A new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine proteinase from Vipera lebetina snake venom

Article abstract of DOI:10.1016/j.biochi.2010.10.004

Vipera lebetina venom contains different metallo- and serine proteinases that affect coagulation and fibrin(ogen)olysis. A novel serine proteinase from V. Lebetina venom having ChymoTrypsin Like Proteolytic activity (VLCTLP) was purified to homogeneity from the venom using Sephadex G-100sf, DEAE-cellulose, heparin-agarose and FPLC on Superdex 75 chromatographies. VLCTLP is a glycosylated serine proteinase with a molecular mass of 41926 Da. It reacts with N-acetyl-l-tyrosine ethyl ester (ATEE) but not with Suc-Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-Pro-Leu-pNA. The complete amino acid sequence of the VLCTLP is deduced from the nucleotide sequence of the cDNA encoding this protein. The full-length cDNA sequence of the VLCTLP encodes open reading frame of 257 amino acid residues that includes a putative signal peptide of 18 amino acids, a proposed activation peptide of six amino acid residues and serine proteinase of 233 amino acid residues. VLCTLP belongs to the S1 (chymotrypsin) subfamily of proteases. The multiple alignment of its deduced amino acid sequence showed structural similarity with other serine proteases from snake venoms. The protease weakly hydrolyses azocasein, Aα-chain and more slowly Bβ-chain of fibrinogen. VLCTLP does not cleave fibrin and has no gelatinolytic activity. Specificity studies against peptide substrates (angiotensin I and II, oxidized insulin B-chain, glucagon, fibrinogen fragments etc.) showed that VLCTLP catalysed the cleavage of peptide bonds after tyrosine residues. VLCTLP is the only purified and characterized serine proteinase from snake venoms that catalyses ATEE hydrolysis. We detected ATEE-hydrolysing activities also in 9 different Viperidae and Crotalidae venoms.

Full text of DOI:10.1016/j.biochi.2010.10.004

Biochimie 93 (2011) 321e330
Contents lists available at ScienceDirect
Biochimie
journal homepage: www.elsevier.com/locate/biochi
Research paper
A new tyrosine-specific chymotrypsin-like and angiotensin-degrading
serine proteinase from Vipera lebetina snake venomq
Ene Siigur a, Külli Tõnismägi a, Katrin Trummal a, Mari Samel a, Heiki Vija a, Anu Aaspõllu a,
,
Gunilla Rönnholm b, Juhan Subbi a, Nisse Kalkkinen b, Jüri Siigur a
*
a National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia
b Protein Chemistry Research Group and Core Facility, Institute of Biotechnology, FIN-00014 University of Helsinki, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
Vipera lebetina venom contains different metallo- and serine proteinases that affect coagulation and fibrin
(ogen)olysis. A novel serine proteinase from V. Lebetina venom having ChymoTrypsin Like Proteolytic
activity (VLCTLP) was purified to homogeneity from the venom using Sephadex G-100sf, DEAE-cellulose,
heparineagarose and FPLC on Superdex 75 chromatographies. VLCTLP is a glycosylated serine proteinase
Received 5 April 2010
Accepted 5 October 2010
Available online 13 October 2010
with a molecular mass of 41926 Da. It reacts with N-acetyl-L-tyrosine ethyl ester (ATEE) but not with Suc-
Keywords:
Vipera lebetina
Snake venom
cDNA cloning
Angiotensin I and II
Chymotrypsin-like serine protease
MALDI-TOF MS
LC-MS/MS
Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-Pro-Leu-pNA. The complete amino acid sequence of the VLCTLP is
deduced from the nucleotide sequence of the cDNA encoding this protein. The full-length cDNA sequence of
the VLCTLP encodes open reading frame of 257 amino acid residues that includes a putative signal peptide of
18 amino acids, a proposed activation peptide of six amino acid residues and serine proteinase of 233 amino
acid residues. VLCTLP belongs to the S1 (chymotrypsin) subfamily of proteases. The multiple alignment of its
deduced amino acid sequence showed structural similarity with other serine proteases from snake venoms.
The protease weakly hydrolyses azocasein, Aa-chain and more slowly Bb-chain of fibrinogen. VLCTLP does
not cleave fibrin and has no gelatinolytic activity. Specificity studies against peptide substrates (angiotensin I
and II, oxidized insulin B-chain, glucagon, fibrinogen fragments etc.) showed that VLCTLP catalysed the
cleavage of peptide bonds after tyrosine residues. VLCTLP is the only purified and characterized serine
proteinase from snake venoms that catalyses ATEE hydrolysis. We detected ATEE-hydrolysing activities also
in 9 different Viperidae and Crotalidae venoms.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
metalloproteinasesereprolysins which need Zn2þ or Ca2þ (or both)
for their lytic activity and are inhibited by metal chelating agents
Snake venoms are rich sources of biologically active components
including enzymes and nonenzymatic proteins and peptides, which
can be used in studying basic mechanisms of haemostasis and
envenomation to enable us to develop diagnostic and therapeutic
agents. Snake venoms, particularly those belonging to the Crotalidae
and Viperidae families, are a rich source of proteolytic enzymes that
have been extensively investigated [reviews 1e6]. Proteomic (ven-
omics) analyses have also proved the presence of various protein-
ases in the venoms of Viperidae and Crotalidae snakes [7e9].
These highly specific proteinases which cleave limited bond(s) in
the blood coagulation factors are usually divided into two groups: 1)
(fibrinolytic enzymes, hemorrhagic proteinases, factor X activator)
[review 10]; 2) serine proteinases [e.g. thrombin-like enzymes that
catalyse the cleavage of fibrinogen, liberating fibrinopeptides A and/
or B (A, B and AB venombins), factor V activator, protein C activator,
plasminogen activator, kinin-releasing enzymes, a- and b-fibrino-
genases]. The snake venom serine proteinases contain a highly
conserved catalytic triad comprising of residues His57, Asp102 and
Ser195 (chymotrypsinogen numbering) [reviews 1,2,5,11]. We have
earlier shown that Vipera lebetina venom contains all four classes of
snake venom metalloproteases [12] and different serine proteases
[13e15] that affect coagulation and fibrin(ogen)olysis. Snake venom
serine proteases usually have trypsin-like and thrombin-like activ-
ities hydrolysing p-toluenesulfonyl-
and N-benzoyl- -arginine ethyl ester (BAEE). Tu et al. [16] tested
chymotrypsin-like activity of thirty-three Crotalidae, Viperidae and
Elapidae venoms using N-benzoyl- -tyrosine ethyl ester (BTEE) and
N-acetyl- -tyrosine ethyl ester (ATEE) but failed to detect tyrosine
L-arginine methyl ester (TAME)
q
The sequences data of VLCTLP and VLBF reported in this paper have been
L
submitted to the GenBank under accession nos. GU570565 and GU570566,
respectively.
* Corresponding author. Tel.: þ372 6398360.
L
L
E-mail address: juri.siigur@kbfi.ee (J. Siigur).
0300-9084/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.biochi.2010.10.004

322
E. Siigur et al. / Biochimie 93 (2011) 321e330
esterase activity in the majority of these venoms except those of
Agkistrodon halys and Bothrops jararaca [16].
Zooplant, Ukraine (A. halys, Agkistrodon blomhoffi, Agkistrodon sax-
atilis, Naja atra), from Sigma, USA (Agkistrodon contortrix contortrix),
from Khimky Serpentarium, Russia (Vipera berus berus).
All other reagents used were of analytical grade.
In this paper we describe the purification and characterize
a novel stable monomeric serine proteinase in V. Lebetina venom
having tyrosine-specific ChymoTrypsin Like Proteolytic activity
(VLCTLP). We also present the complete amino acid sequence of
VLCTLP deduced from cDNA. We detected ATEE-hydrolysing
activities in 9 different Viperidae and Crotalidae venoms.
2.2. Purification of the enzyme
Crude venom (1.5 g) was dissolved in 10 ml of 0.2 M ammonium
acetate, pH 6.7. Insoluble material was removed by centrifugation
(5000ꢀg for 15 min) and the supernatant was applied to the column
(2.2 ꢀ 140 cm) of Sephadex G-100 superfine equilibrated with 0.2 M
ammonium acetate. The elution was carried out with the same
solution at a flow rate of 4 ml/h and 6 ml fractions were collected at
2. Materials and methods
2.1. Materials
2,5-Dihydroxybenzoic acid (DHB),
a
-cyano-4-hydroxycinnamic
4
ꢁC. The absorbance was continuously monitored at 280 nm.
acid, oxidized insulin B-chain from bovine pancreas, glucagon from
hog pancreas, angiotensin I and II, azocasein, bovine fibrinogen,
thrombin, gelatin, Substance P, Cytochrome C, N-Suc-Ala-Ala-
Pro-Leu-pNA and N-Suc-Ala-Ala-Pro-Phe-pNA were from
SigmaeAldrich (USA), trypsin from Promega, USA, PNGase F from
New England Biolabs (USA). Sephadex G-100 (superfine)
was the product of Pharmacia (Uppsala, Sweden), Superdex 75 e
Amersham Biosciences (UK), DEAE-cellulose DE-52 (Whatman,
UK). Heparineagarose was obtained from Kemotex Bio (Tallinn,
Estonia). BAEE and ATEE were from Reanal (Hungary). Oligonucle-
otide primers were ordered from DNA Technology (Aarhus,
Denmark). The studied venoms originate from Tashkent Integrated
Zoo Plant, Uzbekistan (V. lebetina, Vipera ursini, Naja oxiana), from
Latoxan, France (Vipera russellii russellii, Echis ocellatus, Cerastes
cerastes, Bitis arietans, Agkistrodon bilineatus, Calloselasma rhodos-
toma, B. jararaca, B. lanceolatus, Oxyuranus scutellatus), from Astik
Farm, India (Bungarus caeruleus, E. carinatus, N. naja), from Tripolski
Fractions I-VIII were pooled as shown in Fig. 1A and lyophilized.
Combined and concentrated by lyophilization, fraction III from
gel filtration (50 mg in 2 ml of 0.1 M ammonium bicarbonate) was
applied onto the Whatman DE-52 cellulose column (1.5 ꢀ 21 cm)
equilibrated with 0.1 M ammonium bicarbonate. Non-adsorbed
material was washed with the equilibration solution. The column
was eluted with the linear gradient of 0.1e0.5 M ammonium
bicarbonate, flow rate 12 ml/h; fractions of 4.8 ml were collected.
7.7 mg of lyophilized material (fractions with chymotrypsin-like
activity from DEAE-cellulose) was dissolved in 1 ml of 0.05 M ammo-
nium bicarbonate and applied to the column of heparineagarose
(1.5 ꢀ 14 cm) equilibrated with 0.05 M ammonium bicarbonate.
The column was eluted with 0.1 M ammonium bicarbonate and 2 M
ammonium bicarbonate. The flow rate was 6 ml/h; fractions of 3 ml
were collected.
The lyophilized VLCTLP fraction with ATEE-hydrolysing activity
from heparineagarose column (0.5 mg) was dissolved in 0.1 ml of
Fig. 1. Purification of VLCTLP from the Vipera lebetina venom (A) Gel filtration of V. lebetina venom. (B) Ion exchange chromatography on DE-52 cellulose. (C) Heparineagarose
chromatography. (D) Gel filtration on FPLC Superdex 75 column. The measured ATEE activity is shown in bold line (lhs e left hand side; rhs e right hand side; AC e azocasein).

E. Siigur et al. / Biochimie 93 (2011) 321e330
323
0.2 M ammonium acetate and applied to the Superdex 75 (10/300)
column equilibrated with 0.2 M ammonium acetate. The elution
was carried out with the same solution. Protein concentrations
were determined using the Pierce micro BCA kit, and bovine serum
albumin was used as a standard. During the process of column
chromatography the elution profile was followed by the absorbance
at 280 nm.
Analytical isoelectric focusing was performed on 5% poly-
acrylamide gel plates according to the method of Vesterberg [22] in
Multiphor 2117 (LKB, Sweden) apparatus in the pH range of
3.6e9.3. The gels were stained for proteins with Coomassie Brilliant
Blue R250.
2.5. N-terminal sequencing
Five
by reversed phase chromatography on a 1 ꢀ 150 mm Jupiter C4
(5 m, 300 Å, Phenomenex Inc, USA) using a linear gradient of
acetonitrile (0e60% in 60 min) in 0.1% trifluoroacetic acid. Flow rate
was 50 l/min and detection at 214 nm. The protein peak was
mg of lyophilized VLCTLP was dissolved in 10 ml H2O and run
2.3. Protease and esterase activities
m
Esterolytic activity with BAEE (0.25 mM; 0.05 M TriseHCl, pH
8.5) and ATEE (0.5 mM; 0.05 M TriseHCl, pH 8.5) was determined
spectrophotometrically according to Schwert and Takenaka [17]. To
m
collected and used for sequencing by Edman degradation on
a Procise 494A Sequencer (Perkin Elmer, Applied Biosystems Divi-
sion, CA, USA).
1 ml of substrate 20 ml of the enzyme (1 mg/ml) was added and the
increase in absorbance at 253 nm in case of BAEE and the decrease
in absorbance at 237 nm in case of ATEE was recorded. One unit is
defined as the 1.0 change in absorbance in 0.05 M TriseHCl, pH 8.5
at 25 ꢁC per min. The activity with chymotrypsin substrates N-
succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA) and N-
2.6. Tryptic digestion and mass fingerprinting
succinyl-Ala-Ala-Pro-Leu-p-nitroanilide
determined spectrophotometrically. To 1 ml of substrate (0.4 mM in
0.05 M TriseHCl, pH 8.3) 20 l of the enzyme (1 mg/ml) was added
and the change in absorbance at 410 nm was recorded.
Azocaseinolytic activity was measured as described by Siigur
et al. [18].
(Suc-AAPL-pNA)
was
Trypsinolysis was performed in gel as in solution.
After visualization with Coomassie Blue the gel-electrophoresis
bands of protein in interest (native or reduced) were excised from
SDS-polyacrylamide gels, each gel slice was cut into small pieces
(1 mm2), placed into eppendorf tube and treated as described
m
earlier [23]. Equal volumes (0.5
ml) of the peptide mixture and the
VLCTLP-dependent digestion of fibrinogen was assessed with
bovine fibrinogen as substrate. 0.1 ml of 2% fibrinogen solution was
matrix (2,5-dihydroxybenzoic acid, or
a-cyano-4-hydroxycinnamic
acid) were mixed on the MALDI-TOF plate. The mass calibration
standard was substance P.
incubated with 0.1 ml of VLCTLP (10 m
g) at 37 ꢁC in 0.05 M TriseHCl
buffer (pH 7.4) containing 0.1 M NaCl. At various time intervals
0.025 ml of the incubation mixture was withdrawn and added to
0.025 ml of denaturing solution (10 M urea, 4% SDS, 4% 2-mer-
captoethanol). The samples were incubated overnight at 37 ꢁC
before electrophoresis. Specific cleavage of fibrinogen was shown
on 12.5% SDS-polyacrylamide gels. Fibrinolytic activity was deter-
mined by fibrin plate method of Astrup and Müllertz [19].
Gelatinolytic activity was determined according to Bee et al.
[20]. Gelatin was co-polymerised into 10% PAA-gel at final
concentration of 2.5 mg/ml. VLCTLP (5 mg) was mixed with non-
In case of in solution trypsinolysis 5
dissolved in 50 l of 6 M GuanidineeHCl, 0.5 M TriseHCl, 2 mM
EDTA, pH 7.5 and 5 l of 100 mM DTT was added. After keeping the
mixture for 30 min at room temperature, 6 l of 200 mM iodoace-
tamide was added and the mixture was kept at room temperature
for 30 min, then 5 l of 100 mM DTT was added. The alkylated
protein was desalted by reversed phase chromatography on
mg of purified VLCTLP was
m
m
m
m
a 1 ꢀ 20 mm TSK gel TMS-250 column (C1, 10 m, 250 Å,1 ꢀ 20 mm,
m
TOSOH Corporation, Japan) as described for N-terminal sequencing.
The protein peak was collected and dried by vacuum centrifugation.
reducing sample buffer and applied to the gel. After electrophoresis
the SDS was removed by washing the gel for 1 h in 2.5% Triton X-
100 before incubation in 50 mM Trisebuffer (50 mM CaCl2, 0.2 M
NaCl, 0.07% Brij 35), pH 7.6 at 37 ꢁC for 16 h and the gel was stained
with 0.125% Coomassie brilliant blue R250. Clear areas in the gel
indicate regions of enzyme activity.
For trypsin digestion, the dried sample was dissolved in 50
ammonium bicarbonate, 0.05 g trypsin (Sequencing Grade Modi-
fied, V5111, Promega) was added and incubated at 37 ꢁC overnight.
For peptide de novo sequencing, 10 l of the tryptic digest was sub-
jected to LC-MS/MS analysis on a Q-TOF (Micromass, UK) as
described before [24]. The amino acid sequences of the N-terminal
and tryptic peptides were compared with the BLASTP database [25].
ml of 0.1 M
m
m
The thermostability was studied incubating the enzyme solu-
tion (20 ml; 1 mg/ml in 50 mM CH3COONH4, pH 6.5) at various
temperatures (25, 37, 50, 60, 70, 80, 100 ꢁC) for 10 min; then quickly
cooled and ATEE-hydrolysing activities were determined.
2.7. Deglycosylation of VLCTLP
For deglycosylation of VLCTLP PNGase F deglycosylation kit
2.4. Molecular mass determination and isoelectric focusing
(New England Biolabs) was used. 18
(VLCTLP), 1
l of the 10ꢀ glycoprotein denaturing buffer (5% SDS,
0.4 M DTT) and 2 l of H2O were combined and the glycoprotein
was denatured by heating at 100 ꢁC for 10 min. The total reaction
volume was made of 20 l by adding 2
l of 10ꢀ G7 reaction buffer
(0.5 M sodium phosphate pH 7.5), 2 l of 10% n-octylglucoside and
l of PNGase F, the reaction mixture was incubated at 37 ꢁC
overnight (in case of native deglycosylation 4 l of PNGase F and
mg (9 ml) of glycoprotein
m
The molecular masses of the purified proteins were determined
by SDS-PAGE on 12.5% polyacrylamide gels using the method of
Laemmli [21]. The molecular masses were also determined using
a home-built (National Institute of Chemical Physics and Biophysics)
matrix-assisted laser desorption/ionization-time of flight mass
spectrometer (MALDI-TOF-MS) designed for maximum flexibility in
m
m
m
m
2
m
m
use. For MALDI-TOFanalysis the dried samples were dissolved in 5
of 50% ACN, 0.1% TFA. Aliquots of 0.5 l were applied onto the target,
allowed to air dry and 0.5 l of the matrix solution (2,5-dihydrox-
ybenzoic acid e DHB) was applied to the target and allowed to dry in
air. The mass calibration standards were cytochrome C and insulin
B-chain. A nitrogen 337 nm laser (4 ns pulse) was used and at least
30e40 shots were summed up.
m
l
24 h incubation) and subjected to SDS electrophoresis or trypsi-
nolysis. In case of the latter the mixture was once again denatured
m
m
with 2
30 l of 2-iodoacetamide (10 mg/ml, Fluka) was added and the
reaction mixture was held in dark for 1 h. Fifty l of water was
added, followed by 20 l of trypsin solution (Promega Trypsin Gold,
25 ng/
l). The reaction mixture was incubated at 37 ꢁC for 24 h. The
m
l of denaturing buffer and heated at 100 ꢁC for 10 min. Then
m
m
m
m

324
E. Siigur et al. / Biochimie 93 (2011) 321e330
Table 1
Purification of VLCTLP from Vipera lebetina venom.
Purification step
Total protein
(mg)
Specific activity
Total activity
(units)
Purification
(fold)
Recovery
(% initial activity)
(unit/mg)a
Crude venom
Fraction III from gel filtration
DE-52 cellulose
Heparineagarose
Superdex 75
1500.0
113.3
18.1
10.1
6.3
0.2
2.6
6.7
7.4
9.0
300.0
291.3
120.4
74.2
1.0
12.9
33.3
36.8
45.0
100.0
97.1
40.1
24.8
18.9
56.7
a
The activity of the enzyme was measured with N-acetyl-L-tyrosine ethyl ester (ATEE) as substrate.
sample of tryptic peptides was purified using ZipTip C18 and
analyzed with MALDI-TOF MS.
3. Results
3.1. Purification and characterization
VLCTLP was purified from the venom using Sephadex G-100sf,
DEAE-cellulose, heparineagarose and FPLC Superdex 75
chromatographies.
2.8. Synthesis and hydrolysis of oligopeptide substrates
The peptide fragments 408e417 from fibrinogen A chain and
687e693 from pregnancy zone protein, PZP, were synthesized at
the 100 mmol scale on Applied Biosystems 431A Peptide Synthe-
The first step e size exclusion chromatography on Sephadex G-
100sf, e enables to get rid of various enzymes such as phosphodi-
esterase, 50-nucleotidase, phosphomonoesterase,
L-amino acid
sizer using BOC (t-butyl-oxycarbonyl) chemistry as suggested by
the manufacturer. The purity of peptides was assessed by analytical
reverse phase e high performance liquid chromatography (RP-
HPLC) and MALDI-TOF mass spectrometry. The peptide solutions
were directly prepared in 0.05 M NH4HCO3, at concentrations of
about 1e5 mg/ml, and kept frozen at ꢂ20 ꢁC until use.
oxidase [27], apoptosis-inducing protease [28] and factor X activator
[23] (peaks I and II, Fig.1A). The chymotrypsin-like activity (substrate
ATEE) was detected in the third peak with several other components.
The fourth and fifth peaks contain alpha-fibrinogenase [15], factor V
activator [13], phospholipase A2 [29], nerve growth factor [30],
viplebedin-2 [31], and different other components, peak VI contains
lebetase [32]. By ion exchange chromatography on DEAE-cellulose
the chymotrypsin-like activity was detected in the second peak
(Fig. 1B). VLCTLP could be further purified by heparineagarose
chromatography where chymotrypsin-like activity was observed in
the first peak (Fig. 1C). The final purification was obtained using gel
filtration on FPLC Superdex 75 column (Fig.1D). Thefinal preparation
showed 45-fold purification of the enzyme with an 18.9% yield of the
activity and 0.42% of the protein (Table 1). The content of the enzyme
in venom batches is variable, 0.25e0.55% depending on the batch
used. The molecular mass of VLCTLP is 55 kDa by SDS-PAGE (Fig. 2),
41926 Da by MALDI-TOF MS (Fig. 3) and after deglycosylation about
27 kDa by SDS-PAGE (Fig. 4). Deglycosylation of the native enzyme
resulted in heterogeneous preparation due to incomplete process
(data not shown). Isoelectric point of VLCTLP is in the acid region
(ꢃ3.6). The enzyme was stable at 4 ꢁC for several months. The partial
decrease in activity was observed after heating for 10 min at 70 ꢁC
and about 40% of activity was retained at 100 ꢁC. Partial deglycosy-
lation had no effect on enzyme activity (substrate ATEE).
The enzymatic hydrolysis of peptides was carried out in 0.05 M
NH4HCO3, pH 8.3, at 37 ꢁC in an eppendorf tube. In a typical
experiment, 200
ml) in an eppendorf tube was thermally equilibrated to 37 ꢁC in the
thermostated rack. The reaction was started by addition of 15 l of
enzyme solution (1 mg/ml in 0.05 M NH4HCO3). At predetermined
time intervals (0.5e24 h), the aliquot (50 l) was diluted with
500 l of H2O and 10 l of 6 N HCl was added to stop the reaction.
We applied a low enzyme e substrate ratio (1:3e1:70 on a weight
basis) to determine the position of cleavage. 0.5 l of diluted
ml of 0.05 M NH4HCO3 solution of substrate (5 mg/
m
m
m
m
m
mixture was used for MALDI-TOF mass spectrometry analysis.
2.9. Platelet aggregation assay
Platelet aggregation assays were performed in human platelet-
rich plasma according to the previously described protocol [18].
We tested ATEE-hydrolysing activity in 22 Viperidae, Crotalidae
and Elapidae venoms and the activity was observed in 9 snake
2.10. PCR amplification, cloning and sequencing
The cDNA encoding VLCTLP was isolated by PCR amplification as
a byproduct of cloning of the alpha-fibrinogenase-encoding cDNA.
The V. lebetina venom gland cDNA library constructed in the Uni-
ZAP XR vector [26] was used as a source of the template. Using the
antisense primer 50-GGTGTAGCTTTTGTTACTGAG-30 (PI) and T3 as
sense primer, the cDNA fragment corresponding to the 50-terminal
sequence of VLCTLP was amplified. On the basis of this sequence
the primer PII (50-CGAAATCAGGATGAGC-30; sense) was designed
for amplification of the main VLCTLP-coding cDNA fragment with
T7 as antisense primer. The PCR conditions were as follows:
denaturation at 94 ꢁC e 1 min; annealing at 55 ꢁC e 1 min:
extension at 72 ꢁC e 1 min 30 s; 35 cycles. After standard proce-
dures some random clones were sequenced along both strains on
Perkin Elmer ABI Prism Model 310 DNA Sequencer by cycle
sequencing using DYEnamicÔ ET Terminator Cycle Sequencing Kit
(Amersham Biosciences). Three individual VLCTLP clones generated
by independent PCRs were analysed. The full-length cDNA was
constructed from the overlapping fragments.
Fig. 2. SDS-PAGE (12.5% gel) of VLCTLP from different purification steps in reduced (A)
and non-reduced (B) conditions: 1 e V. lebetina venom; 2 e fraction III from gel
filtration; 3 e VLCTLP fraction from DEAE-cellulose; 4 e VLCTLP fraction from hep-
arineagarose; 5 e VLCTLP fraction from Superdex 75 column; 6 e molecular mass
markers (bovine serum albumin 66 kDa, ovalbumin 45 kDa, carboanhydrase 29 kDa,
soybean trypsin inhibitor 20 kDa, cytochrome C 12.3 kDa).

E. Siigur et al. / Biochimie 93 (2011) 321e330
325
Table 2
ATEE esterase activities of snake venomsa.
Species
unit/mg
Echis ocellatus
Vipera lebetina
0.284
0.200
0.120
0.100
0.060
0.044
0.040
0.028
0.020
Echis multisquamatus
Agkistrodon halys
Vipera berus berus
Echis carinatus
Bothrops jararaca
Agkistrodon saxatilis
Agkistrodon blomhoffii
a
To 1 ml of substrate (0.5 mM ATEE in 0.05 M TriseHCl,
l of venom (5 mg/ml) was added and the decrease
in absorbance at 237 nm was recorded.
pH 8.5) 50
m
not degrade the fibrin plate. Some snake venom serine proteinases
(thrombocytin, cerastocytin, serine proteinase PA-BJ) have platelet
aggregating activity [33e35]. VLCTLP has neither aggregation-
inducing nor inhibiting activity.
Fig. 3. MALDI-TOF mass spectrum of VLCTLP. Molecular mass 41926 Da. Matrix was
ferulic acid.
venoms (see Table 2). The highest activities were in E. ocellatus and
V. lebetina venoms. We could not find any chymotrypsin-like
activity in the venoms of the following snakes: A. bilineatus,
A. contortrix contortrix, B. arietans, B. lanceolatus, C. rhodostoma,
C. cerastes, E. ocellatus, V. russellii russellii, V. ursini, B. caeruleus,
O. scutellatus, N. atra, N. oxiana.
The ability of the purified enzyme to hydrolyse several types of
peptides was examined. Masses of peptides and peptide fragments
produced by VLCTLP hydrolysis were detected by MALDI-TOF mass
spectrometry analysis. The oxidized insulin B-chain cleavage by
VLCTLP is different from cleavages with other proteinases in
venom. The enzyme catalysed the cleavage of Tyr16eLeu17 and
Tyr26eThr27 bonds in the oxidized insulin B-chain (Fig. 6A),
Tyr10eSer11, Tyr13eLeu14 and Leu14eAsp15 bonds in glucagon after
overnight incubation and Tyr4eIle5 bond in angiotensins I and II.
Remarkable cleavage of angiotensins was seen already after 5 min
treatment (Fig. 6B).
3.2. Substrate specificity
Substrate specificity of the VLCTLP was studied against different
proteins. The enzyme hydrolyses azocasein very weakly and has no
gelatinolytic activity. PMSF (1 mM, preincubation for 1 h) inhibited
the hydrolytic activity of the enzyme. These experiments confirm
the serine proteinase nature of the enzyme.
We synthesized some tyrosine-containing peptides according to
fibrinogen Aa-chain sequence (fragment 408e417) and pregnancy
zone protein (PZP) sequence (fragment 687e693). The enzyme
catalyses the cleavage of TyreHis bond in the peptide fragment of
fibrinogen 408Glu-Tyr-His-Thr-Glu-Lys-Leu-Val-Thr-Ser417 and very
slowly (after 20 h) TyreVal bond in the peptide fragment of PZP
Incubation of VLCTLP with 1% fibrinogen prolonged the fibrin-
ogen coagulation time by thrombin (Fig. 5B). Cleavage of fibrinogen
is shown in Fig. 5A. The enzyme (50
-chain was cleaved more slowly and the
even after 24 incubation. The fibrinogenolytic activity is
mg/ml) digested A
a-chain, the
687Tyr-Val-Pro-Gln-Leu-Gly-Thr693
.
Specificity studies against
B
b
g-chain was left intact
h
peptide substrates showed that VLCTLP catalysed the cleavage of
peptide bonds preferably after tyrosine residues. It did not cleave
chymotrypsin substrates Suc-Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-
Pro-Leu-pNA confirming the tyrosine specificity. VLCTLP is tyro-
sine-specific chymotrypsin-like enzyme.
remarkably weaker than that of the other fibrinogenases in the
venom. The enzyme does not show fibrinolytic activity since it does
3.3. Sequence of the cDNA encoding VLCTLP and
the deduced amino acid sequence
The VLCTLP gene includes a 50-UTR e nucleotides 1e179,
a proenzyme coding region (nt 180e251), a mature enzyme coding
region (nt 252e953), and a 30-UTR (nt 954e1584). The full-length
cDNA sequence of the VLCTLP encodes open reading frame of 257
amino acid residues that include a putative signal peptide of 18
amino acids, a proposed activation peptide of six amino acid resi-
dues (Gln-Lys-Ser-Ser-Glu-Leu) and mature serine proteinase of
233 amino acid residues (Fig. 7). VLCTLP contains twelve conserved
cysteine residues that form six disulfide bonds.
The calculated molecular mass is 25580 that is considerably
lower than the value obtained by MALDI-TOF MS (41.9 kDa). The
difference could be explained with the heavy glycosylation of the
enzyme. The deduced protein sequence exhibits 5 putative N-
glycosylation sites, NXS/T, at the residues Asn44, Asn100, Asn116,
Asn153 and Asn250 (Fig. 7) but all of them need not to be glyco-
sylated. After deglycosylation the molecular mass of VLCTLP was
reduced to about 27 kDa (Fig. 4). Sugars play important role in
venom toxicology, not only increasing the solubility and stability
of venom glycoproteins but also promoting their target
Fig. 4. Deglycosylation of VLCTLP with PNGase F. 12.5% SDS-PAGE in reduced condi-
tions: 1 e VLCTLP; 2 e deglycosylated VLCTLP and PNGase F (36 kDa); 3 e molecular
mass markers (bovine serum albumin 66 kDa, ovalbumin 45 kDa, carboanhydrase
29 kDa, soybean trypsin inhibitor 20 kDa, cytochrome C 12.3 kDa).

326
E. Siigur et al. / Biochimie 93 (2011) 321e330
Fig. 5. Fibrinogenolytic activity. (A) 0.5 ml of 1% fibrinogen solution was incubated with 0.5 ml of enzyme (50
At various time intervals 50 l of the incubation mixture was withdrawn and added to 50 l of denaturing solution (10 M urea, 4% SDS, 4% 2-mercaptoethanol). The samples were
incubated overnight at 37 ꢁC before electrophoresis. Specific cleavage of fibrinogen was shown on 10% SDS-polyacrylamide gel. 1 e fibrinogen; 2 e 5 min; 3 e 15 min; 4 e 60 min;
5 e 180 min; 6 e 420 min; 7 e 18 h; 8 e molecular mass markers. (B) 2 ml of 1% fibrinogen solution was incubated with 0.5 ml of enzyme (200
g) at 37 ꢁC in 0.05 M TriseHCl buffer
l of thrombin (Sigma) was added. The fibrinogenolytic activity
m
g) at 37 ꢁC in 0.05 M TriseHCl buffer (pH 7.4), containing 0.1 M NaCl.
m
m
m
(pH 7.4), containing 0.1 M NaCl. At various time intervals 300
ml of the incubation mixture was withdrawn and 15 m
was determined indirectly as an increase in the clotting time.
A
1000
1500
2000
2500
3000
3500
m/z
B
1
4
+
(Asp-Tyr+H)
5
8
+
(Angiotensin II+H)+
(Ile -Phe+H)
0
200
400
600
800
1000
1200
m/z
Fig. 6. MALDI-TOF mass-spectra of the cleavage products of peptides after treatment with VLCTLP. Matrix was 2,5-dihydroxybenzoic acid (DHB). (A) Oxidized insulin B-chain
fragments after 3 h treatment. (B) Angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) and cleavage fragments after 5 min treatment. Enzyme concentration 75 g/ml.
m

E. Siigur et al. / Biochimie 93 (2011) 321e330
327
Fig. 7. Complete cDNA and deduced amino acid sequence of the VLCTLP precursor. The nucleotide sequence and the deduced amino acid sequence are numbered from the N-
terminal amino acid of the preproprotein. The cDNA coding regions are shown in uppercase and 50-and 30-untranslated regions in lower-case letters. The N-terminus detected from
protein sequence is in bold italics. Active site serine, histidine and aspartic acid residues are bold. The potential N-glycosylation sites are grey (Asn44; Asn100, Asn116, Asn153,
Asn250). The peptide fragment sequences detected by LC-MS/MS are bold underlined. Tryptic peptide fragments detected by MALDI-TOF MS are underlined. Tryptic peptide
fragments 115e136 and 143e169 were detected after deglycosylation of the enzyme.
recognition and specific binding in vivo [36]. VLCTLP is rather
stable protein due to its 6 disulfide bonds and heavy glycosylation
(5 putative glycosylation sites). It is well known that sugars
increase the solubility and stability of venom glycoproteins.
32 N-terminal amino acid residues are completely identical (Fig. 8).
The rate of identity with other 100 snake venom serine proteinases
covers the range 63e76% (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
These enzymes possess rather different substrate specificities
including fibrinogenases, thrombin-like enzymes, plasminogen
activators, factor V activators, protein C activators, kinin-releasing
enzymes.
3.4. Similarity of VLCTLP to other sequences
Fig. 8 shows the alignment of deduced amino acid sequences of
the precursor of VLCTLP with several homologous sequences from
different snake species.
4. Discussion
The identity of protein sequences is highest between VLCTLP
Snake venom serine proteinases affecting haemostasis belong to
a trypsin-like subfamily of proteinases that share a high degree of
and VLBF, V. lebetina
b-fibrinogenase, about 83%. Their first

Fig. 8. Multiple sequence alignment of precursors of serine proteases from different snake venoms using CLUSTAL W [43]. VLCTLP-Vipera Lebetina chymotrypsin-like protease;
GU570566-VLBF, Vipera Lebetina beta-fibrinogenase; O13063 e [Trimeresurus gramineus]; CAQ72889 e [Echis ocellatus]; Q8JH85 e serine alpha-fibrinogenase precursor [Macro-
vipera lebetina]; AAK12273 e thrombin-like enzyme precursor [Deinagkistrodon acutus]; Q71QJ4 e - serine protease KN4 precursor [Viridovipera stejnegeri], Q71QI1 e serine
protease KN12 precursor [Viridovipera stejnegeri]; CAC00530 e acubin2 [Deinagkistrodon acutus]; Q71QH6 e serine protease KN13 precursor [Viridovipera stejnegeri]. Closed
triangles indicate the amino acid residues (His57, Asp102, Ser195, Chymotrypsinogen numbering.) involved in the catalytic reaction. “*” indicates positions which have a single, fully
conserved residue; “:”indicates that one of the ‘strong’ amino acid groups is fully conserved; “.” indicates that one of the ‘weaker’ groups is fully conserved.

E. Siigur et al. / Biochimie 93 (2011) 321e330
329
sequence identity despite exhibiting remarkably high substrate
selectivity. They are composed of approximately 235 amino acids
and contain 12 conserved cysteine residues paired in six disulfide
bridges. The V. lebetina venom contains several serine proteases,
most of them are specific enzymes: Factor V activator, bradykinin-
between residues 4 and 5 in angiotensin II to release an N-terminal
aspartyl tetrapeptide Ang-(1e4) and a C-terminal tetrapeptide
Ang-(5e8), destroying the biological activity of angiotensin II.
VLCTLP is unique among known snake venom serine proteases
for its substrate specificity. To the best of our knowledge VLCTLP is
the only enzyme isolated from snake venoms that catalyses the
releasing enzyme,
hydrolysis of BAEE. The venom contains also two serine proteinases
that do not catalyse the hydrolysis of BAEE: -fibrinogenase (VLAF)
b-fibrinogenase e these enzymes catalyse the
hydrolysis of the N-acetyl-
L-tyrosine ethyl ester (ATEE).
a
[15] and VLCTLP. We have shown that VLAF effectively hydrolyses
casein, VLCTLP very weakly catalyses the cleavage of azocasein but
differently from other snake venom serine proteinases VLCTLP has
ATEE-hydrolysing activity.
Conflict of interest
The authors declare there are no conflicts of interest.
Acknowledgements
The proteolytic specificity is usually characterized by the
hydrolysis of oxidized insulin B-chain. Differently from arginine
esterases that catalyse mainly the hydrolysis after Arg residue,
VLCTLP cleaves bonds after Tyr residues.
The work was supported by Estonian Science Foundation grant
7251 and by target financing SF0690063s08. Külli Tõnismägi
acknowledges the support by SF0690029s09.
The molecule of VLCTLP contains the amino acids forming
catalytic triad His57, Asp102 and Ser195 (Fig. 8) (chymotrypsinogen
numbering) like all enzymes belonging to the clan SA [37]. The
sequences around those amino acid residues were found to be
highly homologous. The positions of 12 half-cystines are identical
among all the sequences, suggesting that the proteins encoded will
take similar tertiary structure. On the basis of the evolutionary
markers VLCTLP is the member of the S1 (chymotrypsin) subfamily
[38]. Ser195 and Ser214 are both encoded by TCT, and the position
226 is occupied by proline (Fig. 8). In almost all of the active serine
proteinases, the N-terminal residue (Val16, Fig. 8) forms an internal
salt bridge between its amino group and the side chain carboxyl
group of Asp194 (Fig. 8) [39]. Apart from Asp102 and Asp194 (Fig. 8)
there is the third essential aspartate in the molecule of trypsin-like
serine proteinases e Asp189 that is located in the bottom of the
primary specificity pocket near the active site and forms a salt
bridge with the basic residue of the scissile bond [40]. Asp189, six
residues before the catalytic amino acid Ser195 residue, is
conserved in many snake venom serine proteinases, indicating that
it is responsible for the trypsin-like specificity. Substitution of
Asp189 resulted in 105 e fold decrease toward P1 Arg/Lys substrate
[41]. In the case of VLCTLP Asp189 is replaced by Gly189 (Fig. 8) that
points to the possible lack of the trypsin-like substrate specificity
for basic amino acids at the P1 position.
In summary, some mutations in the primary structure may
essentially change the specificity of the enzyme as it happened in
the case of VLCTLP. The point mutation in codon for Asp189 (AeG)
changes it to codon for Gly (Fig. 7, 589G) and deprives it from the
arginine esterase activity. The chymotrypsin-like activity is difficult
to explain. Hedstrom et al. [42] found that Tyr172 residue might be
a specificity determinant because the trypsin Tyr172Trp mutant
showed 2e15% of chymotrypsin activity. The Tyr172 of VLCTLP is
conserved, so there must be some other reason for the chymo-
trypsin-like specificity.
Thus, snake venom serine proteinases share a high degree of
sequence identity (60e80%) and show at the same time a high
substrate specificity. We have found a novel serine proteinase in
V. lebetina venom catalysing the hydrolysis of ATEE. Specificity
studies against tyrosine and phenylalanine residues containing
peptide substrates showed that VLCTLP catalysed the cleavage of
peptide bonds after tyrosine residues.
The best substrates for VLCTLP were angiotensins I and II.
Angiotensin II is an octapeptide that causes vasoconstriction,
increased blood pressure, and release of aldosterone from the
adrenal cortex. Proteinases involved in processing of angiotensin
peptides have been collectively termed “angiotensinases”. Angio-
tensinases are comprised of three groups of peptidases: amino-,
endo- and carboxypeptidases [44]. Similarly to angiotensinase B
(chymotrypsin-like endopeptidase) [45], VLCTLP acts on the bond
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