aspis zinnikeri and Vipera berus berus venom Identification of the origin of a new viper population based on ammodytin I1 heterogeneity Isabelle Guillemin*, Christiane Bouchier†, Thomas
Trang 1Sequences and structural organization of phospholipase A2 genes from
Vipera aspis aspis , V aspis zinnikeri and Vipera berus berus venom Identification of the origin of a new viper population based on ammodytin I1
heterogeneity
Isabelle Guillemin*, Christiane Bouchier†, Thomas Garrigues‡, Anne Wisner§ and Vale´rie Choumet*
Unite´ des Venins, Institut Pasteur, Paris, France
We used a PCR-based method to determine the genomic
DNA sequences encoding phospholipases A2(PLA2s) from
the venoms of Vipera aspis aspis (V a aspis), Vipera aspis
zinnikeri (V a zinnikeri), Vipera berus berus (V b berus)
and a neurotoxic V a aspis snake (neurotoxic V a aspis)
from a population responsible for unusual neurotoxic
envenomations in south-east France We sequenced five
groups of genes, each corresponding to a different PLA2
The genes encoding the A and B chains of vaspin from the
neurotoxic V a aspis, PLA2-I from V a zinnikeri, and the
anticoagulant PLA2 from V b berus are described here
Single nucleotide differences leading to amino-acid
substi-tutions were observed both between genes encoding the same
PLA2 and between genes encoding different PLA2s These
differences were clustered in exons 3 and 5, potentially
altering the biological activities of PLA2 The distribution
and characteristics of the PLA2 genes differed according
to the species or subspecies We characterized for the first
time genes encoding neurotoxins from the V a aspis and
V b berus snakes of central France Genes encoding
ammodytins I1 and I2, described previously in Vipera ammodytes ammodytes(V am ammodytes), were also pre-sent in V a aspis and V b berus Three different ammo-dytin I1 gene sequences were characterized: one from
V b berus, the second from V a aspis, V a zinnikeri and the neurotoxic V a aspis, and the third from the neurotoxic
V a aspis This third sequence was identical with the reported sequence of the V am ammodytes ammodytin I1 gene Genes encoding monomeric neurotoxins of
V am ammodytesvenom, ammodytoxins A, B and C, and the Bov-B LINE retroposon, a phylogenetic marker found
in V am ammodytes genome, were identified in the genome
of the neurotoxic V a aspis These results suggest that the population of neurotoxic V a aspis snakes from south-east France may have resulted from interbreeding between
V a aspisand V am ammodytes
Keywords: ammodytin; neurotoxic; phospholipase A2; vaspin; viper
Phospholipases A2(PLA2s) are major components of snake venoms They catalyze the Ca2+-dependent hydrolysis of the 2-acyl ester bond of 1,2-diacyl-3-sn-phosphoglycerides releasing fatty acids and lysophospholipids These enzymes can be separated into 11 groups Those belonging to group
II have six to eight disulfide bonds and a C-terminal extension not present in group I venom PLA2s [1] They are found in the venoms of Crotalinae and Viperinae snakes and in human platelets, liver and spleen [2,3] Snake venoms contain a large number of PLA2 isoenzymes which differ in neurotoxicity, myotoxicity, cardiotoxicity, anticoagulation and edema-inducing properties [2]
To date, the structures of only six Viperinae PLA2 genes have been studied: ammodytin I1 (DDBJ/EMBL/GenBank accession no AF253048), ammodytin I2 (DDBJ/EMBL/ GenBank accession no X84018), ammodytoxin C (DDBJ/ EMBL/GenBank accession no X76731) and ammodytin L (DDBJ/EMBL/GenBank accession no X84017) from Vipera ammodytes ammodytes(V am ammodytes) and two genes encoding an acidic inhibitor (VP7) (DDBJ/EMBL/ GenBank AC AF373342) and a basic PLA2 protein (VP8) (DDBJ/EMBL/GenBank AC AF373342) from Vipera palaestinae venom [4–6] All these genes are composed of five exons and four introns, like genes encoding human
Correspondence to V Choumet, Unite´ de Biochimie et de Biologie
Mole´culaire des Insectes, 25, Rue du Docteur Roux,
75724 Paris Cedex 15, France.
Fax: + 33 1 40 61 34 71, Tel.: + 33 1 45 68 86 30,
E-mail: vchoumet@pasteur.fr
Abbreviation: PLA2, phospholipase A 2
*Present address: Unite´ de Biochimie et Biologie Mole´culaire des
Insectes, Institut Pasteur, 25, Rue du Dr Roux,
75724 Paris Cedex 15, France.
Present address: Genopole, Institut Pasteur, 28, Rue du Docteur
Roux, 75724 Paris Cedex 15, France.
àPresent address: Unite´ d’Ecologie des Syste`mes Vectoriels,
Institut Pasteur, 25, Rue du Docteur Roux,
75724 Paris Cedex 15, France.
§Present address: Laboratoire de Recherche et de De´veloppement:
Pharmacologie des Re´gulations Neuroendocriniennes, Paris, France.
Note: The nucleotide sequences reported in this paper have been
deposited in the DDBJ/EMBL/GenBank nucleotide sequence
data-bases under accession numbers AY158634, AY158635, AY158636,
AY158637, AY158638, AY158639, AF548351, AY152843,
AY159807, AY159808, AY159809, AY159810, AY159811,
AY243574, AY243575, AY243576, AY243577.
(Received 2 December 2002, revised 4 April 2003,
accepted 22 April 2003)
Trang 2group II PLA2s In contrast, PLA2 genes from Crotalinae
snakes such as Trimeresurus flavoviridis, Trimeresurus
gramineus, Trimeresurus (Ovophis) okinavensis and Crotalus
scutulatus scutulatusare organized into four exons and three
introns, like group I PLA2s [7–10] Despite this difference,
nucleotide sequence analyses have shown that, unusually,
introns are more conserved than exons in both Viperinae
and Crotalinae Moreover, mutations leading to amino-acid
changes are common in the protein-coding regions (but not
in the signal peptide exon), but are limited to the third exon
in V palaestinae [6,11] Thus, the genes encoding the group
II PLA2 of snake venoms evolved by gene duplication,
followed by divergence from a common ancestral gene by
accelerated Darwinian selection, probably as a means of
acquiring new functions [9,11]
In this paper, we extend the study of PLA2 genes to the
French vipers Vipera aspis aspis (V a aspis), Vipera aspis
zinnikeri (V a zinnikeri), and Vipera berus berus (V b
berus) We were prompted to carry out this study by the
recent identification of a distinct, unusually neurotoxic
population of V a aspis in the south-east of France [12]
Several cases of envenomation resulting in symptoms of
neurotoxicity have been reported in recent years in two
French de´partements (Alpes-Maritimes and
Alpes-de-Haute-Provence) Such symptoms have never been observed
after envenomation by V a aspis snakes in other regions of
France The venom of these snakes was detected by ELISA
in the plasma of the patients; it contained PLA2s that
cross-reacted with antibodies to ammodytoxin [12] We
investi-gated gene expression in the venom gland of one of the
snakes captured after one case of human envenomation We
showed, by RT-PCR, that the venom of this snake
contained two neurotoxins One was monomeric and
identical with ammodytoxin B, and the other (vaspin) was
heterodimeric and similar to vipoxin, the toxic complex of
Vipera ammodytes meridionalissnake venom [13] These two
toxins were responsible for the symptoms of neurotoxicity
observed after envenomation [13] We then investigated the
genes encoding the PLA2s present in the venoms of all
the venomous snake species of France On the basis of its
venom PLA2 characteristics and, particularly, sequence
analysis of the ammodytin I1 gene, we suggest a possible
origin for the neurotoxic snake population We also report
high levels of polymorphism, both for individual PLA2
genes and between genes encoding different PLA2s These
polymorphisms may have implications for the structure
and/or function of the enzyme
Experimental Procedures
Snakes were captured in various regions of France:
V a aspis and V b berus in the Puy-de-Doˆme, V a
zinnikeriin the Gironde, and neurotoxic V a aspis in the
Alpes-Maritimes The V a aspis snake captured in the
Alpes-Maritimes was responsible for one case of neurotoxic
envenomation [12] We studied one individual per snake
species Genomic DNA was extracted from snake livers as
previously described [14]
DNA was amplified with a set of primers (Genset Oligos,
Paris, France) targeting conserved regions of the PLA2
genes for which sequences were available in databases The
primer-binding sites were located upstream from the
5¢-UTR (PLA5G) and downstream from the 3¢-UTR (PLA3G) Amplification reactions were carried out in a final volume of 50 lL containing 2.5 lL PLA5G and 2.5 lL PLA3G (10 lMeach), 1 lL dNTPs (dATP, dCTP, dTTP and dGTP, 10 mMeach), 5 lL Taq buffer supplied with the enzyme, 0.25–0.5 lg genomic DNA and 1.5 U rTaqpolymerase (Amersham Biosciences, Orsay, France) The DNA was denatured by heating at 95C for 7 min It was then subjected to 30 amplification cycles as follows: denaturation at 95C for 1 min and annealing coupled with extension at 69C for 6 min A final extension step was carried out, at 72C for 10 min The reaction product was analyzed by agarose gel electrophoresis in 1· Tris/borate buffer
DNA fragments of the expected size (2.1 kb) were purified from the gel with the QIAquick Gel Extraction Kit (Qiagen S.A., Courtabœuf, France), and inserted into the pCR2.1-TOPO vector of the TOPO TA Cloning Kit (Invitrogen SARL, Cergy Pontoise, France) Plasmid DNA was purified with the Montage Plasmid Miniprep96 Kit (Millipore, Saint-Quentin-en-Yvelines, France) Sequen-cing reactions were performed from both ends of the DNA plasmid, using the ABI PRISM BigDye Terminator Cycle Sequencing Ready-Reaction Kit, and a 3700 Genetic Analyzer (Applied Biosystems) The trace files were base-called with Phred [15] Sequences not meeting our production quality criteria (at least 100 bases with a quality over 20) and insert-less vector sequences (detected
by cross-matching; [15]) were discarded Complete nuc-leotide sequences were determined on both strands, with a set of nine primers, designed from database sequences (Table 1)
Results and Discussion
Identification of PLA2 genes
We obtained 96 clones of PLA2 genes per snake species Complete nucleotide sequences were further analyzed for
81 clones of V a aspis, 80 clones of V b berus, 65 of
V a zinnikeri, and 59 of the neurotoxic V a aspis Nuc-leotide and amino-acid sequences were compared with sequences in gene and protein databases, usingBLASTNand
BLASTP, respectively We subsequently amplified the corres-ponding genomic DNA fragments from each snake with primers (Table 1) specific for the PLA2s previously charac-terized in Vipera venoms We also designed primers specific for the Bov-B LINE retroposon, a phylogenetic marker previously identified in some PLA2 genes of Viperidae snakes including V am ammodytes [4,5]
Five groups of snake venom PLA2 genes were sequenced Nucleotide polymorphism was identified in each group Two groups of genes were most similar, in terms of nucleotide sequences, to cDNAs encoding chains A and B
of vaspin (DDBJ/EMBL/GenBank accession no.s AJ459806 and AJ459807, respectively) [13], chains A and
B of vipoxin (Gi numbers: 16974941 and 16974940) from
V am meridionalis[16] and the two subunits of PLA2-I (Gi numbers: 1709547 and 1709548) from V a zinnikeri [17] They also showed a high level of nucleotide identity with cDNAs encoding the presynaptic neurotoxic complex RV4/ RV7 from Daboia russelli formosensis (DDBJ/EMBL/
Trang 3GenBank accession no.s X68385 and X68386, respectively [18]) Two other groups were most similar, in terms of nucleotide sequence, to the ammodytin I1 (DDBJ/EMBL/ GenBank accession no AF253048) and ammodytin I2 (DDBJ/EMBL/GenBank accession no X84017) genes from V am ammodytes, respectively [19] The last group
of genes was most similar to the V b berus PLA2 protein (Gi number: 423975) [20] A list of all the venom PLA2 genes identified in each captured snake is presented in Table 2 Unexpectedly, genes encoding the A and B chains
of vaspin, a heterodimeric neurotoxin, were identified in
V a aspis and V b berus snakes collected in central France (Table 2) These genes are probably either expressed
at a very low level or not expressed at all in the venom of these snakes because no neurotoxic envenomation has ever been reported in the Clermont-Ferrand region (Gabriel Montpied Hospital, personal communication) The expres-sion of these neurotoxin subunits in the venom of the neurotoxic V a aspis may be related to the diet of the snake, but this hypothesis remains to be proven [21] More surprisingly, genes encoding ammodytoxins A, B and C were identified only in the neurotoxic V a aspis snake (Table 2) However, only the ammodytoxin B mRNA was detected in the venom gland of this snake [13] Thus, not all the ammodytoxin genes were expressed Finally, partial sequencing of the PCR product revealed the presence of the Bov-B LINE retrotransposon in intron
D of the ammodytoxin C gene of the neurotoxic V a aspis (Table 2) This feature was also specific to this population of neurotoxic snakes
Table 1 Sequences of primers used for PCR amplification and
sequencing of PLA2 genes F, Forward; R, reverse Y ¼ C or T;
W ¼ A or T.
Primer name Sequences 5¢ )3¢
M13Reverse (F) CCCTATAGTGAGTCGTATTA
T7 Promoter (R) CAGGAAACAGCTATGAC
PLA5G (F) CGGAATTCTGAAGGTGGCCCGCC
AGGTGACAG
PLA3G (R) CGCGGATCCAATCTTGATGGGGC
AGCCGGAGAGG
PLA5G1 (F) AGGAYTCTCTGGATAGTGG
PLA3G1 (R) CTCACCACAGACGATWTCC
PLA5G2 (F) CGGTAAGCCCATAACGCCCA
PLA3G2 (R) CAGGCCAGGATTTGCAGCC
PLA3G4 (R) CATAAACAYGAGCCAGTTGCC
AtxBF b (F) GCCTGCTCGAATTCGGGATG
AtxBrcb(R) CTCCTTCTTGCACAAAAAGTG
AtxACFc(F) CTGCTCGAATTCGGGATG
AtxACrc c (R) GTCYGGGTAATTCCTATATA
Amlrcd(R) CCCTTGCATTTAAACCTCAGGTACAC
a Specific primers used for amplification of the Bov-B LINE
retroposon;bspecific primers used for amplification of the
ammo-dytoxin B gene;cspecific primers used for amplification of the
ammodytoxin A and C genes; d specific primers used for
amplifica-tion of the ammodytin L gene.
Table 2 Characteristics of the venom PLA2 genes of V a aspis, V a zinnikeri, neurotoxic V a aspis and V b berus The PLA2 content of
V am ammodytes venom is as previously reported [2,4].
Snake species
PLA2 genes a
Length of intron D
in ammodytin I1 (bp) AmI1(form) AmI2 Vb VaspA VaspB
Snake species Ammodytin I1 protein sequence b AtxA AtxB AtxC AmL Retroposon
V a zinnikeri L70, S71, E78, L12 – – – – –
Neurotoxic V a aspis + c + c + c – + c (AtxC) 1st group of clones L70, S71, E78, L123
2nd group of clones M70, G71, Q78, F123
V b berus T3 (peptide signal) – – – + c + c (AmL)
N1 K56
V am ammodytes M70, G71, Q78, F123 + + + + + (AtxC, AmL)
a AmI, ammodytin I1; AmI2, ammodytin I2; VaspA, vaspin chain A; VaspB, vaspin chain B; AtxA, ammodytoxin A; AtxB, ammodytoxin B; AtxC, ammodytoxin C; AmL: ammodytin L; b Only amino acids differing between ammodytin I1 molecules are represented The isoform
of ammodytoxin I is indicated in parentheses, as shown in Fig 3.cThe genes were identified by PCR and partially sequenced.
Trang 4Structural organization of PLA2 genes
We report here the first genomic sequences for the genes
encoding vaspin, a heterodimeric neurotoxin from Vipera
snakes, and PLA2 from V b berus The nucleotide
sequences of the genes encoding chains A and B of vaspin
and V b berus PLA2 span about 1.9 kb, as do the
ammodytin I1 and I2 genes They show a similar
organiza-tion to Viperinae PLA2s, with five exons separated by four
introns (Table 3) Exons 3, 4 and the 5¢ part of exon 5
encode the mature protein; exon 1 encodes the 5¢-UTR, and
exon 2 and part of exon 3 encode the signal peptide The 110
nucleotides at the 3¢ end of exon 5 encode the 3¢-UTR The
5¢ donor and 3¢ acceptor splice sites conformed with the
GT/AG rule (Table 3) These features are common to
ViperinaePLA2 genes [1,4]
Regardless of the snake from which the PLA2 gene was
obtained, the lengths of the exons and the 5¢-UTR were
identical, except for exon 5 in the ammodytin I2 gene, which
was three nucleotides shorter than the equivalent exon in the
other PLA2 genes (Table 3) The ammodytoxin C,
dytins I1 and I2 and ammodytin L genes of V am
ammo-dytes, the VP7 and VP8 genes of V palaestinae, and the
PLA2 genes of Crotalinae snakes (Trimeresurus flavoviridis)
were also of similar length [4–6,8] Interestingly, a 476 bp
insertion was observed in the 3¢-UTR of the ammodytin I2
gene from V b berus This fragment was similar to a region
located upstream from the TATA-box-binding protein gene
of T gramineus and T flavoviridis [22], suggesting a
probable common ancestry of V b berus and Trimeresurus species
The length of intron D in the ammodytin I1 gene depended on the species It was 133 bp long in V a zinnikeri and V a aspis whereas it was 259 bp long
in V b berus and V am ammodytes (DDBJ/EMBL/ GenBank accession no AF253048) Interestingly, introns
of both lengths were found in the genome of the neurotoxic
V a aspis, with six of the 10 sequenced clones having the
126 bp deletion as in V a aspis, V a zinnikeri, and the remaining four clones having an intron D similar to that of the V am ammodytes ammodytin I1 gene
All PLA2 genes contained a TAA stop codon, an AATAAA polyadenylation site 80 bp downstream from the stop codon, and a TATA-like box (CATAAAA) 270 bp upstream from the ATG translation initiation codon, as found in other Viperinae and Crotalinae genes [2,22]
Table 3 Structural organization of V a aspis, V a zinnikeri, V b berus and neurotoxic V a aspis PLA2 genes.
PLA2 gene
(no of clones) a Exon
Exon length (bp) Intron
Intron length (bp)
Splice sites 5¢donor/3¢acceptor Ammodytin I1 1 66 A 163 CAGCTgtaag / tccagGTCTG
3 133 C 671–680 GACCGgtaag / tccagCTGCT
4 101 D 133–259 CTGTGgtgag / tgcagGAGGC
5 (3¢-UTR) 140 (110) Ammodytin I2 1 66 A 163 CAGCTgtaag / tccagGTCTG
3 133 C 662–670 GACCGgtaag / tccagCTGCT
5 (3¢-UTR) 137 (110)
5 (3¢-UTR) 140 (110)
3 133 C 667–674 GACCGgtaag / tccagCTGCT
5 (3¢-UTR) 140 (110)
V berus PLA2 1 66 A 163 CAGCTgtaag / tccagGTCTG
5 (3¢-UTR) 140 (110)
a Clones harboring complete sequences of PLA2s are presented.
Fig 1 Alignment of some of the variant genes encoding chain B of vaspin from V a aspis (neurotoxic) and V a zinnikeri Two vaspin chain B gene variants are shown for V a zinnikeri (vp0016B10VAZ and vp0016C06VAZ, DDBJ/EMBL/GenBank accession no.s AY243574 and AY243577, respectively) and V a aspis (vp0015F11VAN and vp0015C10VAN, DDBJ/EMBL/GenBank accesion no.s AY243575 and AY243576, respectively) The nucleotides forming the introns are shown in italics, and those constituting the exons are underlined Stars below the sequence indicate nucleotides conserved in all sequences Dashes correspond to deleted nucleotides Putative transcription factors are boxed.
Trang 5Several putative regulatory sequences were identified
with the TRANSFAC 4.0 databases: binding sites for the
transcription factors Sp1 (CCCGCCA), NF-IL6 (TGGG
GAA), NF-jB (GGGGAAGTCCC) and AP-2 (CCCTG CC) were identified in PLA2 genes (Figs 1 and 2) [22] These trans-acting factors may act as stress-response
Trang 6elements or may be responsible for tissue-specific
regula-tion [23]
Conservation of the nucleotide sequence
We identified variant genes for the same PLA2 in the
genomes of all of the snakes Such variants were identified
for all the sequenced PLA2s Thorough analysis of the
nucleotide sequences showed that, although the
organiza-tion of introns and exons and the sequences of these PLA2
variants were well conserved (with ‡ 84% identity over
1900 bp), single-nucleotide polymorphisms were present
throughout the gene sequence An example is given for
some of the vaspin chain B gene variants identified in
V a zinnikeri and the neurotoxic V a aspis (Fig 1)
Some of the nucleotide mutations were found in both
subspecies whereas others were subspecies-specific These
genes probably resulted from complete duplication events
However, some of the nucleotide polymorphisms observed
may be also accounted for by the error rate of the Taq
polymerase used for DNA amplification, which is
estima-ted by the manufacturers to be 10)4 There were slightly
more nucleotide polymorphisms in introns than in exons
This was particularly true for intron C, which contained
several short deletions and insertions (Fig 1) Two
micro-satellite regions of tandem CA and TCCC repeats were
particularly prone to insertion and deletion (Fig 1), as
reported in previous studies on T flavoviridis and
O okinavensisPLA2 genes [8,10] These features revealed
intragenomic hypervariability within snake PLA2 genes
Exon nucleotide polymorphisms leading to amino-acid
substitutions might result in the creation of PLA2s with
different functions Indeed, PLA2 isoforms, differing by a
few amino acids and in lethal potency or enzymatic
activities, have been isolated from the venoms of individual
Crotalinaesnakes [24]
We then identified a consensus nucleotide sequence for
each of the five PLA2 genes (Fig 2) For the genes encoding
chains A and B of vaspin and PLA2 from V b berus, a
single consensus sequence was obtained, regardless of the
snake species For the ammodytin I1 and I2 genes, however,
two to three consensus sequences were obtained, according
to the snake species or subspecies Only one of the consensus
sequences for the ammodytin I1 and I2 genes is presented in
Fig 2 In contrast with that observed in comparisons of
gene variants encoding the same PLA2 (Fig 1), the
alignment of these consensus sequences showed that
nuc-leotide variations were more common in exons than in
introns (Fig 2) Exons 3 and 5 were the most divergent,
whereas the signal peptide, the 5¢-UTR and 3¢-UTR and the
promoter region were the most highly conserved (Fig 2)
The nucleotide substitutions mostly involved transitions
rather than transversions, in contrast with that observed in
conopeptide genes, thus excluding the involvement of DNA
polymerase V in genomic hypervariability [25] These
observations are not consistent with the neutral evolution
theory, which states that the strong conservation of exons
serves to maintain the function of the mature protein [26]
The protein-coding regions of the PLA2 genes of French
vipers most probably evolved in an accelerated Darwinian
manner, as reported for the PLA2 genes expressed in
Crotalinaeand V am ammodytes venom [2,3,5,7,8]
Deduced amino-acid sequence analysis
We aligned the amino-acid sequences deduced from the consensus nucleotide sequence of each PLA2 (Fig 3) The peptides encoded by exons 3 and 5 were the least well conserved for all the PLA2 genes sequenced, with only 45% and 49% identity, respectively In contrast, the peptide encoded by exon 2 was the most highly conserved, with 76% identity between all PLA2 sequences Similar obser-vations have been reported for the V palaestinae and Trimeresurus PLA2s [6,9] The mature PLA2 proteins displayed a mean of 51% identity in terms of their amino-acid sequences The ammodytin I1 and ammodytin I2 proteins were the most similar, displaying 78% amino-acid sequence identity The B chain of vaspin was the most divergent, displaying 67% identity with the vaspin A chain, and 70% identity with the V b berus PLA2
The signal peptides of all the proteins were 16 amino acids long The mature proteins consisted of 122 amino acids for ammodytin I1, the A and B chains of vaspin and V b berus PLA2, and 121 amino acids for ammo-dytin I2 The amino-acid sequence of the vaspin A chain was identical in all snake species and was 100% identical with that of the acidic subunit of PLA2-I from
V a zinnikeri [17] The deduced amino-acid sequence
of the V b berus anticoagulant PLA2 protein was identical with that of the PLA2 purified from V b berus venom [20] No difference was observed between the ammodytin I2 sequences from the neurotoxic V a aspis snake, V b berus, and V am ammodytes [19] However, for V a aspis, one group of genes (23 of 42) contained a sequence identical with that found in V am ammodytes, whereas another group (19 of 42) had one amino-acid difference (Asn111Ser), corresponding to a mutation in the fifth exon (Fig 3) The sequence of the vaspin B chain was identical in V a zinnikeri and the neurotoxic
V a aspis However, it differed by one residue from the sequence of the B chain of vipoxin from V am merid-ionalis [19], and by three residues from the published
Fig 2 Alignment of the consensus sequences of ammodytin I1 (AmtI1), ammodytin I2 (AmtI2), vaspin chains A and B and V berus PLA2 genes isolated from Frenchvipers The ammodytin I1 consensus sequence was defined from the sequences of V a aspis, V a zinnikeri, neurotoxic
V a aspis isoforms 1 and 2 and V b berus PLA2 (DDBJ/EMBL/ GenBank accession no.s AY159807, AY159810, AY159808, AY159809 and AY159811, respectively) The ammodytin I2 consensus sequence was defined from the sequences of V a aspis, neurotoxic
V a aspis and V b berus (DDBJ/EMBL/GenBank accession no.s AY158637, AY158638 and AY158639, respectively) The vaspin chain
A consensus sequence was defined from the sequences of V a zin-nikeri and neurotoxic V a aspis (DDBJ/EMBL/GenBank accession no.s AY152843 and AF548351) and that of vaspin chain B, from the sequences of V a zinnikeri and neurotoxic V a aspis (DDBJ/EMBL/ GenBank accession no.s AY158635 and AY158634) Dots indicate identity with the ammodytin I1 sequence Asterisks indicate tides conserved within PLA2s Dashes correspond to deleted nucleo-tides if the ammodytin I1 sequence is taken as the reference sequence Italics indicate DNA tandem repeats Putative transcription factor-binding sites are boxed.
Trang 8sequence of the basic subunit of PLA2-I from V a
zin-nikeri [17] Finally, we obtained three different
ammody-tin I1 sequences (In, Ia and Ib) as shown in Table 2 and
Fig 3 The amino-acid sequence of ammodytin In
was identical with that reported for the protein
from V am ammodytes (DDBJ/EMBL/GenBank AC
AF253048) This sequence was identified in four
ammo-dytin I1 clones from the neurotoxic V a aspis
Ammo-dytin Ia accounted for the remaining six clones, and all
the genes of V a aspis and V a zinnikeri Its sequence
was 97% identical (four amino-acid differences:
Met70Leu, Gly71Ser, Gln78Glu and Phe123Leu) with that
of the ammodytin I1 of V am ammodytes (Fig 3) The six
clones of the neurotoxic V a aspis harboring the
ammody-tin Ia sequence were those for which a 126-bp deletion in the
ammodytin I1 gene had been identified (Table 3)
Ammo-dytin Ib was found only in V b berus, and differed from
ammodytin Ia by three amino-acid residues (98% identity:
Ile/Thr, in the signal peptide, and His1Asn, Asn56Lys) In
fact, ammodytin In is a hybrid molecule derived from the
N-terminus of ammodytin Ia and the C-terminus of
ammo-dytin Ib Hybrid PLA2s have been reported in several
venomous species of pit vipers [27] However, this hybrid
could not have been produced by recombination between
ammodytin Ia and Ib genes in the neurotoxic V a aspis
snakes, because there was no gene for ammodytin Ib in the
genome of this snake
These findings provide clues to the evolutionary position
of the neurotoxic V a aspis population with respect to the other snakes studied The genome of one of these neurotoxic snakes displayed features characteristic of V am ammo-dytes(monomeric ammodytins A, B and C, and ammodytin I1n with a 259 bp intron D, the Bov-B LINE retroposon) and of V a aspis (vaspin A and B chains and ammodytin I1a with a 133 bp intron D; Table 2) This suggests possible interbreeding between these two species, leading to a hybrid
V a aspiswith a higher level of polymorphism in venom PLA2 genes in this snake (Table 2) The identification of natural hybrids between V a aspis and V am ammodytes
in Italy is consistent with the hypothesis of horizontal transfer [28] Moreover, immunological analysis of albumin proteins also suggests that V aspis and V ammodytes are closely related species [29] The unusually strong conserva-tion of introns in PLA2 genes may facilitate homologous recombination events between PLA2 genes from different species
Amino-acid substitutions: implications for PLA2 structure and/or function
The amino-acid substitutions due to the variant PLA2 genes are indicated below the protein sequence alignment in Fig 3 Frameshifts were observed in exons 4 and 5 of the ammodytin I1 gene, and in exon 3 of the ammodytin I2 gene
Fig 3 Alignment of V a aspis, V a zinnikeri, V b berus and neurotoxic V a aspis PLA2 protein sequences Dots indicate amino acid residues identical with those of the ammodytin I2 protein Dashes indicate gaps introduced to optimize the alignment, using Renetseder’s numbering system [39] AmI2 (blue) corresponds to ammodytin I2, VaspB (red) corresponds to the vaspin chain B protein and VaspA (green) corresponds to vaspin chain A AmI1n corresponds to ammodytin from the neurotoxic V a aspis AmI1a corresponds to the ammodytin I1 of V a aspis, V a zinnikeri and the neurotoxic V a aspis AmI1b corresponds to ammodytin I1 from V b berus VB (black) corresponds to V b berus PLA2 The cysteine residues involved in disulfide bridges are indicated in yellow indicates residue Asp49 Amino acid substitutions resulting from nucleotide polymorphisms are indicated below the alignment, in the color used for the PLA2.
Trang 9(data not shown) There was also a single nonsense
mutation leading to insertion of the TAA stop codon in
exon 3 of one of the vaspin A chain genes of V a zinnikeri
(data not shown) These four clones are inactive The
existence of pseudogenes has also been reported in the
genome of O okinavensis [10] As previously shown by Kini
& Chan [11], the highest mutation frequencies corresponded
to the exposed regions of the molecule, particularly those
involved in the pharmacological activities of PLA2 These
mutations may affect the enzymatic activity, stability and
toxicity of the PLA2s
Enzymatic activity and stability of PLA2s
Key residues involved in or required for PLA2 structure and
catalysis were well conserved (Fig 3) Most of the amino
acids forming the hydrophobic channel and its opening are
located in the N-terminal helix and helix a3 [14,30,31] These
amino acids were conserved in the five PLA2s, with only
rare substitutions observed (Tyr22His; Ala102Val;
Ala103-Val; Fig 3) The amino-acid residues that form part of the
calcium-binding loop, including Tyr28, Gly30, Gly32 and
Asp49, were very well conserved among PLA2s
Neverthe-less, the Tyr28His, Gly30Asp, and Asp49Asn substitutions
were found in some genes (Fig 3), and these substitutions
may affect Ca2+binding, which is necessary for the catalytic
activity of PLA2 enzymes In group II PLA2s, 12–16
cysteine residues are involved in the formation of six to eight
disulfide bridges that stabilize the structure of the molecule
Six of these residues were substituted (Fig 3), probably
decreasing the stability of the molecule by preventing the
formation of disulfide bonds
Toxicity and heterocomplex formation
Little is known about the toxicity of ammodytin I1 Komori
et al [32] purified three PLA2s from V aspis venom
(PLA2-I, PLA2-II and PLA2-III) and determined their
biological activities The N-terminal sequence of PLA2-III
is identical with that deduced here from the nucleotide
sequence of ammodytin I1 Thus, if PLA2-III corresponds
to ammodytin I1, it is not lethal Ammodytin I2 is a
nontoxic PLA2 [33] Vaspin is a neurotoxin [13,17] and the
PLA2 of V b berus has potent anticoagulant activity [20]
Mutations leading to amino-acid substitutions were most
common in exon 4 They were clustered in an exposed
region defined as the b-wing and in a short segment defined
as the anticoagulant region [34] Such mutations were also
found in exons 3 and 5, specifically in the first 16 residues of
exon 3 and at the 3¢ extremity of exon 5 (Fig 3) It has been
suggested that the b-wing region and the region between
amino acids 106 and 128 in exon 5 are involved in PLA2
toxicity [30,35,36] If this is indeed the case, then
substitu-tions occurring in these areas may affect the neurotoxicity or
anticoagulant effect of the PLA2
The substitutions in the A and B chains of vaspin, in the
inhibitor and PLA2 subunits, respectively, should be
considered together, as these proteins associate in a
het-erodimeric complex to exert their neurotoxic effects The
formation of this complex involves intermolecular
inter-actions [17,37,38] The inhibitor subunit stabilizes the
unstable PLA2 subunit through hydrophobic, ionic and
electrostatic interactions, and hydrogen bonds [17,37,38] Substitution of the residues involved in these interactions would therefore be expected to impair the formation or stabilization of the complex, or both, thereby affecting the toxicity of the protein This is probably the case for changes
in the residues involved in the interaction between the two subunits: Asn1Ser, Phe3Leu in the PLA2 subunit and Gln34His in the inhibitor subunit (Fig 3) [17,37]
Conclusion Several groups of genes encoding Vipera venom PLA2s (chains A and B of vaspin, ammodytin I1, ammodytin I2 and an anticoagulant PLA2 from V b berus venom) were sequenced in this study Nonsynonymous mutations were observed in these genes, demonstrating a high level of genetic variability in Viperinae PLA2s Some of these mutations led to amino-acid changes, most commonly in the sequences encoded by the third and fifth exons, which are involved in the biological functions of PLA2 Some genes were pseudogenes, inactivated by frameshifts or by mutations leading to the presence of a stop codon in the sequence The level of expression of the functional genes
is probably controlled by stress-responsive promoters Analysis of venom gland PLA2 cDNAs is currently underway to determine which of these genes are expressed The presence in the neurotoxic snake of two ammodytin I1 isoforms (In and Ia) and of several neurotoxin-encoding genes, some specific to V am ammodytes venom, and of the Bov-B LINE retroposon, which was isolated from the
V am ammodytes genome but not from the V a aspis genome, leads us to conclude that the new population of neurotoxic V a aspis is of ÔhybridÕ origin Phylogenetic and evolutionary analyses are underway to confirm this hypothesis
Acknowledgements
I G holds a postdoctoral fellowship from the Direction des Programmes Transversaux de Recherche (PTR) of the Pasteur Institute This work was funded by the Direction des PTR of the Pasteur Institute.
We also thank Stephane Ferris and Eliana Ochoa from the genomics platform Genopole-IP for technical assistance We are grateful to
Y Doljanski, O Grosselet and A Teynie´ for capturing the snakes and for carrying out the herpetological survey.
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