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Snake venomics of the South and Central American
Bushmasters. Comparison of the toxin composition of Lachesis
muta gathered from proteomic versus transcriptomic analysis
Libia Sanza, José Escolanoa, Massimo Ferrettib, Mirtha J. Biscoglioc,
Elena Riverad, Ernesto J. Crescentie, Yamileth Angulo f, Bruno Lomonte f,
José María Gutiérrezf, Juan J. Calvetea,⁎
aInstituto de Biomedicina de Valencia, C.S.I.C., Valencia, Spain
bDepartment of Agricultural Biotechnology, University of Padova, Padova, Italy
cDepartamento de Quimica Biologica, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Buenos Aires, Argentina
dLaboratorio de Radioisotopos, Catedra de Fisica, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Buenos Aires, Argentina
eInstitute of Immunooncology, Buenos Aires, Argentina
fInstituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica
A R T I C L E I N F O A B S T R A C T
Article history:
Received 25 September 2007
Accepted 19 October 2007
We report the proteomic characterization of the venoms of two closely related pit vipers of
the genus Lachesis, L. muta (South American Bushmaster) and L. stenophrys (Central
American Bushmaster), and compare the toxin repertoire of the former revealed through
a proteomic versus a transcriptomic approach. The protein composition of the venoms of
Lachesis muta and L. stenophrys were analyzed by RP-HPLC, N-terminal sequencing, MALDI-
TOF peptide mass fingerprinting and CID-MS/MS. Around 30–40 proteins of molecular
masses in the range of 13–110 kDa and belonging, respectively, to only 8 and 7 toxin families
were identified in L. muta and L. stenophrys venoms. In addition, both venoms contained a
large number of bradykinin-potentiating peptides (BPP) and a C-type natriuretic peptide
(C-NP). BPPs and C-NP comprised around 15% of the total venom proteins. In both species,
themost abundant proteinswere Zn2+-metalloproteinases (32–38%) and serine proteinases
(25–31%), followed by PLA2s (9–12%), galactose-specific C-type lectin (4–8%), L-amino acid
oxidase (LAO, 3–5%), CRISP (1.8%; found in L. muta but not in L. stenophrys), and NGF (0.6%).
On the other hand, only six L. muta venom-secreted proteinsmatched any of the previously
reported 11 partial or full-length venom gland transcripts, and venom proteome and
transcriptome depart in their relative abundances of different toxin families. As expected
from their close phylogenetic relationship, the venoms of L. muta and L. stenophrys share (or
contain highly similar) proteins, in particular BPPs, serine proteinases, a galactose-specific
C-type lectin, and LAO. However, they dramatically depart in their respective PLA2
complement. Intraspecific quantitative and qualitative differences in the expression of
PLA2molecules were found when the venoms of five L. muta specimens (3 from Bolivia and
2 from Peru) and the venom of the same species purchased from Sigma were compared.
These observations indicate that these class of toxins represents a rapidly-evolving
Keywords:
Snake venomics
Venom proteomics
Snake toxins
Lachesis muta
Lachesis stenophrys
Bushmaster
Pit viper
Mass spectrometry
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⁎ Corresponding author. Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain. Tel.: +34 96 339 1778; fax: +34 96
369 0800.
E-mail address: jcalvete@ibv.csic.es (J.J. Calvete).
1874-3919/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jprot.2007.10.004
ava i l ab l e a t www.sc i enced i r ec t . com
www.e l sev i e r. com/ loca te / j p ro t
Author's personal copy
gene family, and suggests that functional differences due to structural changes in PLA2s
molecules among these snakes may have been a hallmark during speciation and
adaptation of diverging snake populations to new ecological niches, or competition for
resources in existing ones. Our data may contribute to a deeper understanding of the
biology and ecology of these snakes, and may also serve as a starting point for studying
structure–function correlations of individual toxins.
© 2007 Elsevier B.V. All rights reserved.
1. Introduction
Venom toxins likely evolved from proteins with a normal
physiological function and appear to have been recruited into
the venom proteome before the diversification of the ad-
vanced snakes, at the base of the Colubroidea radiation [1–4].
Given the central role that diet has played in the adaptive
radiation of snakes [5], venoms represent the critical innova-
tion that allowed advanced snakes to transition from a
mechanical (constriction) to a chemical (venom) means of
subduing and digesting prey larger than themselves, and as
such, venom proteins have multiple functions including
immobilizing, paralyzing, killing and digesting prey. Venoms
produced by snakes of the family Viperidae (vipers and pit
vipers) contain proteins that interfere with the coagulation
cascade, the normal haemostatic system and tissue repair,
and human envenomations are often characterized by clotting
disorders, hypofibrinogenemia and local tissue necrosis [6,7].
In spite of the fact that viperid venoms may contain well over
100 protein components [8], venom proteins belong to only a
few major protein families, including enzymes (serine protei-
nases, Zn2+-metalloproteinases, L-amino acid oxidase, group II
PLA2) and proteinswithout enzymatic activity (disintegrins, C-
type lectins, natriuretic peptides, myotoxins, CRISP toxins,
nerve and vascular endothelium growth factors, cystatin and
Kunitz-type proteinase inhibitors). However, snake venoms
depart from each other in the composition and the relative
abundance of toxins [1,2,8–14].
In addition to understanding how venoms evolve, char-
acterization of the protein/peptide content of snake venoms
also has a number of potential benefits for basic research,
clinical diagnosis, development of new research tools and
drugs of potential clinical use, and for antivenom production
strategies [15]. Within- and between-species heterogeneity of
venoms may also account for differences in the clinical
symptoms observed in accidental envenomations. In order
to explore the putative venom components, several labora-
tories have carried out transcriptomic analyses of the venom
glands of viperid (Bitis gabonica [16], Bothrops insularis [17], Bo-
throps jararacussu [18], Bothrops jararaca [19], Agkistrodon acutus
[20,21], Echis ocellatus [22], and Lachesis muta [23]), elapid
(Oxyuramus scutellatus [24]), and colubrid (Philodryas olfersii
[25]) snake species. Transcriptomic investigations provide
catalogues of partial and full-length transcripts that are
synthesized by the venom gland. Here, we report a proteomic
analysis of L. muta venom, which complements the study of
snake venom gene transcriptional activity (transcriptome) in
the same species [23] by showing the relative abundance of the
various protein families that are actually secreted into the
venoms.
Lachesis is a genus of venomous pit vipers widely dis-
tributed in remote, lowland tropical forested areas in Central
and South America, and the only neo-tropical pit viper that
lays eggs. Three species are currently recognized in this genus,
L. muta (South American bushmaster), L. stenophrys (Central
American bushmaster), and L. melanocephala (Black-headed
bushmaster) (http://www.reptile-database.org) . The bush-
masters are the largest of all pit vipers and the longest
venomous snakes in the western hemisphere. Adults grow to
an average of 2 to 2.5 m, although 3 m is not too unusual.
Lachesis melanocephala is endemic of the pacific versant
of Costa Rica, where it is confined to the southwestern Osa
Peninsula [26]. L. muta is found in South America in the
equatorial forests east of the Andes, ranging from Colombia,
eastern Ecuador, Peru, northern Bolivia, eastern and southern
Venezuela, to Guyana, Surinam, French Guiana and much of
northern Brazil. Lachesis stenophrys, is found in the Atlantic
lowlands of southern Nicaragua, Costa Rica and the Atlantic
and Pacific lowlands of central and eastern Panama. In South
America it occurs in the Pacific lowlands of Colombia and
northwestern Ecuador, the Caribbean coast of northwestern
Colombia and inland along the Magdalena and Cauca river
valleys.
Human envenoming by Lachesis (muta or stenophrys) are
infrequent but rather severe and characterized by conspic-
uous local tissue damage (edema, hemorrhage and necrosis),
nausea, coagulopathies, hypotension, shock and renal dis-
turbances [27]. Brown [28] mentions a venom yield of 200–
411 mg from L. muta and gives the following LD50% values for
mice: 1.5 mg/kg (i.v.), 1.6–6.2 mg/kg (i.p.), 6.0 mg/kg (s.c.).
Paradoxically, although bites can be deadly, snake venoms
also contain components of theraupeutic value. The venom of
L. muta has attracted medical interest because its reported
protective effect in rats subjected to high cytostatic doses,
when administered at low (4 ng/ml) concentration in combi-
nation with Mg, Se, Zn (4 μg/ml each) [29]. It has been also
reported that administration of this formulation to nude mice
that had developed tumors by inoculation of PANC-1 cells,
inhibited tumor growth and angiogenesis, induced apoptosis,
and modulated the activity of antioxidant enzymes [30].
Ongoing research from our laboratories, which will be
published elsewhere, indicates that daily subcutaneous
administration of L. muta venom (0.5 ml, 4 ng/ml) significantly
(pb0.05) increased the survival of N-nitroso-N-methylurea-
induced tumor-bearing rats (103 days) compared to non-Lm-
treated animals (66 days). Furthermore, the venom provoked
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Table 1 – Assignment of the reverse-phase fractions of Lachesis muta venom (Santa Cruz de la Sierra, Bolivia), isolated as in
Fig. 1, to protein families by N-terminal Edman sequencing, mass spectrometry, and collision-induced fragmentation by
nESI-MS/MS of selected peptide ions from in-gel digested protein bands (separated by SDS-PAGE as in Fig. 2)
HPLC
fraction
N-terminal
sequencing
Isotope-averaged
molecular
mass
Peptide
ion
MS/MS-derived
sequence
Protein family
Lm- m/z z
1 N.D. 384.0 2 Ac-TPPAGPD(+41) [Q27J49]
465.6 2 TPPAGPDVGP(+91) BPP-C-NP precursor
581.8 2 DHHAVGGGGGGGGA(+91)
2 N.D. 480.6 2 ZKKWPPGH [Q27J49]
3 N.D. 532.1 2 TPPAGPDVGPR [Q27J49]
482.0 2 PPAGPDVGPR
768.5 1 AGPDVGPR
697.4 1 GPDVGPR
543.5 1 DVGPR
4, 5 n.p.
6 N.D. 702.6 2 ZKKWPPGHHIPP [Q27J49]
764.1 1 ZKKWPP
7 N.D. 597.2 2 ZKKWPPGHHI [Q27J49]
549.0 2 ZKKWPPGHH
582.9 2 (308.4)KWPPGHH
8 N.D. 725.5 3 HHIPPVVVQEWPPGHHIPP [Q27J49]
9 N.D. 639.3 2 ZEWPPGHHIPP [Q27J49]
10 N.D. 623.1 2 ZKWDPPPISPP [Q27J49]
544.1 2 WDPPPISPP
945.5 1 ZKWDPPPI
1032.5 1 ZKWDPPPIS
11 N.D. 868.7 3 ZKWDPPPISPPLLKPHES
PAGGTTA
[Q27J49]
845.1 3 ZKWDPPPISPPLLKPHE
SPAGGTT
758.7 3 ZKWDPPPISPPLLKPHESPAG
653.0 3 ZKWDPPPISPPLLKPHE
611.5 3 ZKWDPPPISPPLLKPH
688.7 3 PPPISPPLLKPHESPAGGTTA
578.6 3 PPPISPPLLKPHESPAG
12 N.D. 715.8 3 ZKWDPPPISPPLLKPHESP [Q27J49]
679.3 2 ZKWDPPPISPPL
13 GDGCFGLKLDRIGSMSGLGC 1983.4 C-NP [Q27J49]
GCFGLKLDRIGSMSGLGC 1811.6
14 Blocked 12041, 12154 unknown
15 Blocked 16 kDa ■▼ 556.2 2 NPNPVPTGCR Nerve growth factor
632.1 2 IDAACVCISR
682.1 2 ALTMEGNQASWR
16 HLLQFGDLINKIARRNGIS- 13932.6 649.6 2 HLLQFGDLINK PLA2
-YYGFYGCYCGL 605.3 3 EICECDRDAAICFR
17 HLLQFGDLINKIARRNGIS- 13916.3 649.1 2 HLLQFGDLINK PLA2
-YYGFYG 605.3 3 EICECDRDAAICFR
490.7 2 EICECDR
548.7 2 DNLDTYDNK
752.7 2 CCFVHDCCYGK
512.7 2 YWFFHPK
507.9 2 GRPQDATDR
18 m: HLLQFEQLIRKIAGRSGFRYYGFY-
-GCYCGLGGQGRPQDA 14008.6 PLA2
M: SVDFDSESPRKPEIQNKIVDLHNSL 24683.9 CRISP
19 VFGGDECNINEHRSLVVLFNSS 30 kDa ■ 2-chain Ser-proteinase
TSTTLCAGILEGGKDSCHGDSG [Q27J47]
SVDFDSESPRKPEIQ 24683.8 CRISP
20 IIGGDECNINEHRSL 28 kDa ■ 647.5 2 XNXXDYEVCR Ser-proteinase
763.8 2 IIGGDECNINEHR
VFGGDECNINEHRSL 26 kDa ■ 608.1 2 KVPNKDEETR Ser-proteinase [Q27J47]
714.9 2 SLPSSPPSVGSVCR
773.4 2 VFGGDECNINEHR
SVDFDSESPRKPEIQ 24 kDa ■ CRISP
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Table 1 (continued)
HPLC
fraction
N-terminal
sequencing
Isotope-averaged
molecular
mass
Peptide
ion
MS/MS-derived
sequence
Protein family
Lm- m/z z
21 VFGGDECNINEHRSL
VVLFNSS
27 kDa ■ Ser-proteinase [Q27J47]
22 M: NNCPQDWLPMNG
LCYKIFD
28 kDa ■/
14 kDa▼
Gal-lectin [Q9PSM4]
m: VIGGDECNINEHR
FLVALY
29 kDa▼ Ser-proteinase [P33589]
23 M: VIGGDECNINEHRFLVALYDG- 30 kDa▼ Ser-proteinase [P33589]
-LSGTFLCG Venombin A
m: VFGGDECNINEHRSLVVLFNS- 30 kDa▼ Ser-proteinase [Q27J47]
-SGFLCAGT
23–29 NNCPQDWLPMNGL
CYKIFD
28 kDa ■ 639.6 2 LWNDQVCESK Gal-lectin [Q9PSM4]
14 kDa▼ 620.9 2 DFSWEWTDR
644.3 2 SCTDYLTWDK
665.8 2 AWEDAEmFCR
786.8 2 EFCVELVSLTGYR
639.3 3 YGESLEIAEYISDYHK
24 IVGGDECNINEHRFL
VALYDP
30 kDa▼ Serine proteinase
25 VIGGDECNINEHRF
LVALYD
30 kDa▼ Serine proteinase [P33589]
26, 27 VIGGDECNINEHRSL
VALYD
38 kDa▼ 575.5 2 NVKFDDEQR Serine proteinase Venombin A
756.9 2 VIGGDECNINEHR (S35689)
867.3 2 VLCAGVLEGGIDTCNR
772.8 2 SLPSNPPSEDSVCR
VFGGDECNINEHR
SLVVLFN
31 kDa▼ 773.9 2 VFGGDECNINEHR Serine proteinase (P84036)
711.5 2 AIYPEFGLPATSR Plasminogen-activator
28 VFGGDECNINEHRSLVV 31 kDa▼ 791.4 2 CANINLLDYAVCR Serine proteinase
744.6 2 (261.2)PSSPPSVGSVCR
28–38 29 kDa ■
14 kDa▼ 459.8 3 GHCYKPFNEPK C-type lectin-like
29 ADDRNPLGECFRETD
YEEFLEIAK
58 kDa ■▼ 583.1 2 KFWEDDGIR L-amino acid oxidase
641.0 2 SAGQLYEESLGK
744.1 2 ETDYEEFLEIAK
30 (NFPPYEANIMRV) 26 kDa ■▼ 781.3 2 VHEIVNTLNGFYR PI-metalloproteinase(P22796)
590.1 2 TFGEWRER Hemorrhagic factor LHFII
641.1 2 NSVGIVQDHSPK
869.5 2 YIELVVVADHGmFTK
31 Blocked 26 kDa ■▼ 781.3 2 VHEIVNTLNGFYR PI-metalloproteinase (P22796)
590.1 2 TFGEWRER Hemorrhagic factor LHFII
641.1 2 NSVGIVQDHSPK
869.5 2 YIELVVVADHGmFTK
32, 33 Blocked 37 kDa ■▼ 518.8 2 SVGIVQDYR PIII-Metalloproteinase
672.2 3 LTPGSQCADGECCDQCR
671.3 2 YIELVLLADHR
26 kDa ■▼ 781.3 2 VHEIVNTLNGFYR PI-metalloproteinase
(P22796)
590.1 2 TFGEWRER Hemorrhagic factor LHFII
34 Blocked 58 kDa ■▼ 752.6 2 XFCEFNNFPCR PIII-Metalloproteinase
650.0 2 YVEXVVVADHR
35 Blocked 27 kDa ■▼ 577.6 2 ZVVTAEQQR PI-Metalloproteinase
604.6 2 QGAQCAEGLCCDQCR
530.3 2 XACEPQDVK
627.2 2 PQCXXQQXPR
635.6 2 LYCFPSSPATK
36 Blocked 110 kDa ■ 761.9 2 SAAADTXEAFADWR (PIII-Metalloproteinase)2
49 kDa▼ 594.9 3 (581.5)VVVADHN(467.0)
(continued on next page)
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50–70% cell proliferation inhibition of cultured human neo-
plasia-derived cell lines MDA-MB-231 and MCF-7 (breast
cancer), PANC-1 (pancreatic carcinoma), WM35 and HT168
(melanoma), and U937 (histiocytic lymphoma).
Otero and co-workers [31] have reported quantitative
differences in toxic and enzymatic activities along with subtle
variations in the electrophoretic patterns of L. muta and L.
stenophrys venoms from Brazil, Colombia, and Costa Rica,
although experimental envenomation by these venoms
induced a qualitatively similar pathophysiological profile.
Here, we report the proteomic characterization of the toxin
composition of L. muta and L. stenophrys venoms, and compare
the toxin repertoire of the former revealed through a
proteomic versus a transcriptomic approach. Venom toxin
composition provides a comprehensible catalogue of the
venom-secreted proteins, which may contribute to a deeper
understanding of the biology and ecology of the snake, the
biological effects of the venom, and may also serve as a
starting point for studying structure–function correlations of
individual toxins.
2. Experimental section
2.1. Isolation and relative quantitation of venom proteins
Venom of L. stenophrys (Central American Bushmaster) was
pooled from specimens collected in Costa Rica and kept at the
serpentarium of the Instituto Clodomiro Picado, University of
Costa Rica in San José. L. muta (South American Bushmaster)
venom samples were obtained from 3 wild caught specimens
and were kindly provided by SICAE' s.r.l., a snakes farm
located in the nature reserve Potrerillos del Guendá (Santa
Cruz de la Sierra, Bolivia, P.O. Box 1615) (www.sicae-online.
com). Venoms from 2 specimens of L. muta from Peru (Iquitos,
Departamento de Loreto or Cenepa, Mamayaque) were also
analyzed. A further sample of L. muta venom was purchased
from Sigma–Aldrich (Alcobendas, Madrid, Spain; catalog no.
V7376). The source of this venom was not provided by the
vendor. For reverse-phase HPLC separations, 2–5 mg of crude,
lyophillized venom of L. mutawere dissolved in 100 μl of 0.05%
trifluoroacetic acid (TFA) and 5% acetonitrile, and insoluble
material was removed by centrifugation in an Eppendorff
centrifuge at 13,000 ×g for 10 min at room temperature.
Proteins in the soluble material were separated using an
ETTAN™ LC HPLC system (Amersham Biosciences) and a
Lichrosphere RP100 C18 column (250×4mm, 5 μmparticle size)
eluted at 1 ml/min with a linear gradient of 0.1% TFA in water
(solution A) and acetonitrile (solution B) (5% B for 10 min,
followed by 5–15% B over 20 min, 15–45% B over 120 min, and
45–70% B over 20 min). Protein detection was at 215 nm and
peaks were collected manually and dried in a Speed-Vac
(Savant). Given that the wavelength of absorbance for a
peptide bond is 190–230 nm, protein detection at 215 nm
allows to estimate the relative abundances (expressed as per-
centage of the total venom proteins) of the different protein
families from the relation of the sum of the areas of the
reverse-phase chromatographic peaks containing proteins
from the same family to the total area of venom protein
peaks in the reverse-phase chromatogram. In a strict sense,
and according to the Lambert–Beer law, the calculated relative
amounts correspond to the “% of total peptide bonds in the
sample”, which is a good estimate of the % byweight (gr/100gr)
of a particular venom component.
2.2. Characterization of HPLC-isolated proteins
Isolated protein fractions were subjected to N-terminal
sequence analysis (using a Procise instrument, Applied
Biosystems, Foster City, CA, USA) following the manufac-
turer's instructions. Amino acid sequence similarity searches
were performed against a non-redundant protein sequence
databank (comprising all non-redundant GenBank CDS trans-
lations+RefSeq Proteins+PDB+SwissProt+PIR+PRF) (http://
www.ncbi.nlm.nih.gov/BLAST/blastcgihelp.shtml#protein_data
bases) using the BLAST program [32] implemented in the WU-
BLAST2 search engine at http://www.bork.embl-heidelberg.de.
The molecular masses of the purified proteins were deter-
mined by SDS-PAGE (on 12–15% polyacrylamide gels) and by
electrospray ionization (ESI) mass spectrometry using an
Applied Biosystems QTrap™ 2000 mass spectrometer [33]
operated in Enhanced Multiple Charge mode in the range m/z
600–1200.
2.3. In-gel enzymatic digestion and mass fingerprinting
Protein bands of interest were excised from a Coomassie
Brilliant Blue-stained SDS-PAGE and subjected to automated
Table 1 (continued)
HPLC
fraction
N-terminal
sequencing
Isotope-averaged
molecular
mass
Peptide
ion
MS/MS-derived
sequence
Protein family
Lm- m/z z
37 Blocked 27 kDa ■▼ 627.2 2 PQCXXQQXPR PI-Metalloproteinase
635.6 2 LYCFPSSPATK
38 Blocked 48 kDa ■▼ 657.1 2 YXEXVVVADHR PIII-Metalloproteinase
504.8 2 (182.6)YNQPSK
X, Ile or Leu; Z, pyrrolidone carboxylic acid; Ac-, N-acetyl;m, methionine sulphoxide. Unless other stated, for MS/MS analyses, cysteine residues
were carbamidomethylated. Molecular masses of native proteins were determined by electrospray-ionization (±0.02%) or MALDI-TOF (⁎) (±0.2%)
mass spectrometry. Apparent molecular mass determined by SDS-PAGE of non-reduced (■) and reduced (▼) samples; n.p., non-peptidic
material found. M andm, denote mayor andminor products co-eluting in the same HPLC fraction. Previously reported proteins are identified by
their databank accession codes.
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reduction with DTT and alkylation with iodoacetamide,
and digestion with sequencing grade bovine pancreas trypsin
(Roche) using a ProGest™ digestor (Genomic Solutions) follow-
ing the manufacturer's instructions. The tryptic pep-
tide mixtures were dried in a Speed-Vac and redissolved in
5 μl of 70% acetonitrile and 0.1% TFA. Digests (0.65 μl) were
spotted onto aMALDI-TOF sample holder,mixedwith an equal
volume of a saturated solution of α-cyano-4-hydroxycinnamic
acid (Sigma) in 50%acetonitrile containing 0.1%TFA, dried, and
analyzed with an Applied Biosystems Voyager-DE Pro MALDI-
TOF mass spectrometer, operated in delayed extraction and
reflectormodes. A tryptic peptidemixture of Cratylia floribunda
seed lectin (SwissProt accession code P81517) prepared and
previously characterized in our laboratory was used as mass
calibration standard (mass range, 450–3300 Da).
2.4. CID-MS/MS
For peptide sequencing, the protein digest mixture was loaded
in a nanospray capillary column and subjected to electrospray
ionization mass spectrometric analysis using a QTrap 2000
mass spectrometer (Applied Biosystems) [33] equipped with a
nanoelectrospray source (Protana, Denmark). Doubly- or triply-
charged ions of selected peptides from the MALDI-TOF mass
fingerprint spectra were analyzed in Enhanced Resolution MS
mode and the monoisotopic ions were fragmented using the
Enhanced Product Ion tool with Q0 trapping. Enhanced Resolu-
tion was performed at 250 amu/s across the entire mass range.
Settings for MS/MS experiments were as follows: Q1-unit
resolution; Q1-to-Q2 collision energy — 30–40 eV; Q3 entry
barrier— 8 V; LIT (linear ion trap) Q3 fill time— 250 ms; and Q3
scan rate— 1000 amu/s. CID spectra were interpretedmanually
or using a licensed version of the MASCOT program (http://
www.matrixscience.com) against a private database containing
927 viperid protein sequences deposited in the SwissProt/
TrEMBL database (Knowledgebase Release 12 of July 2007;
http://us.expasy.org/sprot/). MS/MS mass tolerance was set to
±0.6 Da. Carbamidomethyl cysteine and oxidation of methione
were fixed and variable modifications, respectively.
2.5. Variation in venom composition between Lachesis taxa
We used similarity coefficients to estimate the similarity
of venom proteins between taxa. These coefficients are similar
to the bandsharing coefficients used to compare individual
genetic profiles based on multilocus DNA fingerprints [34]. We
defined the Protein Similarity Coefficient (PSC) between two
species “a” and “b” in the following way: PSCab=[2×(no.
of proteins shared between a and b)/(total number of distinct
proteins in a+total number of distinct proteins in b)]×100.
We judged two proteins (listed in Tables 1 and 2) as being
different when they met one or more of these criteria: 1) Had
different N-terminal sequences and/or distinct internal pep-
tides sequences (derived from MS/MS data) corresponding
to homologous regions; 2) had different peptide mass finger-
prints; 3) were of different sizes (judged by MALDI-TOF MS
or SDS-PAGE). For these comparisons, two proteinswere judged
to differ in size if they differed bymore than our estimate of the
95% confidence interval for particular sizing techniques (0.01%
for ESI-QTrap MS; 0.4% for MALDI-TOF MS-derivedmasses, and
+1.4 kDa for SDS-PAGE-determined masses); or 4) eluted in
different reverse-phase HPLC peaks. We emphasize that these
measures will give only minimum estimates of the similarities
between the venom profiles. We suspect that a number of the
proteins that we judge to be the same using the above criteria
would be found to differ at one or more of these criteria if more
complete information were available.
3. Results and discussion
3.1. Characterization of bushmaster venom proteomes
The crude venoms of L. muta and L. stenophrys were fractio-
nated by reverse-phase HPLC (Figs. 1 and 3), followed by
analysis of each chromatographic fraction by SDS-PAGE
(Figs. 2 and 4) and N-terminal sequencing. Molecular masses
of purified proteins were determined by ESI-MS or MALDI-TOF
mass spectrometry (Tables 1 and 2). Fig. 5A displays an
example of electrospray-ionization mass spectrum of the
25 kDa protein isolated in fraction 18 (Figs. 1 and 2) and
identified as a member of the CRISP family (Table 1). Protein
fractions showing single electrophoretic band, molecular
mass, and N-terminal sequence were straightforwardly
assigned by BLAST analysis (http://www.ncbi.nlm.nih.gov/
BLAST) to a known protein family, indicating that representa-
tive members of most snake venom toxin families are present
amongst the 927 viperid protein sequences deposited to date
in the SwissProt/TrEMBL database. Protein fractions showing
heterogeneous or blocked N-termini were analyzed by SDS-
PAGE and the bands of interest are subjected to automated
reduction, carbamidomethylation, and in-gel tryptic digestion
in a ProGest digestor (Genomic Solutions). The resulting
tryptic peptides are then analyzed by MALDI-TOF mass
fingerprinting followed by amino acid sequence determina-
tion of selected doubly- and triply-charged peptide ions by
collision-induced dissociation tandem mass spectrometry.
Product ion spectra were manually or using either the on-
line form of the MASCOT program (searching against the non-
redundant MSDB database) or a licensed version of this
program against a private snake venom database comprising
927 sequence entries (212 in SwissProt, 715 in TrEMBL) plus the
previously assigned peptide ion sequences from snake
venomics projects carried out in our laboratory [9–14]. Fig. 5B
illustrates the de novo sequencing of a doubly-charged tryptic
peptide ion (m/z 774.1) from protein Lm29 (Figs. 1 and 2)
identified as an L-amino acid oxidase (Table 1). The outlined
snake venomics approach allowed us to assign unambigu-
ously all the isolated venom toxins representing more than
0.05% of the total venom proteins (i.e. less than 50 ng in 100 μg
of venom proteins) to known protein families (Tables 1 and 2).
Supporting the view that venom proteomes are mainly
composed of toxins belonging to a few protein families [4,9–
14,16–24], the proteins found in the venoms of L. muta and
L. stenophrys cluster, respectively, in 8 and 7 different families
(bradykinin-potentiating peptides, NGF, PLA2, serine protei-
nase, cysteine-rich secretory proteins (CRISP; only found in
L. muta), C-type lectins, L-amino acid oxidase (LAO), and Zn2+-
dependent metalloproteinases) (Fig. 6), whose relative abun-
dances are listed in Table 3.
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Table 2 – Assignment of the reverse-phase fractions of Lachesis stenophrys venom (Costa Rica), isolated as in Fig. 3, to protein
families by N-terminal Edman sequencing, mass spectrometry, and collision-induced fragmentation by nESI-MS/MS of
selected peptide ions from in-gel digested protein bands (separated by SDS-PAGE as in Fig. 4)
HPLC fraction N-terminal sequencing Isotope-
averaged
molecular
mass
Peptide
ion
MS/MS-derived
sequence
Protein family
Ls- m/z z
1 N.D. 480.6 2 ZKKWPPGH [Q27J49]
2 N.D. 532.4 2 TPPAGPDVGPR [Q27J49]
482.1 2 PPAGPDVGPR
3, 4 n.p.
5 N.D. 702.8 2 ZKKWPPGHHIPP [Q27J49]
710.5 2 QKKWPPGHHIPP
549.0 2 ZKKWPPGHH
530.1 2 WPPGHHIPP(+41)
6 N.D. 639.1 2 ZEWPPGHHIPP [Q27J49]
622.1 2 PRPQIPPLVVQ
7 N.D. 748.4 2 SHKGWPPRPQIPP [Q27J49]
572.9 2 GWPPRPQIPP
649.8 2 WPPRPQIPPLV
8 N.D. 623.1 2 ZKWDPPPISPP [Q27J49]
543.4 2 WPPRPQIPP
9 GDGCFGLKLDRIGSMSGLGC 1983.1 C-NP [Q27J49]
10 Blocked 16 kDa ■▼ 556.2 2 NPNPVPTGCR Nerve growth factor
632.1 2 IDAACVCISR
682.1 2 ALTMEGNQASWR
11 N.D. 15 kDa▼ 649.6 2 HLLQFGDLIDK PLA2
12 HLLQFGDLIDKIAGR 14052 649.7 2 HLLQFGDLIDK PLA2
13 HLLQFGDLIDKIAGR 13898 649.7 2 HLLQFGDLIDK PLA2
753.3 2 CCFVHDCCYGK
605.4 3 EICECDRDAAICFR
14 IIGGDECNINEHRFL 37 kDa ■▼ 647.7 2 XNXXDYEVCR Serine proteinase
763.8 2 IIGGDECNINEHR
13 kDa▼
15 (V/I)(V/I)GGDECNINEHRFL 34 kDa ■▼ 647.7 2 XNXXDYEVCR Serine proteinase
763.8 2 IIGGDECNINEHR
621.9 2 TGXWGXR
16 (V/I)(V/I)GGDECNINEHRFL 32 kDa ■▼ 756.8 2 VIGGDECNINEHR Serine proteinase
683.4 2 (292.2)PEFGLPATSR
715.3 2 SXPSSPPSVGSVCR
17 M: NNCPQDWLPMNGLCY 28 kDa ■ 670.6 3 NNCPQDWLPMNGLCYK Gal-lectin [Q9PSM4]
14 kDa▼ 639.6 3 YGESLEIAEYISDYHK
786.8 2 EFCVELVSLTGYR
736.8 2 YKPGCHLASFHR
701.3 2 GQAEVWIGLWDK
765.4 2 GQAEVWIGLWDKK
657.7 2 AWEDAEMFCR
621.1 2 DFSWEWTDR
685.3 2 KDFSWEWTDR
800.8 2 KYKPGCHLASFHR
m: VIGGDECNINEHRFL 34 kDa▼ Serine proteinase
18 IVGGDECNINEHRFL 34+26 kDa▼ 756.9 2 IVGGDECNINEHR Serine proteinase
711.5 2 AIYPEFGLPATSR
19 N.D. 29 kDa ■▼ 690.4 2 (306.4)PEFGLPATSR Serine proteinase
20 VLGGDECNINEHRFL 33+24 kDa▼ 715.3 2 SLPSSPPSVGSVCR Serine proteinase
647.7 2 XNXXDYEVCR
21 VIGGDECNINEHRSLVALYD 38 kDa ■▼ 575.5 2 NVKFDDEQR Ser-proteinase Venombin A
756.9 2 VIGGDECNINEHR (S35689)
867.3 2 VLCAGVLEGGIDTCNR
710.9 2 SLMNIYLGMHNK
772.8 2 SLPSNPPSEDSVCR
VFGGDECNINEHRSLVVLFN 31 kDa ■▼ 773.9 2 VFGGDECNINEHR Ser-proteinase (P84036)
711.5 2 AIYPEFGLPATSR Plasminogen-activator
22 VFGGDECNINEHRSLVV 32 kDa▼ 715.3 2 SLPSSPPSVGSVCR Serine proteinase
647.7 2 XNXXDYEVCR
690.4 2 (306.4)PEFGLPATSR
773.9 2 VFGGDECNINEHR
VVGGDECNINEHR 31 kDa▼ 749.9 2 VVGGDECNINEHR Serine proteinase
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Except for the absence of a CRISP molecule in the venom of
L. stenophrys, the two Lachesis species investigated show very
similar overall venom toxin compositions (Table 3). However,
comparison of the chromatographic separations of the venom
proteins from L. muta and L. stenophrys (Figs. 1 and 3) and the
tryptic peptide mass fingerprints of their individual protein
bands (Tables 1 and 2), evidenced both, a number of very
similar (or identical) proteins but also toxins from the same
family showing a large degree of structural divergence.
Identical L. muta and L. stenophrys venom components
include a number of bradykinin-potentiating peptides (BPPs)
and a C-type natriuretic peptide released from the 239-amino-
acid precursor protein Q27J49 (Fig. 7). BPPs found in fractions
Ls7, Lm9/Ls6, Lm6/Ls5, and Lm10/Ls8 have been previously
identified byMALDI-TOFMS in the crude venomof a specimen
kept in captivity at the serpentarium of the Fundação Ezequiel
Dias (Belo Horizonte, Brazil) [35], indicating that expression of
these peptides appear not to exhibit geographical variation.
BPPs have been described as snake venom inhibitors of the
angiotensin-converting enzyme, a dipeptidylcarboxypepti-
dase expressed in endothelial, epithelial and neuroepithelial
cells, which converts inactive angiotensin I into the potent
vasoconstrictor angiotensin II, and degrades bradykinin into
bradykinin (1–7) or bradykinin (1–5) [36]. BPPs prevent the
hypertensive effect of the angiotensin II and potentiate the
hypotensive effect of the circulating bradykinin. C-natriuretic
peptides elicit natriuretic, diuretic, and vasorelaxant activ-
ities. Lachesis protein Q27J49 encodes BPPs and a C-natriuretic
peptide, combining in one precursor molecule two kinds of
vasoactive molecules. Vasodilatation and hypotension con-
tribute synergistically to overall venom toxicity evoking the
rapid diffusion of toxic substances in the circulatory system
and a hypotensive shock, which is a major cause of death of
the prey or victim induced by viper snake bites. The BPPs were
also essential for the development of the first commercial ACE
inhibitor, captopril, for the treatment of human hypertension
[37]. Although L. muta and L. stenophrys express similar relative
amounts of BPPs into their venoms, each snake showed
Table 2 (continued)
HPLC fraction N-terminal sequencing Isotope-
averaged
molecular
mass
Peptide
ion
MS/MS-derived
sequence
Protein family
Ls- m/z z
23 N.D. 32 kDa▼ 488.2 2 ETYPNVPR Serine proteinase
618.8 2 XNXXDYAVCR
744.6 2 (261.2)PSSPPSVGSVCR
791.8 2 CANXNXXDYXVCR
824.8 2 NDTEWDKDXMXXR
24,25 ADDRNPLGECFRETDYEEFL 58 kDa ■▼ 583.1 2 KFWEDDGIR L-amino acid oxidase
641.0 2 SAGQLYEESLGK
744.0 2 ETDYEEFLEIAK
26 N.D. 110+52 kDa ■▼ 532.3 2 YNGNXNTXR PIII-metalloproteinase
27 N.D. 52 kDa ■▼ 532.3 2 YNGNXNTXR PIII-metalloproteinase
27 kDa▼ 605.3 2 DYYEMFXTK PI-metalloproteinase
640.8 2 NSVGXVQDHSPK
15 kDa▼ 621.3 2 DFSWEWTDR Gal-lectin [Q9PSM4]
786.9 2 EFCVELVSTGYR
28–31 Blocked 27 kDa▼ 802.6 2 VHEXVNTXNVFYR PI-metalloproteinase
605.3 2 DYYEMFXTK ∼ (P22796)
540.3 2 TFGEWRER
640.8 2 NSVGXVQDHSPK
861.5 2 YXEXVVVADHGMFTK
532.8 2 YNGNXNTXR
683.4 3 TLLIAVTMAHELGHNLGMK
29 Blocked 52 kDa ■▼ 526.7 2 GNYYGYCR PIII-metalloproteinase
801.3 2 MYEXANTVNDXYR
684.8 3 XTVKPEAGYTXNAFGEWR
30 Blocked 110 kDa ■ 635.8 2 XYCFPSSPATK (PIII-Metalloproteinase)2
48 kDa▼
31 Blocked 52 kDa ■▼ 506.2 2 FTSAGNVCR PIII-metalloproteinase
650.2 2 YVEXVVVADHR
752.4 2 XFEFNNFPCR
32 N.D. 110 kDa ■ 752.4 2 XFEFNNFPCR (PIII-Metalloproteinase)2
52 kDa▼ 506.2 2 FTSAGNVCR
X, Ile or Leu; Z, pyrrolidone carboxylic acid; Ac-, N-acetyl;m, methionine sulphoxide. Unless other stated, for MS/MS analyses, cysteine residues
were carbamidomethylated. Molecular masses of native proteins were determined by electrospray-ionization (±0.02%) or MALDI-TOF (⁎) (±0.2%)
mass spectrometry. Apparent molecular mass determined by SDS-PAGE of non-reduced (■) and reduced (▼) samples; n.p., non-peptidic
material found. M andm, denote mayor andminor products co-eluting in the same HPLC fraction. Previously reported proteins are identified by
their databank accession codes.
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distinct complements of Q27J49-derived peptides, suggesting
that the processing steps required to form themature BPPs are
overlapping though not identical in the two Lachesis species.
However, whether the occurrence of distinct sets of BPPs re-
flects an evolutionary adaptation or merely a neutral conse-
quence of speciation deserves further detailed investigations.
Through the pathophysiological consequences of the
presence of large amounts of BPPs in Lachesis venoms
deserve further and detailed consideration, the large content
of BPPs in the two Lachesis venoms investigated may be
associated with the conspicuous hypotension of very rapid
onset which characterizes bushmaster envenomation
cases, an effect that is likely to contribute to hemody-
namic complications leading to cardiovascular shock [27].
Fig. 2 –SDS-PAGE of reverse-phase separated fractions from the venom of Lachesis muta (Santa Cruz de la Sierra, Bolivia).
SDS-PAGE showing the protein composition of the reverse-phase HPLC separated venom protein fractions run under
non-reduced (panel A) and reduced (panel B) conditions. Molecular mass markers (in kDa) are indicated at the left of each gel.
Protein bands were excised and characterized by mass fingerprinting and CID-MS/MS. The results are shown in Table 1.
Fig. 3 –Reverse-phase HPLC separation of the venomproteins
of Lachesis stenophrys. Twomilligrams of L. stenophrys venom
(Costa Rica) were applied to a Lichrosphere RP100 C18
column, which was then developed as in Fig. 1. Fractions
were collected manually and characterized by N-terminal
sequencing, ESI mass spectrometry, tryptic peptide mass
fingerprinting, and CID-MS/MS of selected doubly- or
triply-charged peptide ions. The results are shown in Table 2.
Fig. 1 –Reverse-phase HPLC separation of the Lachesis muta
venom proteins. Two milligrams of Lachesis muta venom
(Santa Cruz de la Sierra, Bolivia) were applied to a
Lichrosphere RP100 C18 column, which was then developed
with the following chromatographic conditions: isocratically
(5% B) for 10min, followed by 5–15% B for 20min, 15–45% B for
120 min, and 45–70% B for 20 min. Fractions were collected
manually and characterized by N-terminal sequencing, ESI
mass spectrometry, tryptic peptide mass fingerprinting, and
CID-MS/MS of selected doubly- or triply-charged peptide
ions. The results are shown in Table 1.
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Furthermore, the low molecular mass of these peptides is
likely to confer them with very low antigenicity, with the
consequent implications for antivenom development. It
would be relevant to assess whether antivenoms are
effective at binding and neutralizing BPPs present in Lachesis
venoms.
Other very similar or identical proteins in L. muta and
L. stenophrys venoms, based on their similar chromatographic
Fig. 5 –Mass spectrometric characterization of isolated proteins and tryptic peptides. (A) Electrospray-ionizationmass spectrum
of the major protein isolated in HPLC fraction 18 (Figs. 1 and 2, Table 1). From the series of ions (M+16H)16+–(M+21H)21+ an
isotope-averaged molecular mass of 24683.9±2.1 Da, and was identified by N-terminal sequencing as a member of the CRISP
family. (B) MS/MS spectrum of the doubly-charged tryptic parent ion ofm/z 744.1 (encircled) from the 58 kDa protein isolated in
fraction Lm29 of Lachesis muta venom HPLC separation (Figs. 1 and 2, Table 1). Ions of the major sequence-specific y-ion series
and of a minor series of the complementing b-ions, from which the sequence (231.2) DYEEFXEXAK sequence tag was deduced,
are labelled. This sequence is present as ETDYEEFLEIAK in the N-terminal sequence of the 58 kDa parent protein identified as
an L-amino acid oxidase (Table 1).
Fig. 4 –SDS-PAGE of reverse-phase separated fractions from the venom of Lachesis stenophrys (Costa Rica). SDS-PAGE showing
the protein composition of the reverse-phase HPLC separated venom fractions (see Fig. 3) run under non-reduced (panel A) and
reduced (panel B) conditions. Molecular mass markers (in kDa) are indicated at the left of each gel. Protein bands were excised
and characterized by mass fingerprinting and CID-MS/MS. The results are shown in Table 2.
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retention time and molecular masses and by sharing tryptic
ions include the nerve growth factor isolated in fractions
Lm15 and Ls10; the serine proteinase Q27J47 isolated
in fractions Lm20 and Ls14/15, as well as the thrombin-like
enzime venombin A [S35689] and the plasminogen-activating
proteinase P84036, both eluted in HPLC fractions Lm26
and Ls21; the galactose-specific lectin Q9PSM4; the L-amino
acid oxidase characterized in fraction Lm29 and Ls24; and
the major PI-metalloproteinase, hemorrhagic factor LHFII
[P22796].
Based on phylogenetic hypothesis, published morphologi-
cal and behavioral differences, and the allopatric distribu-
tions of distinctive population groups, Zamudio and Green
elevated L. muta and L. stenophrys to species level in 1997 [38].
They estimated that the Central and South American forms
diverged 18–6 Mya, perhaps due to the uplifting of the Andes.
As judged from the protein chemical and mass spectrometric
data listed in Tables 1 and 2, each Lachesis venom may
contain 24–26 different gene products. Using a similarity
coefficient (PSC), we estimate that L. muta and L. stenophrys
share only 8 proteins. Such a low figure (PSC=30–32%) high-
light the rapid structural diversification of venom toxins of
closely related congeneric taxa.
3.2. Proteomic vs. transcriptomic of L. muta venom
Comparison of the protein composition of the venom of
L. muta determined using a proteomic (this work) and a trans-
criptomic approach [23] shows clear differences, both in the
relative occurrence of protein families (expressed as percen-
tages of the total HPLC-separated proteins) (Fig. 6) and in the
identity of the polypeptides of each protein family. It is worth
to notice that only 6 venom proteins matched any of the
previously reported 11 partial or full-length venom gland
transcripts [23]. On the other hand, 16 L. muta venom com-
ponents correspond to proteins not reported in the database-
deposited transcriptome of the same species [23]. This set of
novel proteins comprise both minor components, i.e. the
17 kDa nerve growth factor (Lm15) and the C-type lectin(s)
spread in fractions 28–38, and relatively abundant proteins,
such as all the venom-secreted PLA2 molecules (Lm16–18), the
CRISP molecule found in venom fractions Lm18–20, several
serine proteinases (Lm20, Lm24, and Lm28), the single 52 kDa
L-amino acid oxidase found in fraction Lm29, and a number of
snake venom metalloproteinases of the PI and PIII classes of
the reprolysin family (Lm32–38) (Table 1). The low degree of
venom composition accordance between the proteomic and
the transcriptomic approaches has been also reported for
B. gabonica [13], and clearly indicate that the cDNA library
lacked many transcripts encoding venom-expressed proteins.
On the other hand, in some cases the lack of correspondence
between the proteomic and the transcriptomic data may be
due to the unavailability of the cDNA-deduced protein se-
quences in the public-accessible databases. Hence, Junqueira-
de-Azevedo et al. [23] reported the cloning of two almost
identical cDNA clusters coding for an L-amino acid oxidases
(comprising 3.7% of the total toxin-coding ESTs), which
matched LAOs from other Viperidae species over the entire
2705 bp extension and which may correspond to the L. muta
LAO identified earlier by Sanchez and Magalhães [39]. Sim-
ilarly, Junqueira-de-Azevedo et al. [23] also found clusters
coding for single forms of nerve growth factor (NGF; 0.3%
of total toxin-coding ESTs) and snake venom vascular
Table 3 – Overview of the relative occurrence of proteins of
the different toxin families in the venoms of Lachesis muta
and Lachesis stenophrys
Protein family % of total venom proteins
Lachesis muta Lachesis stenophrys
BPP/C-NP 14.7 14.6
Nerve growth factor 0.6 0.4
PLA2 8.7 12.3
CRISP 1.8 –
Serine proteinase 31.2 25.6
Gal-lectin/C-type lectin-like 7.9 3.6
L-amino acid oxidase 2.7 5.3
Zn2+-metalloproteinase 31.9 38.2
Fig. 6 –Proteomics and transcriptomics of Lachesis venoms. Comparison of the protein composition of the venoms of Lachesis
muta (A) and Lachesis stenophrys (C) determined using a proteomic (this work) and a transcriptomic (panel B) approach [23]. BPP,
bradykinin-potentiating/C-natriuretic peptide; NGF, nerve growth factor; LAO, L-amino acid oxidase; PLA2, phospholipase A2;
SVMP, snake venom metalloproteinase; svVEGF, snake venom vascular endothelial growth factor; CRISP, cysteine-rich
secretory protein; Gal-lectin, galactose-specific lectin; Ser-Prot, serine proteinase; 3FTx, three-finger toxin; DPP,
dipeptidylpeptidase.
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endothelial growth factor (svVEGF) in their reported L. muta
transcriptome, although their sequences are not available
in the non-redundant SwissProt/TrEML and NCBI data-
banks. However, in those venoms in which LAO and growth
factors have been identified, these toxins appear to be ex-
pressed as single components, suggesting that they may rep-
resent products of single copy genes, and thus that the
proteomic and the transcriptomic approach may match the
same venom components.
Transcripts encoding putative secreted toxin classes [23],
which were not be found in our proteomic analysis include
three finger-like toxins [Q27J50], ohanin-like protein [Q27J48],
svVEGF, and dipeptidyl peptidase. In addition, neither L. muta
nor L. stenophrys venom contained detectable amounts of
lachesin, a medium-size disintegrin [P31990] isolated from
lyophilized venom of L. muta of non-reported origin purchased
from Miami Serpentarium Laboratories (Salt Lake City, UT)
[40]. The occurrence of non-venom-secreted toxins suggests
that these messengers could exhibit an individual or a
temporal expression pattern over the life time of the snake.
Sex-based individual variation of snake venom proteome
among B. jararaca siblings have been reported [41]. In addition,
ontogenetic and geographical variations have been noticed in
the venomproteomes of other snakes, i.e. Crotalus viridis viridis
[42], C.v. oreganus [43,44], Bothrox atrox [45,46], and Bothrops
asper [46], and might represent a common phenomenon in
many other species (see below). Alternatively, the non-
venom-secreted or very low abundance (b0.05% of the total
venom proteins), toxins may play a hitherto unrecognized
physiological function in the venom gland, or may simply
represent a hidden repertoire of orphanmolecules which may
eventually become functional for the adaptation of snakes to
changing ecological niches and prey habits. Clearly, although
further work is needed to clarify this point, overall, our results
emphasize the relevance of detailed proteomic studies for a
thorough characterization of the venom composition.
3.3. Intraspecific variation in venom-secreted
PLA2 molecules
The PLA2 molecules isolated in fractions 16–18 (Fig. 1, Table 1)
display high N-terminal sequence similarity to the hemolytic
and platelet aggregation inhibitory Lm-PLA2-I and Lm-PLA2-II
characterized by Fully et al. [47] from L. muta venom provided
by Sigma or Fundação Ezequiel Dias (FUNED, Brazil), to LMPA1
(P84651) from the same source, and to two basic neurotoxic
Asp49 PLA2 molecules (LmTX-I and LmTX-II) isolated from
L. muta venom purchased from Sigma [48,49] (Table 4). In
particular, the N-terminal sequence of the PLA2 isolated in
fraction Lm18 (Table 1) seems to be identical to that of LM-
PLA2-II, except for the striking lack of the serine residue
at position 16. This residue is absolutely conserved in the
structures of all known myotoxic PLA2 molecules [50], strong-
ly suggested that a gap at position 16 may represent a
sequencing or a typographical error. Regardless of that, none
of the other PLA2 isoenzymes reported in the literature could
be matched to any of the PLA2 molecules found in the venom
of L. muta from the nature reserve Potrerillos del Guendá
(Santa Cruz de la Sierra, Bolivia) sampled here (Table 4). This
prompted us to investigate if the lack of identity could be due
to geographic variations of L. muta venoms. To this end, the
venoms of 5 specimens (3 from Bolivia and 2 from Peru) and
the venom purchased from Sigma (unknown origin) were
compared. Noteworthy, the 6 reverse-phase HPLC separations
were essentially superimposable, except for quantitative and
qualitative differences in PLA2 expression (Fig. 8, Table 4).
Thus, each venom exhibited a distinct combination, and/or
concentration, of the same three PLA2molecules (Lm16, Lm17,
and Lm18) listed in Table 1. The most abundant PLA2
molecules in Bolivian specimens were Lm16 and Lm18,
whereas Lm17 was the predominant PLA2 in Peruvian
L. muta venoms (Fig. 8). The venom purchased from Sigma
(of non-declared origin) displayed the “Bolivian PLA2
Fig. 7 –Bradykinin-potentiating peptides and C-natriuretic peptides. Mapping of the bradykinin-potentiating peptides and the
C-natriuretic peptide found in the venoms of L. muta (Lm-) and L. stenophrys (Ls-) onto the cDNA-deduced amino acid sequence
of the precursor protein Q27 J49. Peptides shared by both venoms are in boldface and underlined, while those distinctly
expressed in L. muta and L. stenophrys are displayed in italics and in boldface on a gray background, respectively. Venom
fractions in which the peptides were recovered are identified by their numbering in Tables 1 and 2. Asterisks indicated that the
peptide isolated from the venom contained N-terminal pyroglutamic acid.
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signature” (Lm16+Lm18) but departed from the Bolivian and
Peruvian venom-secreted PLA2 profiles in expressing a unique
PLA2 molecule (labelled 16a in Fig. 8) with identical N-terminal
sequence and very similar isotope-averaged molecular mass
(13723 Da) as myotoxic PLA2 molecules from B. jararacussu
(AAO27453; 13714 Da) [18], B. pirajai (1QLL_A; 13744 Da), and
B. asper (P24605; 13,725 Da). As a whole, these results point out
to a high degree of intraspecific variability in the expression of
phospholipases A2 in L. muta venoms. Intraspecific variability
of PLA2 loci has been reported in other viperid (Vipera palestinae
[51]) and crotalid (B. asper [52]; Trimeresurus flavoviridis [53])
species, and this phenomenon is often linked to differences in
diet among populations [53,54].
Snake venom phospholipase A2 genes are members of a
large, rapidly-evolving multigene family with many diverse
functions. Positive Darwinian selection is common in group-II
viperid snake venom PLA2 genes and is associated with the
evolution of new toxin functions and speciation events [55].
Adaptive evolution of group-I phospholipases in elapids is also
associated with speciation events [56], suggesting adaptation
of the phospholipase arsenal to novel prey species after niche
shifts.
3.4. Concluding remarks
Reports of envenomations by species of Lachesis are scarce,
probably due to the fact that these species are distributed in
remote tropical rainforests. The recorded cases [27,57–60] are
characterized by local and systemic manifestations typical of
viperid venoms. Local effects include pain, edema, and
hemorrhage, with the development of blisters. Systemic
manifestations include coagulopathy and widespread bleed-
ing. In some cases, a ‘parasympathomimetic-like’ effect has
been described, with bradicardia, abdominal colics, nausea
and vomiting [60]. These latter effects have been described in
South American cases, but not in those occurred in Central
America. Our detailed proteomic analysis of the venoms of
Lachesis species supports the hypothesis that snake venom
proteomes are composed of proteins belonging to only a few
toxin families exhibiting structural divergence and distinct
relative abundances in even closely related species. However,
there does not appear to be a simple relationship between
venom composition and the presence or absence of the
described ‘parasympathomimetic-like’ effect. Because spe-
cies-specific effects of venom components are largely
unknown, it is difficult to assign a functional role unequi-
vocally to the variation we observed in Lachesis venoms.
Subtle functional differences in some venoms components,
which may confer distinct pharmacological activities to
proteins from the same family but distinctly expressed in
L. muta and L. stenophrys, may be responsible for species-
specific effects. Our proteomic analysismay serve as a starting
point for studying structure–function correlations of indivi-
dual toxins aiming at the development of new research tools
and drugs of potential clinical use [61–63]. It is also worth to
notice that though intraspecific variation in venom toxins
may inform us about evolutionary processes acting at the
species or population level, it represents also a source of
Fig. 8 – Intraspecific variation in PLA2 isoenzyme venom
composition. Panels A–F, details of the reverse-phase HPLC
chromatograms of the venoms from Lachesis muta from
Bolivia (A–C), Peru (D and E), and purchased from Sigma (F).
Numbers refer to the reverse-phase HPLC separation shown
in Fig. 1. Table 3 displays the N-terminal sequences and
molecular masses of the PLA2 molecules from the different
sources. The peak 16a marked with asterisk in F
corresponds to a PLA2 molecule specifically found in the
venom sold by Sigma.
Table 4 – Comparison and occurrence of PLA2 molecules
characterized in the venom proteomes of Lachesis muta
from different sources
N-terminal sequence (Mass) Bolivia Peru Sigma
1 2 3 1 2
HLLQFGDLINKIARRNGISYYG
(13,932 Da)
☻(16) ☻ ☻ ☻ ☻
HLLQFGDLINKIARRNGISYYG
(13,916 Da)
☻(17) ☻ ☻
HLLQFEQLIRKIAGRSGFRYYG
(14,008 Da)
☻(18) ☻ ☻ ☻
SLFELGKMILQETGKNPAKSY
(13,723 Da)
☻(16a)
HLLQFGDLIDKIAGRSGFWYYG
PA2_LACMU (13,889 Da)
HLLQFGDLIDKIAGRSGFWYYG
LM-PLA2-I
HLLQFEQLIRKIAGRGFRYYGF
LM-PLA2-II
HLLQFNKMIKFETRKNAIPFYAF
LM-TX-I (14245 Da)
HLLQFNKMIKFETRKNAIPFYAF
LM-TX-II (14186 Da)
Bolivia-1 corresponds to the venom analyzed in detailed in Table 1.
Numbers in parentheses indicate the reverse-phase HPLC fraction
of Figs. 1 and 7 containing the corresponding PLA2 molecules.
N-terminal sequences and, when available, the molecular mass of
PLA2 proteins characterized previously from venoms of L. muta
specimens kept in captivity at the Fundação Ezequiel Dias (Belo
Horizonte, Brazil) (PA2_LACMU, LM-PLA2-I, and LM-PLA2-II) or
isolated from L. muta venom purchased from Sigma (LmTX-I and
LmTX-II), are displayed.
58 J O U R N A L O F P R O T E O M I C S 7 1 ( 2 0 0 8 ) 4 6 – 6 0
Author's personal copy
concern in antivenom production strategies. Broad spectrum
antivenoms may thus be prepared using pooled venoms. On
the other hand, as knowledge of a particular group increases,
its categorisation may need to be re-assessed. At this res-
pect, the occurrence of intraspecific toxin composition
variation in separated snake populations, as revealed in
this and other works, suggests that polytypic species with a
number of subspecies or races are more widespread than
previously thought. Along with analysis of morphological
traits, a detailed proteomic characterization of snake
venoms may aid in establishing a taxonomic subdivision of
snake species.
Acknowledgement
This study has been financed by grant BFU2004-01432/BMC
from theMinisterio de Educación y Ciencia, Madrid, Spain, and
Acciones Integradas CSIC-UCR 2006CR0010.
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