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0582-9879 Acta Biochim et Biophysica Sinica 2004, 36(1):27-32 CN 31-1300/Q
Purification, Gene Cloning and Expression
of an Acidic Phospholipase A2 from Agkistrodon
shedaoensis Zhao
Qian JIN#,
Li-Xia YANG1#,
Hao-Mang JIAO, Bin LU, Yu-Qun WU1, and Yuan-Cong ZHOU*
( Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology,
Shanghai Institutes for BiologicalSciences, the Chinese Academy of Sciences,
Shanghai 200031, China;
1Institute
of Snakes and Snake Venom, Dalian She Dao Hospital, Dalian 116041, China )
Abstract A protein with the activity of phospholipase A2, named
asAPLA2, was
purified to homogeneity from the venom of Agkistrodon shedaoensis Zhao
through DEAE-Sepharose CL-6B anion exchange column, Source S, and Mono Q FPLC.
Its molecular weight was estimated to be 19 kD by SDS-PAGE, and its pI was
about 3.5 by IEF analysis. It inhibited the platelet aggregation that was
induced by 1 μmol/ L ADP, and the IC50 was determined to be 6 μmol/L. Degenerating
primer was designed and synthesized according to the Nterminal amino acid
sequence of asAPLA2. Its full-length cDNA was cloned by RT-PCR from the total RNA extracted
from the snake venom gland. Its molecular weight and the pI are determined to
be 13,649.36 and 4.393 respectively as caculated by DNAclub and DNAstar
software according to the deduced amino acid sequence. Then the gene was cloned
into the expression plasmid pET-40b(+) and expressed in E. coli BL21(DE3).
Western blot analysis indicated that the expressed protein cross-reacted with
the antibody against the native enzyme.
Key words Agkistrodon shedaoensis Zhao; APLA2;
purification; cloning; gene expression
Phospholipase A2 (PLA2, EC 3.1.1.4) is abundant in the venom of snake and
scorpion, or the pancreas of mammals. It catalyzes the hydrolysis of the Sn-2
ester bond of 1,2- diacyl-3-phosphoglycerides to produce lysophosphatidylcholine
and fatty acid. In addition to this enzyme activity, PLA2 from snake
venom also possesses a wide variety of pharmacological activities, such as
neurotoxicity [1], myotoxicity [2], cardiotoxicity [3], hemolytic activity [4],
inhibiting effect on platelet aggregation [5] and some other activities. It was
also found that PLA2 can block the development of malaria parasite in the mosquito midgut [6].
Usually there are several kinds of phospholipase A2 coexisting in the venom
of an individual source. These isoenzymes may react with each other or with
other proteins in snake venom to exert actions on the prays that the snakes
captured.
Three kinds of PLA2 have been purified from
the venom of Agkistrodon halys Pallas. They are designated as acidic PLA2 (APLA2), neutral
PLA2 (NPLA2) and basic
PLA2 (BPLA2) according
to their isoelectric points, which are 4.5, 6.9, and 9.3, respectively. These
isoenzymes show different pharmacological activities. APLA2 can inhibit
platelet aggregation, NPLA2 is a presynaptic neurotoxin, and BPLA2 possesses the
ability to hemolyze erythrocytes. In previous works, we paid more attention on
the studing of APLA2 than the other two isoenzymes. The researches of APLA2 mainly
focused on the purification of the protein, the determination of its amino acid
sequence, the analysis of its configuration in solution as well as its crystal
structure [7], its mechanism in inhibiting the platelet aggregation [8], the
cloning and expression of its cDNA, and so on [9].
In this paper, a new acidic PLA2 from the venom of Agkistrodon
shedaoensis Zhao which lives only in Dalian She Dao (Snake Island) of
China, has been purified. The cloning and expressing of its cDNA were also
performed to provide more information on the structure-function relationship of
this acidic PLA2.
Materials and Methods
Materials
Live snake of Agkistrodon shedaoensis Zhao and its
snake venom were provided by Institute of Snakes and Snake Venom, Dalian She
Dao Hospital (Dalian, China). AKTA FPLC system, DEAE-Sepharose CL-6B, Source S
and Mono Q were purchased from Amersham. E. coli DH16B, BL21(DE3) and
expression vector pET-40b(+) were kept in our laboratory. pMD18-T vector, DNA
restriction enzymes, Taq DNA polymerase, T4 DNA ligase were the products
of TaKaRa. Trizol total RNA isolation kit and MMLV first strand cDNA synthesis
kit were purchased from Sangon. Human blood was obtained from healthy
volunteers. Anti-APLA2 antibody was prepared in our laboratory. All other chemicals were local
products of analytic grade.
Total RNA extraction and cDNA synthesis
The snake was sacrificed by decapitation. Venom glands
were removed immediately, homogenized, and quickly suspended in Trizol reagent
(Sangon). The extraction of total RNA and the cDNA synthesis were performed
according to the manufacturer’s protocol (Sangon).
Purification of asAPLA2 and its N-terminal
sequence determination
Crude venom was applied onto DEAE-Sepharose CL- 6B
column, and the active fractions with PLA2 activity were collected and
further purified by Source S FPLC and Mono Q FPLC. The active fraction was
collected, lyophilized, and stored at –20 .
The determination of N-terminal amino acid sequence was
performed on an ABI-491A protein sequencer.
Primer synthesis
Primer 1 was designed according to the N-terminal
sequence of asAPLA2 and was an oligonucleotide mixture with all possible sequences
corresponding to the amino acid sequence. Primers were synthesized by Sangon.
Primer 1, 5‘-GTAGTACTAGCCTGGT(TCA)CA(GA)T(CT)GAGAC(AT)C-3‘;
Primer 2, Oligo (dT)18.
PCR, cloning and DNA sequence analysis
PCR for amplifying asAPLA2 cDNA with total cDNA as
the template was performed for 35 cycles with denaturation for 1 min at 94 ,
annealing for 1 min at 42 , and elongation for 1.5 min at 72 . Then PCR product
was subcloned into the pMD18-T vector to transform E. coli DH16B.
The gene sequence was analyzed by the dideoxy
chaintermination method using RV-M and M13-47 universal primers. The analysis
and comparison of the sequences were performed with DNAstar and DNAclub
software.
Gene expression and Western blot
The assembled gene was subcloned into the expression
vector pET-40b(+) and transferred into E. coli BL21(DE3). Bacterial
cultures were grown in LB medium at 37 . Cells were harvested by centrifugation
at 5000 g for 10 min when A600 reached 0.6.
Antibodies against native APLA2 from Agkistrodon
halys Pallas were prepared in our laboratory. Western blot was performed as
described previously [10].
AsAPLA2 enzymatic activity assay
PLA2 activity was assayed by estimating the fatty acids
released from phosphatidylcholine (PC) according the method of Novak [11]. The
substrate was freshly prepared according to Kawauchi et al. [12]. The
reaction mixture contained 1 mmol/L PC, 1 mmol/L deoxycholate, 1 mmol/ L CaCl2, 50 mmol/L
NaCl and 100 μg enzyme. The pH of reaction solution should be maintained at about 8.20
by replenishing 0.02 mol/L KOH. PLA2 activity is expressed as the amount of KOH (mmol)
consumed per mg protein per min.
Platelet aggregation inhibiting activity assay
Blood from healthy donors with no medications in the last
two weeks was mixed with 3.8% sodium citrate in 9:1 (V/V), then
the mixture was centrifuged for 10 min at 100 g at room temperature. The
supernatant is platelet-rich plasma (PRP). Residual blood was subsequently
centrifuged at 1000 g for 30 min to obtain the platelet-poor plasma
(PPP). PRP was mixed with PPP to about 16.63 × 1012 platelets/L.
The platelet aggregation assay was performed in an aggregometer (Shanghai Biochemical
Apparatus Factory) at 37 with stirring (900 r/min). AsAPLA2 was dissolved
in PBS at pH 7.4 immediately before use, then incubated with PRP for 3 min.
Then the platelet aggregation of the mixture was stimulated by 1 μmol/ L ADP. The
inhibition of platelet aggregation was assessed by comparison with the maximal
aggregation induced by the control dose of ADP (1 μmol/ L). IC50 value was
determined from dose-effect curves. All experiments were performed in
triplicate.
Results
AsAPLA2 purification, N-terminal sequencing and
characterization
After DEAE-Sepharose CL-6B, Source S and Mono Q FPLC, a
homogeneous PLA2 preparation (asAPLA2) ascertained by SDS-PAGE was obtained (Fig. 1). Its N-terminal
10 amino acid residues were SLVQFETLIM, and molecular weight was estimated to
be around 19 kD by SDS-PAGE (Fig. 2). Its isoelectric point was about 3.5 (Fig.
3).
Fig. 1 Chromagraphy of asAPLA2 on Mono Q FPLC
The column (HR 5/5) was developed with 0.02 mol/L, pH 7.1 Tris-HCl buffer,
containing a liner gradient of increasing NaCl concentration from 0 to 0.5
mol/L. The fraction desired was indicated by arrow.
Fig. 2 The SDS-PAGE analysis of asAPLA2
1, asAPLA2; 2,
marker.
Fig. 3 The IEF analysis of asAPLA2
AsAPLA2 showed high enzymatic activity with PC as the substrate.
0.02 mol/L KOH was replenished into the reaction mixture to neutralize the
fatty acid released to maintain the reaction solution pH around 8.20, 5 μl every time. The
enzymatic activity of asAPLA2 on PC was determined to be 5.62 mmol/(L・min・mg ) after
three times repeating. Beside the enzymatic activity, the platelet aggregation
inhibiting activity of asAPLA2 was also assayed with 1 μmol/L ADP as the stimulator. The inhibition effect of
asAPLA2 on platelet
aggregation was assessed with pure 1 μmol/L ADP stimulated aggregation as the control. The IC50 value
determined from dose-effect curves, in which the dose of asAPLA2 consumed was
the abscissa and the platelet aggregation inhibition was the ordinate, was
determined to be 6 μmol/ L (Fig. 4 and Fig.5).
Fig. 4 Inhibition of ADP-induced platelet aggregation
Coagulation time was recorded three minutes after the protein preincubated
with the plasma. 1, control; 2, 6.96 mmol/L; 3, 9.94 mmol/L; 4, 34.8 mmol/L.
Fig. 5 Concentration dependence of ADP-induced platelet
aggregation inhibition
The IC50 value determined from
the curve is 6 μmol/L. All experiments
were performed three times.
Cloning and sequence determination of asAPLA2
Total RNA was extracted from the venom gland of Agkistrodon
shedaoensis Zhao according to the directions in the kit, and the isolated
total RNA was reversely transcribed with oligo(dT)18 primer. 35 cycles PCR
was conducted to amplify the asAPLA2 cDNA with the total cDNA as templates, Taq DNA
polymerase, and primer 1 and primer 2 [oligo(dT)18]. The PCR products were
identified
to be about 400 bp as expected by agarose gel
electrophoresis, then subcloned into the pMD-18T vector (Fig. 6). Plasmids
purified from positive clones were used for nucleotide sequencing (Fig. 7).
Both the molecular weight and the isoelectric point of asAPLA2 were
calculated by software according to the deduced amino acid sequence of asAPLA2, and
determined to be 13,649.36 and 4.393 respectively. The mature asAPLA2 covered an
open reading frame of 366 nucleotides, encoded 122 amino acid residues and
contained seven disulfide bonds. It is very similar to the APLA2 of Agkistrodon
halys Pallas, and their homology reaches 79%. APLA2 from Agkistrodon
halys
Pallas can also inhibit the platelet aggregation, so the
research on asAPLA2 would provide important information for further studying
structure-function relationship of APLA2 or other PLA2s.
Fig. 6 The 1% agarose gel analysis of the amplified
products with voltage at 10 V/cm in 0.5×TBE buffer
1, the amplified products; 2, DNA marker.
Fig. 7 The cDNA and deduced amino acid sequence of asAPLA2
Expression vector designing
PLA2 has seven disulfide bonds, so the correct disulfide bonds
formation should be considered in this experiment. The expression vector
pBLMVL2 contained temperatureregulated cIts857 gene has been
successfully used in the expression of several PLA2 genes from Agkistrodon
halys Pallas and Agkistrodon acutus in our previous works. However,
the expression products of this vector were all
in inclusion body forms and needed to be renaturated.
Consequently, we used a secretion expression vector pET- 40b(+) with a T7lac
promoter for high-level expression of peptide sequences, and the expression
product was fused with a DsbC・Tag of 236
amino acid. DsbC with di-sulfide bond isomerase activity may enhance the target
protein’s solubility and facilitate disulfide bond formation.
Typically, a DsbC fusion protein can be purified by His・Bind metal chelation chromatography since it carries six
successive His residues in its C-terminus.
Expression of asAPLA2
The gene asAPLA2 was inserted into the
expression plasmid to obtain pET-asAPLA2 , and then the
recombinant plasmid was transferred into E. coli strain BL21(DE3). The E.
coli culture containing pET-asAPLA2 expressed the protein
after being induced by IPTG as shown in SDS-PAGE analysis (Fig. 8). The
temperature of culturing can affect the output of active protein. In lower
culturing temperature, the protein synthesis was slower so that the protein
folding effiency increased. In our experiement, it was found that the growth at
37 caused the partial formation of inclusion bodies, while incubation at 28–30 ℃ led to soluble and active protein.
Fig. 8 The SDS-PAGE analysis of the expression of the asAPLA2 gene in E. coli
1, total proteins after induction; 2, total proteins before induction; 3,
marker. The arrow indicates the position of expressed recombinant protein.
The expressed asAPLA2 cross-reacted with
antibodies against native APLA2 in Western blot as expected (Fig.9). The expression
product was confirmed to be with platelet aggregation inhibiting activity by
the ADP-induced platelet aggregation test.
For comparison, native asAPLA2 and its expression
product were assayed under the same conditions. The expressed asAPLA2 showed high
enzymatic activity with PC as the substrate (Table 1).
Fig. 9 Western blot analysis of expression products in E.
coli
The expressed asAPLA2 cross-reacted with antibodies against native APLA2. The arrow indicates
the position of expressed recombinant protein.
Table 1 Relative enzymatic activity of asAPLA2
|
Sample |
Native asAPLA2 |
Expressed asAPLA2 |
|
Relative enzymatic |
100 |
80 |
Discussion
The structure-function researches on PLA2 were mainly
focused on the regions relating to the enzyme activity and the platelet aggregation
inhibition activity. The studies on APLA2 crystal structure [7]
and structure-function relationship [13] have shown that there is an aromatic
cluster on the surface of APLA2 molecule formed by Phe20, Trp21, Tyr113 and Trp119 with two
acidic residues Glu6 and Asp115 surrounding the cluster, and this structure may be of importance related
to the inhibition of platelet aggregation activity as the binding or reaction
site (Fig. 10). AsAPLA2 purified from Agkistrodon shedaoensis Zhao
containing the same region was also found to inhibit platelet aggregation,
which proved once again that this unusual structure may be of great importance
for platelet aggregation inhibition activity.
Fig. 10 The amino acid sequence of PLA2
1, asAPLA2 from
Agkistrodon shedaoensis Zhao; 2, APLA2 from Agkistrodon halys Pallas;
3, bovine pancreatic phospholipase A2. The amino acid residues are numbered
essentially according to the common numbering proposed by Renetseder et
al. [14].
Although the molecular weight of APLA2 is only about
14 kD, it has seven disulfide bonds. Therefore the expression system with high
efficiency should be chosen. PLA2s expressed in eukaryotes, such as yeast, may form proper
disulfide bonds, but the yield was low. In contrast, E. coli is a
high-level expression system for recombinant proteins.
Usually, the expression products formed inclusion body,
which could have certain PLA2 enzymatic activity after renaturation. pET-40b(+) is a
secretion vector with a powerful T7lac promoter and DsbC tag (236 amino
acid). DsbC with disulfide bond isomerase activity may facilitate disulfide
bond formation to obtain a proper folded structure, and the 6 His residues in
the tag could help thepurification, and improve the purity.
Agkistrodon shedaoensis Zhao only lives in
Dalian She Dao (Snake Island) of China. Previous researches mostly focused on
its form, taxonomy, distribution and habit. Few researches on its biochemical aspects
are performed. Our study on PLA2 from Agkistrodon shedaoensis Zhao could help to
provide more information on the structure-function relationship of PLA2s.
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Received: August 29, 2003 Accepted: October 31, 2003
The novel nucleotide sequence data have been submitted to the GenBank data
bank and are available under Accession No. 577565
# These
authors contributed equally to this work
* Corresponding author: Tel, 86-21-54921273; E-mail,[email protected]