50.00 6.01 ±1.15
A549 18.03 ±2.09 4.40 ±0.03 4.42 ±0.87
MPanc-96 22.75 ±2.25 - 4.70 ±0.87
CaCo-2 4.21 ±0.96 1.82 ±0.14 4.90 ±1.10
253J-BV 4.22 ±1.41 3.00 ±1.98 5.45 ±1.16
HeLa 6.14 ±1.12 1.27 ±0.20 5.75 ±1.07
PC3 6.95 ±1.19 - 3.33 ±0.96
The crude venoms of both snakes show a similar activity against breast (MDA-MB-231), colon
(Caco-2) and bladder (253J-BV) cancer cell lines. V. a. montandoni shows a high cytotoxicity against four
cell lines (HEK-293, U-87 MG, A549 and HeLa) out of the eight tested (see Figure 8). The determination
of the IC
50
shows strong differences of the closely related vipers against the MCF-7 breast cancer
cells and the A549 lung cancer cells. The V. a. transcaucasiana venom was found to have an IC
50
of
21.75 ±2.45 µg/mL and 18.03 ±2.09 µg/mL against MCF-7 and A549 cells, respectively. In contrast,
the V. a. montandoni venom has an IC
50
>50.00
µ
g/mL and 4.40
±
0.03
µ
g/mL against MCF-7 and
A549 cells, respectively (see Table 3). Interestingly, the V. a. transcaucasiana crude venom exhibited
the highest cytotoxic effect (1.84
±
0.76
µ
g/mL) against the receptor triple negative breast cancer
cells MDA-MB-231 in comparison to V. a. montandoni and the 2.6-fold less active positive control
(
4.80 ±1.10 µg/mL
), while both venoms are less toxic to the second tested breast cancer cells MCF-7
(ER negative, PR positive, HER2 positive) with IC
50
values of 21.75
±
2.45
µ
g/mL and >50.00
µ
g/mL
to Parthenolide (6.01
±
1.15
µ
g/mL). The subtypes of breast cancer have been generally identified
based on the presence of three receptors: estrogen receptor (ER), progesterone receptor (PR) and
human epidermal growth factor receptor-2 (HER-2) [67].
Toxins 2018,10, 23 16 of 24
Toxins 2018, 10, x FOR PEER REVIEW 15 of 23
Figure 8. MTT human cell viability after 48 h crude venom treatment. Cytotoxicity of: V. a.
transcaucasiana (red) (top); and V. a. montandoni (blue) (bottom) crude venom in three concentrations
(50, 5 and 0.5 μg/mL) against different human cell lines. Cell viability was measured by MTT assay
after 48 h at 570 nm. Noncancerous human cells: HEK-293 (human embryonic kidney). Cancerous
human cells: U-87 MG (epithelial-like glioblastoma-astrocytoma); SH-SY5Y (neuroblastoma); MDA-
MB-231 (breast epithelial adenocarcinoma); A549 (lung adenocarcinoma); MPanc-96 (pancreas
adenocarcinoma); MCF-7 (epithelial breast adenocarcinoma); CaCo-2 (epithelial colorectal
adenocarcinoma); 253-JBV (bladder carcinoma); HeLa (epithelial cervical carcinoma); PC-3 (prostate
adenocarcinoma). Not tested cell lines by n/a and error in mean ± SD.
Based on these markers, a classification of breast cancer tumors as hormone receptor positive,
HER-2/Neu amplified tumors, and those which do not express ER, PR and do not have a HER-2/Neu
amplification were defined as triple-negative breast cancer (TNBC). This might have a significant role
for patient-tailored treatment strategies, as TNBC demonstrates approximately 10–15% of all breast
cancers and patients with TNBC have an unsuccessful outcome compared to the other breast cancer
subtypes [68]. Unfortunately, the tested crude venoms also exhibit a comparable cytotoxic effect
against the tested non-cancerous kidney cells with values of 1.34 ± 0.72 and 3.55 ± 0.61 μg/mL. The
comparison of further non-cancerous to attributed cancerous cell lines is part of ongoing due to
restricted sample size. Nevertheless, we suspect that the high toxic effect on non-cancerous cell lines
can be overcome by developing a targeted drug delivery system for potential treatment followed by
detailed studies for each drug candidate.
According to the MTT assay, V. a. transcaucasiana venom
showed active venom fractions against the human breast adenocarcinoma epithelial cells (MDA-MB-
231) (see Figure S14). Fractions 1, 17, 18 and 19 exhibited the strongest cytotoxic effect on the triple
negative MDA-MB-231 breast cancer cells with IC
50
of 2.96 μg/mL to 9.22 μg/mL (see Figure 9 and
Table 3).
Figure 8.
MTT human cell viability after 48 h crude venom treatment. Cytotoxicity of:
V. a. transcaucasiana
(red)(
top
); andV. a. montandoni (blue) (
bottom
) crude venominthreeconcentrations(50, 5 and 0.5
µ
g/mL)
against different human cell lines. Cell viability was measured by MTT assay after 48 h at 570 nm.
Noncancerous human cells: HEK-293 (human embryonic kidney). Cancerous human cells: U-87 MG
(epithelial-like glioblastoma-astrocytoma); SH-SY5Y (neuroblastoma); MDA-MB-231 (breast epithelial
adenocarcinoma); A549 (lung adenocarcinoma); MPanc-96 (pancreas adenocarcinoma); MCF-7 (epithelial
breast adenocarcinoma); CaCo-2 (epithelial colorectal adenocarcinoma); 253-JBV (bladder carcinoma);
HeLa (epithelial cervical carcinoma); PC-3 (prostate adenocarcinoma). Not tested cell lines by n/a and
error in mean ±SD.
Based on these markers, a classification of breast cancer tumors as hormone receptor positive,
HER-2/Neu amplified tumors, and those which do not express ER, PR and do not have a HER-2/Neu
amplification were defined as triple-negative breast cancer (TNBC). This might have a significant
role for patient-tailored treatment strategies, as TNBC demonstrates approximately 10–15% of all
breast cancers and patients with TNBC have an unsuccessful outcome compared to the other breast
cancer subtypes [
68
]. Unfortunately, the tested crude venoms also exhibit a comparable cytotoxic
effect against the tested non-cancerous kidney cells with values of 1.34
±
0.72 and 3.55
±
0.61
µ
g/mL.
The comparison of further non-cancerous to attributed cancerous cell lines is part of ongoing due
to restricted sample size. Nevertheless, we suspect that the high toxic effect on non-cancerous cell
lines can be overcome by developing a targeted drug delivery system for potential treatment followed
by detailed studies for each drug candidate. According to the MTT assay, V. a. transcaucasiana
venom showed active venom fractions against the human breast adenocarcinoma epithelial cells
(MDA-MB-231) (see Figure S14). Fractions 1, 17, 18 and 19 exhibited the strongest cytotoxic effect
on the triple negative MDA-MB-231 breast cancer cells with IC
50
of 2.96
µ
g/mL to 9.22
µ
g/mL
(see Figure 9and Table 3).
Toxins 2018,10, 23 17 of 24
Toxins2018,10,23 16of23
Figure9.MTThumancellviabilityafter48hV.a.transcaucasianavenomfractiontreatment.
CytotoxicityofselectedV.a.transcaucasianavenomHPLCfractions(F1,17–19)inthreeconcentrations
(20μg/mL,10μg/mLand2μg/mL)againstMDA‐MB‐231breastadenocarcinomaepithelialcellline.
CellviabilitywasmeasuredbyMTTassayafter48hat570nm.Doxorubicinwasusedasreference
compoundanderrorinmean±SD.
Table3.IC50valuesofselectedV.a.transcaucasianaHPLCfractionsagainstMDA‐MB‐231cells.The
halfmaximalinhibitoryconcentration(IC50inμg/mL)fortheHPLCfractions1,17,18and19ofVat
venomagainstMDA‐MB‐231breastadenocarcinomaepithelialcellline.Doxorubicinwasusedas
referencecompoundanderrorinmean±SD.
SamplesIDV.ammodytestranscaucasianaFraction Doxorubicin
117 18 19
IC
50
(μg/mL)5.92±0.143.98±0.852.96±0.389.22±0.62>20.00
Massspectrometricanalysisofthebioactivefractionsidentifiedfraction17asanAmmodytin
I2(A)variant,fraction18asaVipoxinchainBorVaspinbasicsubunitvariantandfraction19asa
cysteine‐richvenomprotein.Fraction1isamixtureofvarioussmallpeptideswithstillunknown
sequence.Theeffectivityandspecificityofsnakevenomphospholipasesagainstcancercellsare
known[69].ThecytotoxicityoftheCRISPinfraction19isduetotheblockingabilityagainstseveral
ionchannels[70,71].
3.Conclusions
Here,wereportonthefirstproteomiccharacterizationofthevenomsfromViperaammodytes
transcaucasianaandViperaammodytesmontandonibyusingacombinedmassspectrometry‐guided
approach.Theinitialintactmassprofilingofthevenomsfacilitatedthedetectionof~50venom
componentsforV.a.transcaucasianaand~59venomcomponentsforV.a.montandoni.Additionally,
theintactmassprofilingrevealedthepresenceoftwotripeptidemetalloproteaseinhibitorsandtheir
precursorsinthevenoms,whichwouldnothavebeendetectedbythebottom‐upapproach.However,
duetothelimitedapplicabilitytohighmolecularmasscompoundstheanalysiswasfurther
expandedtothebottom‐upapproach.Thedenovosequencingshowedforbothsnakevenomsthe
presenceof11majorViperidaetoxinfamilieswiththeexceptionofKunitztypeproteinaseinhibitors
andhyaluronidases,whicharenotpresentineithervenom.
Thecomparativeanalysisbyinitialmassprofilingincombinationwiththewell‐established
bottom‐upprotocolrevealedstrongsimilaritiesinthevenomcompositionofthetwostudiedsnake
venomsofV.a.transcaucasianaandV.a.montadoni.Whilerelatedtoxinfamiliesorevenidentical
proteinscouldbeidentifiedprovingacloserelationshipoftheproteomeandunderconsiderationof
intraspecificvenomvariation,smalldifferencesinthevenomcompositionsinturncouldbe
parametersforadifferentiationintosubspecies.
Insummary,themassspectrometry‐guidedcomparativeanalysisofthevenomproteome
extendedbyintactmassprofilesprovidesanexcellentmethodtohighlightcloserelationshipsof
Figure 9.
MTT human cell viability after 48 h V. a. transcaucasiana venom fraction treatment.
Cytotoxicity of selected V. a. transcaucasiana venom HPLC fractions (F1,17–19) in three concentrations
(20
µ
g/mL, 10
µ
g/mL and 2
µ
g/mL) against MDA-MB-231 breast adenocarcinoma epithelial cell line.
Cell viability was measured by MTT assay after 48 h at 570 nm. Doxorubicin was used as reference
compound and error in mean ±SD.
Table 3.
IC
50
values of selected V. a. transcaucasiana HPLC fractions against MDA-MB-231 cells. The half
maximal inhibitory concentration (IC
50
in
µ
g/mL) for the HPLC fractions 1, 17, 18 and 19 of Vat venom
against MDA-MB-231 breast adenocarcinoma epithelial cell line. Doxorubicin was used as reference
compound and error in mean ±SD.
Samples ID V. ammodytes transcaucasiana Fraction Doxorubicin
1 17 18 19
IC50 (µg/mL) 5.92 ±0.14 3.98 ±0.85 2.96 ±0.38 9.22 ±0.62 >20.00
Mass spectrometric analysis of the bioactive fractions identified fraction 17 as an Ammodytin
I2(A) variant, fraction 18 as a Vipoxin chain B or Vaspin basic subunit variant and fraction 19 as a
cysteine-rich venom protein. Fraction 1 is a mixture of various small peptides with still unknown
sequence. The effectivity and specificity of snake venom phospholipases against cancer cells are
known [
69
]. The cytotoxicity of the CRISP in fraction 19 is due to the blocking ability against several
ion channels [70,71].
3. Conclusions
Here, we report on the first proteomic characterization of the venoms from Vipera ammodytes
transcaucasiana and Vipera ammodytes montandoni by using a combined mass spectrometry-guided
approach. The initial intact mass profiling of the venoms facilitated the detection of ~50 venom
components for V. a. transcaucasiana and ~59 venom components for V. a. montandoni. Additionally,
the intact mass profiling revealed the presence of two tripeptide metalloprotease inhibitors and
their precursors in the venoms, which would not have been detected by the bottom-up approach.
However, due to the limited applicability to high molecular mass compounds the analysis was further
expanded to the bottom-up approach. The de novo sequencing showed for both snake venoms the
presence of 11 major Viperidae toxin families with the exception of Kunitz type proteinase inhibitors
and hyaluronidases, which are not present in either venom.
The comparative analysis by initial mass profiling in combination with the well-established
bottom-up protocol revealed strong similarities in the venom composition of the two studied snake
venoms of V. a. transcaucasiana and V. a. montadoni. While related toxin families or even identical
proteins could be identified proving a close relationship of the proteome and under consideration of
Toxins 2018,10, 23 18 of 24
intraspecific venom variation, small differences in the venom compositions in turn could be parameters
for a differentiation into subspecies.
In summary, the mass spectrometry-guided comparative analysis of the venom proteome
extended by intact mass profiles provides an excellent method to highlight close relationships of
venomous snakes at the proteome level and especially in combination with top-down venomics
this would give you an even more detailed picture of venom diversity. Hence, the investigation of
venoms for the phylogenetic analysis, using a combination of chromatographic, electrophoretic and
different mass spectrometric techniques, is very sensitive and fast. In this context, reliable databases
are an important basis for the de novo identification and the variation of the venom composition by
differences in age and food supply should always be considered as a critical point [
72
]. The future
transcriptomic analysis of the venom glands represents a valuable technique, which could support
the proteomic analysis and elucidate the venom proteome in its entirety. The complete analysis of the
venom proteome of Vipera ammodytes transcaucasiana in connection with mitochondrial DNA could
finally clarify the controversial question about the taxonomic status.
Finally, the preliminary
in vitro
cytotoxicity screening against various cancer cell lines for the
crude venoms and components for V. a. transcaucasiana demonstrated significant cytotoxic effects
on the triple negative MDA MB 231 breast cancer cells with IC
50
of 2.96
µ
g/mL to 9.22
µ
g/mL for
an Ammodytin I2(A) variant, a Vipoxin chain B or Vaspin basic subunit variant and a cysteine-rich
venom protein. Nevertheless, the high toxic effect also against non-cancerous cell lines requires the
development of a targeted drug delivery system for potential treatment.
4. Materials and Methods
4.1. Collection and Preparation of Venom Samples
Venom samples of the V. a. transcaucasiana were collected in June 2015 from four specimens
in total, one from I¸sık Mountain (Çerke¸s district, Çankırı Province, Turkey) and three from Çavu¸s
Mountain (Sivas Province, Turkey). Vipera ammodytes montandoni venom samples were collected from
one individual in the Tekirda˘g province and one in the Kırklareli province (Turkish Thrace) in April
2016. Crude venoms were extracted, using a paraffin-covered laboratory beaker without exerting
pressure on the venom glands, pooled for each subspecies and lyophilized. Ethical permission (Ege
University, Animal Experiments Ethics Committee, 2010#43) and special permission (2011#7110) for
field studies from the Republic of Turkey, Ministry of Forestry and Water Affairs were received.
4.2. Determination of Protein Content
Protein concentrations were determined from diluted venom sample (1 or 2 mg/mL) in
ultrapure water by Micro-BCA (Bicinchoninic Acid) Protein Assay using a UV/Vis spectrophotometer
(Thermo-Scientific, Darmstadt, Germany) at a wavelength of
λ
= 595 nm. Bovine serum albumin (BSA)
was used as a reference.
4.3. Cell Culture and In Vitro Cytotoxicity Assay
The following human cell lines were used for determination of cytotoxicity: noncancerous
cells: HEK293 (human embryonic kidney); cancerous human cells: U87MG (epithelial-like
glioblastoma-astrocytoma); SHSY5Y (neuroblastoma); MDA-MB-231 (breast epithelial adenocarcinoma);
A549 (lung adenocarcinoma); MPanc-96 (pancreas adenocarcinoma); MCF-7 (epithelial breast
adenocarcinoma); CaCo-2 (epithelial colorectal adenocarcinoma); 253-JBV (bladder carcinoma); HeLa
(epithelial cervical carcinoma); PC-3 (prostate adenocarcinoma). All cell lines were purchased from
the US American Type Culture Collection (ATCC, Manassas, VA, USA) except for 253J-BV cells, which
were obtained from Creative Bioarray (Shirley, NY, USA). All cells were cultivated in Dulbecco’s
modified Eagle’s medium F12 (DMEM/F12), supplemented with 10% fetal bovine serum (FBS),
2 mM/L glutamine, 100 U/mL of penicillin and 100 mg/mL of streptomycin (Gibco, Visp, Switzerland).
Toxins 2018,10, 23 19 of 24
The
in vitro
cytotoxicity testing with crude venoms and fractions was performed according to the
protocol of Nalbantsoy and Hempel et al. [
15
]. The morphological changes of the cells after treatment
with crude venom or its fractions were observed with an inverted microscope (Olympus, Tokyo, Japan)
compared to the control group following for 48 h.
4.4. Determination of Half Maximal Inhibitory Concentration (IC50)
The IC
50
values were calculated by fitting the data to a sigmoidal curve and using a four-parameter
logistic model and presented as an average of three independent measurements. The IC
50
values
were reported at 95% confidence interval, and calculations were performed using Prism 5 software
(GraphPad5, San Diego, CA, USA). The values of the blank wells were subtracted from each well of
treated and control cells and half maximal inhibition of growth (IC
50
) were calculated in comparison
to the untreated controls.
4.5. Preparation of Venom Samples for Intact Mass Profiling
The crude venoms were dissolved in aqueous 1% (v/v) formic acid (HFo) to a final concentration
of 10 mg/mL, and centrifuged at 20,000
×
gfor 5 min to spin down insoluble content. Dissolved
venoms were then mixed with 30
µ
L of citrate buffer (0.1 M, pH 4.0). The samples were mixed with
an equal volume of 1% aqueous formic acid and centrifuged at 20,000
×
gfor 5 min. Subsequently,
samples were submitted to HPLC-high-resolution (HR) ESI-MS/MS measurements.
4.6. Intact Mass Profiling
The intact mass profiling was performed by LC-ESI-HR-MS experiments on an LTQ Orbitrap XL
mass spectrometer (Thermo, Bremen, Germany) coupled to an Agilent 1260 HPLC system (Agilent,
Waldbronn, Germany) using a Supelco Discovery 300 Å C18 (2
×
150 mm, 3
µ
m particle size)
column. The instrument settings for the HPLC system and ESI-MS were adopted from Nalbantsoy and
Hempel et al. [15]
. The intact mass profiles were inspected with the Xcalibur Qual Browser (Thermo
Xcalibur 2.2 SP1.48, Thermo Fisher Scientific, Waltham, MA, USA) and deconvolution of isotopically
resolved spectra was carried out by using the XTRACT algorithm of Xcalibur Qual Browser. The protein
assignment was done by comparison to the retention time of the HPLC run and corresponding
LC-MS/MS information from SDS-PAGE trypsin digests.
4.7. Bottom-Up Venomics
The lyophilized crude venoms (4 mg) were dissolved to a final concentration of 20 mg/mL in
aqueous 3% (v/v) ACN with 1% (v/v) HFo and centrifuged at 20,000
×
gfor 5 min to spin down
insoluble content. The supernatant was loaded onto a semi-preparative reversed-phase HPLC with a
Supelco Discovery BIO wide Pore C18-3 column (4.6
×
150 mm, 3
µ
m particle size) using an Agilent
1260 Low Pressure Gradient System (Agilent, Waldbronn, Germany). The column was operated with
a flow rate of 1 mL/min and performed using ultrapure water with 0.1% (v/v) HFo (buffer A) and
ACN with 0.1% (v/v) HFo (buffer B). The technical settings and bottom-up workflow was performed
according to the protocol of Nalbantsoy and Hempel et al. [
15
] with the following amendments.
After the chromatographic separation of the crude venoms, the vacuum-dried peak fractions were
submitted to a SDS-PAGE with a content of 15% polyacrylamide. Afterwards the coomassie-stained
band were excised and reduced, via in-gel trypsin digestion, with freshly prepared dithiothreitol
solution (100 mM DTT in 100 mM ammonium hydrogencarbonate, pH 8.3, heated for 30 min at 56
◦
C)
and alkylated with iodoacetamide (55 mM IAC in 100 mM ammonium hydrogencarbonate, pH 8.3,
stored for 30 min at 25
◦
C in the dark). The peptides were extracted with 100
µ
L aqueous 30% (v/v)
ACN just as 5% (v/v) HFo for 15 min at 37
◦
C. The supernatant was vacuum dried (Thermo speedvac,
Bremen, Germany), re-dissolved in 20
µ
L aqueous 3% (v/v) ACN with 1% (v/v) HFo and submitted to
HPLC-MS/MS analysis.
Toxins 2018,10, 23 20 of 24
The bottom-up analysis was performed with an Orbitrap XL mass spectrometer (Thermo,
Bremen, Germany) via an Agilent 1260 HPLC system (Agilent Technologies, Waldbronn, Germany)
using a reversed-phase Grace Vydac 218MSC18 (2.1
×
150 mm, 5
µ
m particle size) column.
The pre-chromatographic separation was performed with the following settings: After an isocratic
equilibration (5% B) for 1 min, the peptides were eluted with a linear gradient of 5–40% B for 10 min,
40–99% B for 3 min, washed with 99% B for 3 min and re-equilibrated in 5% B for 3 min.
LC-MS/MS data files (.raw) were converted to mgf-files using MSConvert GUI of the ProteoWizard
Software Foundation (ProteoWizard package, version 3.0.10577, Los Angeles, CA, USA) and annotated
by DeNovo GUI [
73
] (ProteoWizard package, version 1.15.8, Los Angeles, CA, USA) with the following
settings: fixed modifications: carbamidomethyl Cys (+57.02 Da); variable modifications: acetylation
of Lys (+42.01 Da) and phosphorylation of Ser and Thr (+79.97 Da). The deduced amino acid
sequences were squared against a non-redundant protein NCBI database of Viperidae (taxid:8689)
using BLASTP [74] (http://blast.ncbi.nlm.nih.gov).
4.8. Data Accessibility
Mass spectrometry proteomics data (.mgf, .raw and output files) have been deposited with the
ProteomeXchange Consortium [
75
] (http://proteomecentral.proteomexchange.org) via the MassIVE
partner repository under Project Name “Venomics of the V. a. transcaucasiana and V. a. montandoni”
and data set identifier PXD007609.
4.9. Relative Toxin Quantification
The quantification of venom composition is based on the RP-HPLC peak integration (UV
214nm
)
in comparison to the total integral of all analyzed peaks based on the protocol of Juarez et al. [
76
].
In the case of HPLC co-eluting toxins components, the SDS-PAGE band ratio of optical intensities and
densities was, respectively, used for emphasis of peak integral portion [17,22,23].
Supplementary Materials:
The following are available online at www.mdpi.com/2072-6651/10/1/23/s1,
Figures S1–S5: Tripeptide precursor fragments of V. a. montandoni and V. a. transcaucasiana, Figures S6–S9:
Intact masses of V. a. transcaucasiana, Figures S10–S13: Intact masses of V. a. montandoni, Figures S14: Cytotoxicity
analysis of fractionated components from Vipera a. transcaucasiana, Table S1: Venom proteins and peptides
identified from Vipera ammodytes transcaucasiana, Table S2: Venom proteins and peptides identified from Vipera
ammodytes montandoni.
Acknowledgments:
We thank Daniel Petras (University of California) and Rashed Al Toma (TU Berlin) for helpful
discussion on the manuscript. We acknowledge support by the German Research Foundation and the Open
Access Publication Funds of Technische Universität Berlin.
Author Contributions:
B.G., M.K. and M.A.O. chatched and milked the snakes. B.-F.H., A.N. and R.D.S. conceived
and designed the experiments, B.-F.H. and A.N. performed the experiments, B.-F.H., A.N. and M.D. analyzed
the data. All authors contributed reagents/materials/analysis tools, B.-F.H., A.N. and M.D. wrote the paper.
All contributors critically read and revised the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
King, G.F. Venoms as a platform for human drugs: Translating toxins into therapeutics. Expert Opin. Biol. Ther.
2011,11, 1469–1484. [CrossRef] [PubMed]
2.
Oldrati, V.; Bianchi, E.; Stöcklin, R. Spider Venom Components as Drug Candidates. In Spider Ecophysiology;
Nentwig, W., Ed.; Springer: Berlin/Heidelberg, Germany, 2013; pp. 491–503.
3.
Oldrati, V.; Arrell, M.; Violette, A.; Perret, F.; Sprüngli, X.; Wolfender, J.-L.; Stöcklin, R. Advances in venomics.
Mol. Biosyst. 2016,12, 3530–3543. [CrossRef] [PubMed]
4.
Koh, D.C.I.; Armugam, A.; Jeyaseelan, K. Snake venom components and their applications in biomedicine.
Cell. Mol. Life Sci. 2006,63, 3030–3041. [CrossRef] [PubMed]
5.
Vetter, I.; Davis, J.L.; Rash, L.D.; Anangi, R.; Mobli, M.; Alewood, P.F.; Lewis, R.J.; King, G.F. Venomics: A new
paradigm for natural products-based drug discovery. Amino Acids 2011,40, 15–28. [CrossRef] [PubMed]
Toxins 2018,10, 23 21 of 24
6.
Calvete, J.J. Venomics: Integrative venom proteomics and beyond. Biochem. J.
2017
,474, 611–634. [CrossRef]
[PubMed]
7.
Xie, B.; Huang, Y.; Baumann, K.; Fry, B.G.; Shi, Q. From Marine Venoms to Drugs: Efficiently Supported by a
Combination of Transcriptomics and Proteomics. Mar. Drugs 2017,15, 103. [CrossRef] [PubMed]
8.
Ménez, A. Perspectives in Molecular Toxinology; John Wiley & Sons: New York, NY, USA, 2002;
ISBN 978-0471495031.
9.
Prashanth, J.R.; Lewis, R.J.; Dutertre, S. Towards an integrated venomics approach for accelerated
conopeptide discovery. Toxicon 2012,60, 470–477. [CrossRef] [PubMed]
10.
Heather, J.M.; Chain, B. The sequence of sequencers: The history of sequencing DNA. Genomics
2016
,107,
1–8. [CrossRef] [PubMed]
11.
Lomonte, B.; Calvete, J.J. Strategies in ‘snake venomics’ aiming at an integrative view of compositional,
functional, and immunological characteristics of venoms. J. Venom. Anim. Toxins Incl. Trop. Dis.
2017
,23, 26.
[CrossRef] [PubMed]
12.
Fox, J.W.; Ma, L.; Nelson, K.; Sherman, N.E.; Serrano, S.M.T. Comparison of indirect and direct approaches
using ion-trap and Fourier transform ion cyclotron resonance mass spectrometry for exploring viperid
venom proteomes. Toxicon 2006,47, 700–714. [CrossRef] [PubMed]
13.
Georgieva, D.; Arni, R.K.; Betzel, C. Proteome analysis of snake venom toxins: Pharmacological insights.
Expert Rev. Proteom. 2008,5, 787–797. [CrossRef] [PubMed]
14.
Göçmen, B.; Heiss, P.; Petras, D.; Nalbantsoy, A.; Süssmuth, R.D. Mass spectrometry guided venom profiling
and bioactivity screening of the Anatolian Meadow Viper, Vipera anatolica.Toxicon 2015,107, 163–174.
15.
Nalbantsoy, A.; Hempel, B.-F.; Petras, D.; Heiss, P.; Göçmen, B.; I˘gci, N.; Yildiz, M.Z.; Süssmuth, R.D.
Combined venom profiling and cytotoxicity screening of the Radde’s mountain viper (Montivipera raddei)
and Mount Bulgar Viper (Montivipera bulgardaghica) with potent cytotoxicity against human A549 lung
carcinoma cells. Toxicon 2017,135, 71–83. [CrossRef] [PubMed]
16.
Calvete, J.J.; Juárez, P.; Sanz, L. Snake venomics. Strategy and applications. J. Mass Spectrom.
2007
,42,
1405–1414. [CrossRef] [PubMed]
17.
Calvete, J.J.; Petras, D.; Calderón-Celis, F.; Lomonte, B.; Encinar, J.R.; Sanz-Medel, A. Protein-species
quantitative venomics: Looking through a crystal ball. J. Venom. Anim. Toxins Incl. Trop. Dis.
2017
,23.
[CrossRef] [PubMed]
18.
Calvete, J.J. Proteomic tools against the neglected pathology of snake bite envenoming. Expert Rev. Proteom.
2011,8, 739–758. [CrossRef] [PubMed]
19.
Petras, D.; Heiss, P.; Süssmuth, R.D.; Calvete, J.J. Venom Proteomics of Indonesian King Cobra, Ophiophagus
hannah: Integrating Top-Down and Bottom-Up Approaches. J. Proteome Res.
2015
,14, 2539–2556. [CrossRef]
[PubMed]
20.
Petras, D.; Heiss, P.; Harrison, R.A.; Süssmuth, R.D.; Calvete, J.J. Top-down venomics of the East African
green mamba, Dendroaspis angusticeps, and the black mamba, Dendroaspis polylepis, highlight the complexity
of their toxin arsenals. J. Proteom. 2016,146, 148–164. [CrossRef] [PubMed]
21.
Melani, R.D.; Skinner, O.S.; Fornelli, L.; Domont, G.B.; Compton, P.D.; Kelleher, N.L. Mapping Proteoforms
and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics.
Mol. Cell Proteom. 2016,15, 2423–2434. [CrossRef] [PubMed]
22.
Calderón-Celis, F.; Diez-Fernández, S.; Costa-Fernández, J.M.; Encinar, J.R.; Calvete, J.J.; Sanz-Medel, A.
Elemental Mass Spectrometry for Absolute Intact Protein Quantification without Protein-Specific Standards:
Application to Snake Venomics. Anal. Chem. 2016,88, 9699–9706. [CrossRef] [PubMed]
23.
Calvete, J.J. Next-generation snake venomics: Protein-locus resolution through venom proteome
decomplexation. Expert Rev. Proteom. 2014,11, 315–329. [CrossRef] [PubMed]
24.
Cox, N.; Chanson, J.; Stuart, S. The Status and Distribution of Reptiles and Amphibians of the Mediterranean Basin;
The World Conservation Union (IUCN): Gland, Swizerland, 2006.
25.
Mallow, D.; Ludwig, D.; Nilson, G. True Vipers: Natural History and Toxinology of Old World Vipers; Krieger
Publishing: Malabar, FL, USA, 2003.
26. O’Shea, M. Venomous Snakes of the World, 2nd ed.; New Holland: London, UK, 2008.
27.
Fox, J.W.; Serrano, S.M.T. Exploring snake venom proteomes: Multifaceted analyses for complex toxin
mixtures. Proteomics 2008,8, 909–920. [CrossRef] [PubMed]
28. Mackessy, S.P. Handbook of Venoms and Toxins of Reptiles; CRC Press: Boca Raton, FL, USA, 2010.
Toxins 2018,10, 23 22 of 24
29.
Stoyanova, V.; Aleksandrov, R.; Lukarska, M.; Duhalov, D.; Atanasov, V.; Petrova, S. Recognition of Vipera
ammodytes meridionalis neurotoxin vipoxin and its components using phage-displayed scFv and polyclonal
antivenom sera. Toxicon 2012,60, 802–809. [CrossRef] [PubMed]
30.
Lamb, T.; de Haro, L.; Lonati, D.; Brvar, M.; Eddleston, M. Antivenom for European Vipera species
envenoming. Clin. Toxicol. 2017,55, 557–568. [CrossRef] [PubMed]
31.
Boulenger, G.A. On the Geographical Variations of the Sand-Viper, Vipera ammodytes.Proc. Zool. Soc. Lond.
1903,73, 185–186. [CrossRef]
32.
Boulenger, G.A. On the sand viper of Romania (Vipera ammodytes, var. montandoni). Ann. Mag. Nat. Hist.
1904,7, 134–135.
33.
Boulenger, G.A. On the geographical races of Vipera ammodytes.Ann. Mag. Nat. Hist.
1913
,8, 283–287.
[CrossRef]
34. Bruno, S. Sulla Vipera ammodytes (Linnaeus, 1758) in Italia. Mem. Mus. Civ. Stor. Nat. 1968,15, 289–336.
35. Sochurek, E. Vipera ammodytes gregorwallneri n. spp. Herpet. Blatt. 1974,1, 1–4.
36.
Nilson, G.; Tuniyev, B.; Andrén, C.; Orlov, N.L. Vipers of Caucasus: Taxomonic Considerations; Joger, U., Ed.;
Kaupia: Darmstadt, Germany, 1999.
37.
Tomovi´c, L. Systematics of the Nose-horned Viper (Vipera ammodytes, Linnaeus, 1758). Herpetol. J.
2006
,16,
191–201.
38.
Heckes, U.; Gruber, H.-J.; Stumpel, N. Schlangen (Serpentes) III: Viperidae. In Handbuch der Reptilien und
Amphibien Europas.: Schlangen (Serpentes) III, Viperidae; Joger, U., Stümpel, N., Böhme, W., Eds.; AULA: Graz,
Austria, 2006; pp. 81–151.
39.
Ursenbacher, S.; Schweiger, S.; Tomovi´c, L.; Crnobrnja-Isailovi´c, J.; Fumagalli, L.; Mayer, W. Molecular
phylogeography of the nose-horned viper (Vipera ammodytes, Linnaeus (1758)): Evidence for high genetic
diversity and multiple refugia in the Balkan peninsula. Mol. Phylogenetics Evol.
2008
,46, 1116–1128.
[CrossRef] [PubMed]
40.
Göçmen, B.; Mulder, J.; Karis, M.; Mebert, K. New Locality Records of Vipera ammodytes transcaucasiana
(Boulenger, 1913) in Turkey. South-West. J. Hortic. Biol. Environ. 2015,2, 91–98.
41.
Georgieva, D.; Risch, M.; Kardas, A.; Buck, F.; von Bergen, M.; Betzel, C. Comparative analysis of the venom
proteomes of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis.J. Proteome Res.
2008
,7, 866–886.
[CrossRef] [PubMed]
42.
Frangides, C.Y.; Koulouras, V.; Kouni, S.N.; Tzortzatos, G.V.; Nikolaou, A.; Pneumaticos, J.; Pierrakeas, C.;
Niarchos, C.; Kounis, N.G.; Koutsojannis, C.M. Snake venom poisoning in Greece. Experiences with 147
cases. Eur. J. Intern. Med. 2006,17, 24–27. [CrossRef] [PubMed]
43.
Brvar, M.; Kurtovi´c, T.; Grenc, D.; Lang Balija, M.; Križaj, I.; Halassy, B. Vipera ammodytes bites treated with
antivenom ViperaTAb: A case series with pharmacokinetic evaluation. Clin. Toxicol.
2017
,55, 241–248.
[CrossRef] [PubMed]
44.
Archundia, I.G.; de Roodt, A.R.; Ramos-Cerrillo, B.; Chippaux, J.-P.; Olguín-Pérez, L.; Alagón, A.; Stock, R.P.
Neutralization of Vipera and Macrovipera venoms by two experimental polyvalent antisera: A study of
paraspecificity. Toxicon 2011,57, 1049–1056. [CrossRef] [PubMed]
45.
Kato, H.; Iwanaga, S.; Suzuki, T. The isolation and amino acid sequences of new pyroglutamylpeptides from
snake venoms. Experientia 1966,22, 49–50. [CrossRef] [PubMed]
46.
Francis, B.; Kaiser, I.I. Inhibition of metalloproteinases in Bothrops asper venom by endogenous peptides.
Toxicon 1993,31, 889–899. [CrossRef]
47.
Huang, K.F.; Hung, C.C.; Wu, S.H.; Chiou, S.H. Characterization of three endogenous peptide inhibitors for
multiple metalloproteinases with fibrinogenolytic activity from the venom of Taiwan habu (Trimeresurus
mucrosquamatus). Biochem. Biophys. Res. Commun. 1998,248, 562–568. [CrossRef] [PubMed]
48.
Munekiyo, S.M.; Mackessy, S.P. Presence of peptide inhibitors in rattlesnake venoms and their effects on
endogenous metalloproteases. Toxicon 2005,45, 255–263. [CrossRef] [PubMed]
49.
Yee, K.T.; Pitts, M.; Tongyoo, P.; Rojnuckarin, P.; Wilkinson, M.C. Snake Venom Metalloproteinases and Their
Peptide Inhibitors from Myanmar Russell’s Viper Venom. Toxins 2016,9, 15. [CrossRef] [PubMed]
50.
Wagstaff, S.C.; Favreau, P.; Cheneval, O.; Laing, G.D.; Wilkinson, M.C.; Miller, R.L.; Stöcklin, R.; Harrison, R.A.
Molecular characterisation of endogenous snake venom metalloproteinase inhibitors. Biochem. Biophys.
Res. Commun. 2008,365, 650–656. [CrossRef] [PubMed]
Toxins 2018,10, 23 23 of 24
51.
Latinovi´c, Z.; Leonardi, A.; Šribar, J.; Sajevic, T.; Žužek, M.C.; Frangež, R.; Halassy, B.; Trampuš-Bakija, A.;
Pungerˇcar, J.; Križaj, I. Venomics of Vipera berus berus to explain differences in pathology elicited by Vipera
ammodytes ammodytes envenomation: Therapeutic implications. J. Proteom.
2016
,146, 34–47. [CrossRef]
[PubMed]
52. Tu, A.T. Venoms: Chemistry and Molecular Biology, 1st ed.; John Wiley & Sons: New York, NY, USA, 1977.
53. Tu, A.T. Handbook of Natural Toxins; Dekker: New York, NY, USA, 1991.
54.
Mackessy, S.P. Evolutionary trends in venom composition in the western rattlesnakes (Crotalus viridis sensu
lato): Toxicity vs. tenderizers. Toxicon 2010,55, 1463–1474. [CrossRef] [PubMed]
55.
Angulo, Y.; Escolano, J.; Lomonte, B.; Gutiérrez, J.M.; Sanz, L.; Calvete, J.J. Snake venomics of Central
American pitvipers: Clues for rationalizing the distinct envenomation profiles of Atropoides nummifer and
Atropoides picadoi. J. Proteome Res. 2008,7, 708–719. [CrossRef] [PubMed]
56.
Fernández, J.; Lomonte, B.; Sanz, L.; Angulo, Y.; Gutiérrez, J.M.; Calvete, J.J. Snake venomics of Bothriechis
nigroviridis reveals extreme variability among palm pitviper venoms: Different evolutionary solutions for
the same trophic purpose. J. Proteome Res. 2010,9, 4234–4241. [CrossRef] [PubMed]
57.
Gibbs, H.L.; Sanz, L.; Sovic, M.G.; Calvete, J.J. Phylogeny-based comparative analysis of venom proteome
variation in a clade of rattlesnakes (Sistrurus sp.). PLoS ONE 2013,8, e67220. [CrossRef] [PubMed]
58.
Chijiwa, T.; Deshimaru, M.; Nobuhisa, I.; Nakai, M.; Ogawa, T.; Oda, N.; Nakashima, K.; Fukumaki, Y.;
Shimohigashi, Y.; Hattori, S.; et al. Regional evolution of venom-gland phospholipase A2 isoenzymes of
Trimeresurus flavoviridis snakes in the southwestern islands of Japan. Biochem. J.
2000
,347, 491–499. [CrossRef]
[PubMed]
59.
Daltry, J.C.; Wüster, W.; Thorpe, R.S. Diet and snake venom evolution. Nature
1996
,379, 537–540. [CrossRef]
[PubMed]
60.
Gibbs, H.L.; Mackessy, S.P. Functional basis of a molecular adaptation: Prey-specific toxic effects of venom
from Sistrurus rattlesnakes. Toxicon 2009,53, 672–679. [CrossRef] [PubMed]
61.
Mackessy, S.P. Venom Ontogeny in the Pacific Rattlesnakes Crotalus viridis helleri and C. v. oreganus.Copeia
1988,1988, 92–101. [CrossRef]
62.
Pimenta, D.C.; Prezoto, B.C.; Konno, K.; Melo, R.L.; Furtado, M.F.; Camargo, A.C.M.; Serrano, S.M.T.
Mass spectrometric analysis of the individual variability of Bothrops jararaca venom peptide fraction. Evidence
for sex-based variation among the bradykinin-potentiating peptides. Rapid Commun. Mass Spectrom.
2007
,
21, 1034–1042. [CrossRef] [PubMed]
63.
Tashima, A.K.; Sanz, L.; Camargo, A.C.M.; Serrano, S.M.T.; Calvete, J.J. Snake venomics of the Brazilian
pitvipers Bothrops cotiara and Bothrops fonsecai. Identification of taxonomy markers. J. Proteom.
2008
,71,
473–485. [CrossRef] [PubMed]
64.
Mancheva, I.; Kleinschmidt, T.; Aleksiev, B.; Braunitzer, G. Sequence homology between phospholipase
and its inhibitor in snake venom. The primary structure of the inhibitor of vipoxin from the venom of the
Bulgarian viper (Vipera ammodytes ammodytes, Serpentes). Hoppe-Seyler’s Z. Physiol. Chem.
1984
,365, 885–894.
[CrossRef] [PubMed]
65.
Krizaj, I.; Liang, N.S.; Pungercar, J.; Strukelj, B.; Ritonja, A.; Gubensek, F. Amino acid and cDNA sequences of
a neutral phospholipase A2 from the long-nosed viper (Vipera ammodytes ammodytes) venom. Eur. J. Biochem.
1992,204, 1057–1062. [CrossRef] [PubMed]
66.
Almeida, L.M.; Resende, J.R.; Watanabe, R.K.; Corassola, V.C.; Huancahuire-Vega, S.; da S Caldeira, C.A.;
Coutinho-Neto, A.; Soares, A.M.; Vale, N.; de C Gomes, P.A.; et al. Snake venom peptides and low mass
proteins: Molecular tools and therapeutic agents. Curr. Med. Chem.
2017
,24, 3254–3282. [CrossRef] [PubMed]
67.
Brenton, J.D.; Carey, L.A.; Ahmed, A.A.; Caldas, C. Molecular classification and molecular forecasting of
breast cancer: Ready for clinical application? J. Clin. Oncol. 2005,23, 7350–7360. [CrossRef] [PubMed]
68.
Subik, K.; Lee, J.-F.; Baxter, L.; Strzepek, T.; Costello, D.; Crowley, P.; Xing, L.; Hung, M.-C.; Bonfiglio, T.;
Hicks, D.G.; et al. The Expression Patterns of ER, PR, HER2, CK5/6, EGFR, Ki-67 and AR by
Immunohistochemical Analysis in Breast Cancer Cell Lines. Breast Cancer
2010
,4, 35–41. [CrossRef] [PubMed]
69.
Khunsap, S.; Khow, O.; Buranapraditkun, S.; Suntrarachun, S.; Puthong, S.; Boonchang, S. Anticancer
properties of phospholipase A2 from Daboia siamensis venom on human skin melanoma cells. J. Venom. Anim.
Toxins Incl. Trop. Dis. 2016,22. [CrossRef] [PubMed]
70.
Yamazaki, Y.; Morita, T. Structure and function of snake venom cysteine-rich secretory proteins. Toxicon
2004
,
44, 227–231. [CrossRef] [PubMed]
Toxins 2018,10, 23 24 of 24
71.
Wang, Y.-L.; Kuo, J.-H.; Lee, S.-C.; Liu, J.-S.; Hsieh, Y.-C.; Shih, Y.-T.; Chen, C.-J.; Chiu, J.-J.; Wu, W.-G.
Cobra CRISP functions as an inflammatory modulator via a novel Zn2+- and heparan sulfate-dependent
transcriptional regulation of endothelial cell adhesion molecules. J. Biol. Chem.
2010
,285, 37872–37883.
[CrossRef] [PubMed]
72.
Alape-Girón, A.; Sanz, L.; Escolano, J.; Flores-Díaz, M.; Madrigal, M.; Sasa, M.; Calvete, J.J. Snake venomics
of the lancehead pitviper Bothrops asper: Geographic, individual, and ontogenetic variations. J. Proteome Res.
2008,7, 3556–3571. [CrossRef] [PubMed]
73.
Muth, T.; Weilnböck, L.; Rapp, E.; Huber, C.G.; Martens, L.; Vaudel, M.; Barsnes, H. DeNovoGUI: An open
source graphical user interface for de novo sequencing of tandem mass spectra. J. Proteome Res.
2014
,13,
1143–1146. [CrossRef] [PubMed]
74.
Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol.
1990,215, 403–410. [CrossRef]
75.
Vizcaíno, J.A.; Deutsch, E.W.; Wang, R.; Csordas, A.; Reisinger, F.; Ríos, D.; Dianes, J.A.; Sun, Z.; Farrah, T.;
Bandeira, N.; et al. ProteomeXchange provides globally coordinated proteomics data submission and
dissemination. Nat. Biotechnol. 2014,32, 223–226. [CrossRef] [PubMed]
76.
Juárez, P.; Sanz, L.; Calvete, J.J. Snake venomics: Characterization of protein families in Sistrurus barbouri
venom by cysteine mapping, N-terminal sequencing, and tandem mass spectrometry analysis. Proteomics
2004,4, 327–338. [CrossRef] [PubMed]
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
