Isolation and biochemical characterization of a gamma-type phospholipase A2 inhibitor from Macropisthodon rudis snake serum

Accepted Manuscript Isolation and biochemical characterization of a gamma-type phospholipase A2 inhibitor from Macropisthodon rudis snake serum Lipeng Zhong, Chunhong Huang PII:

S0041-0101(16)30274-4

DOI:

10.1016/j.toxicon.2016.09.011

Reference:

TOXCON 5461

To appear in:

Toxicon

Received Date: 21 March 2016 Revised Date:

15 July 2016

Accepted Date: 14 September 2016

Please cite this article as: Zhong, L., Huang, C., Isolation and biochemical characterization of a gammatype phospholipase A2 inhibitor from Macropisthodon rudis snake serum, Toxicon (2016), doi: 10.1016/ j.toxicon.2016.09.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Toxicon j o u r n a l h o m e p a g e : www.elsevier.com/locate/toxicon

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Isolation and biochemical characterization of a gamma-type phospholipase A2 inhibitor from Macropisthodon rudis snake serum Lipeng Zhong ∗a, Huang Chunhong b

a Department of clinical laboratory, The Fourth Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330003, P.R. China; [email protected] b

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Department of Biochemistry, College of Basic Medical Science, Nanchang University, Nanchang, Jiangxi 330006, P.R. China. [email protected].

ABSTRACT

A novel phospholipaseA2 (PLA2) inhibitory protein (PLI) was purified from the serum of Macropisthodon rudis, a non-

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venomous snake mainly found in southern China. The molecular mass of the purified PLI was 160 kDa as determined by Superdex 200HR; however, the PLI protein had only one subunit of 25.4 kDa as determined by 12% SDS-PAGE, indicating an oligomeric protein. PLI cDNA obtained by PCR from the liver of Macropisthodon rudis, revealed 549 bps coding for a mature protein of 183 amino acid residues. Based on an amino acid sequence alignment with venomous and non-venomous snakes, this inhibitor was determined to be in the γ type family of PLI. In vitro experiments showed that PLIγ inhibited enzymatic,

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antibacterial

2016 Elsevier Ltd. All rights reserved.

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Keywords: Agkistrodon acutus; snake venom phospholipaseA2; phospholipaseA2 inhibitors; Macropisthodon rudis; inflammation;

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inflammatory, and antibacterial activities of snake venom PLA2 isolated from Agkistrodon acutus.

——— ∗Correspondence: [email protected]; Tel.: +11-86-0791-87029477; Fax: +11-86-0791-87029477

2 1.

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Introduction

the three-finger motifs (Samy et al., 2015). PLI has co-evolved Phospholipase A2 (PLA2s, EC. 3.1.1.4) catalyzes the hydrolysis with SVPLA2 as a means of protecting the snake from its own of the 2-acyl ester bond of 3-sn-phospholipids, producing venom by neutralizing PLA2. Hence, research into endogenous arachidonic acid (AA) and lysophospholipid (Murakami et al., PLIs may yield drug targets for the treatment of venomous snakebites. dependent enzyme, and is a vital component of snake venom. In

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2015). Snake venom phospholipaseA2 (SVPLA2) is a calcium-

Macropisthodon rudis, known as the false viper, is widespread addition, SVPLA2 exhibits a wide variety of pharmacological

in southern China. In the present study, we successfully purified

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effects such as myotoxicity, cardiotoxicity, neurotoxicity and

a novel PLIγ from the serum of Macropisthodon rudis, which hemorrhage (Damico et al., 2008). There is currently no reliable

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showed a highly inhibitory effect on Agkistrodon acutus way to treat poisonous snakebites in the impoverished and

(Hundred-pace snake) PLA2 in vitro.

remote mountains area.

2. Materials and Methods

Since the first PLA2 inhibitor (PLI) was isolated from

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Trimeresurus flavivurudls serum (Kihara, 1976), there have been

2.1 Materials

Six species of snake (Elaphe carinata, Bungarus multicintus,

of snakes such as Viperidae and Elapidae (Ohkura et al., 1997;

Natrix annularis, Macropisthodon rudis, Naja naja atra, and

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an increasing number of endogenous PLIs identified in the sera

Sinonatrix annularis, n=3) were supplied by Huangshan snake

potential treatments for poisonous snakebites. To date, three

farm (Huangshan, Anhui Province, China). Snake venom PLA2

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Okumura et al., 1999). These proteins could be developed as

types PLI proteins have been isolated: PLIα, PLIβ and PLIγ.

(1 mg/ml) was purified previously from Agkistrodon acutus.

PLIα has a carbohydrate recognition domain (CRD) of Ca2+-

Blood was harvested from the snake tail vein in tubes containing

dependent (C-type) lectin (Estevao-Costa et al., 2016). PLIβ has

0.38% sodium citrate and then centrifuged at 1,000 × g for 20

homology with human leucine-rich alpha (2)-glycoprotein (LRG)

min at room temperature. CNBr-activated Sepharose 4B,

(Shirai et al., 2010). PLIγ is composed of highly conserved

Millipore and Superdex 200HR 10/30 column were purchased

structural units of two tandem cysteine repeats, characteristic of

from GE. Molecular markers and PCR SuperMix were purchased

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from Transgen. All other chemicals are of analytical grade (AR)

The molecular mass of the native inhibitor was as determined

using a Superdex 200HR 10/30 column, with Transferrin (Mr =

and purchased in China. 2.2 Screening of anti-PLA2 activity

81,000), Human Ig (Mr = 150,000), and Ferritin (Mr = 440,000) as molecular weight markers. The protein concentration of PLI

activity (Gimenes et al., 2014). This method uses an agarose gel

was 1.2 mg/ml using the Bradford method. The isoelectric point

containing yolk lecithin. When SVPLA2 (1 mg/ml) was added to

(pI) of PLI was estimated by IEF. The inhibitor was assessed by

the sampling well, the activity was measured by the color change

SDS-PAGE and reverse phase HPLC using a Hipore C18 column

resulting from the hydrolysis of the yolk lecithin. Each sampling

of 250 × 4.6mm (Bio-Rad) equilibrated with solvent A (5%

well contained 20 µl SVPLA2 and either 20 µl snake serum or 20

acetonitrile, 0.1% trifluoroacetic acid), and eluted with a

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µl saline. Reactions were performed at 37°C for 8 h.

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A transparent circle method was employed to measure SVPLA2

concentration gradient of solvent B (90% acetonitrile, 0.1% trifluoroacetic acid). The column was run at a flow rate of 1

2.3 Purification of PLI protein

ml/min and absorbance was monitored at 280 nm. After

CNBr-activated Sepharose 4B per the manufacturers protocol.

electrophoresis by native PAGE (20% gel) and SDS-PAGE (12%

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Agkistrodon acutus PLA2 (20 mg) was coupled to 5 ml of

gel), protein bands were visualized with Coomassie Blue R250.

(Buffer A) and poured in a 1.0 × 5.0 cm plastic column.

Western blot analysis of the purified protein was performed with

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The resin was equilibrated with 0.05 M phosphate buffer, pH 7.2

antibodies obtained from recombinant PLIγ from Sinonatrix

Buffer A and loaded on the column at a flow rate of 1.0 ml/min.

annulari (Le et al., 2015).

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Macropisthodon rudis plasma (15 ml) was diluted 1:1 with

After loading the serum and washing with buffer A, the bound

2.4 cDNA Cloning and Sequencing

protein was eluted with 0.05 M glycine-HCl buffer, pH 3.0

Total RNA was isolated from a Macropisthodon rudis liver

(Buffer B). The eluted protein was buffer-exchanged and

using an RNAprep pure Tissue Kit (TIANGEN, CHINA)

concentrated using an Amicon Ultra-15ml (3kDa) Centrifugal

according to the manufacturer’s instruction. cDNA was

Filter Unit with successive washings with 0.05 M phosphate

synthesized using EasyScript First-Strand cDNA Synthesis

buffer, pH 8.0.

SuperMix (TransGen Biotech, CHINA). To obtain cDNA

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Toxicon ACCEPTED MANUSCRIPT by the neighbor-joining algorithm. The degrees of confidence for

designed from a published PLIγ nucleotide sequence. A pair of

internal lineages in the phylogenetic tree were determined by the

degenerate primers was used for amplification. The forward

bootstrap confidence using Kimura’s method to compute a

primer is CRCTCATGTAMWTTTGTCACAA, where R, M and

distance matrix with 1000 replicates.

W denote A/G, A/C, A/T, respectively. The reverse primer is

2.6 Inhibitory activities to SVPLA2 in vitro

TTATTCAGAAGGTGTARTTTTGG, where R denotes A/G.

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encoding PLIγ, PCR was performed with oligonucleotides

The RAW 264.7 cell line was obtained from China Center for Type Culture Collection (CCTCC). Cells were cultured in

2.5 Amino acid sequence alignments and Phylogenetic Analysis

DMEM supplemented with 10% FBS, 100 µg/ml of streptomycin, and 100 units/ml of penicillin. The cells were maintained in 5%

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Multiple alignments were performed with Genedoc Software

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The expected length of amplified products is 550bps.

CO2 at 37°C. RAW 264.7 cells were incubated with different

obtained from Expasy were: I6PG79 (Sinonatrix annularis

concentrations of purified PLIγ from Macropisthodon rudis

PLIγA), P82144 (Agkistrodon blomhoffii siniticus PLIγA),

snake serum and SVPLA2 isolated from Agkistrodon acutus for

Q9PWI4 (E. quadrivirgata PLIγA), C0STK8 (E.climacophora

18 h. Enzyme activity was assayed by determining the

PLIγA), D9N4B6 (Trimeresurus elegans PLIγA), A8I4K9

concentration of AA, a reaction product, by ELISA kit

(Bothrops erythromelas PLIγA), Q7LZI1 (Naja kaouthia PLIγ),

(Elabscience

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(http://www.nrbsc.org/gfx/genedoc/). The sequences of PLIγ

Biotechnology

C),

per

the

manufacturer’s

instruction.

Q9PTC4 (Notechis ater), A8I4L6 (Bothrops jararaca), Q78CF8

Antimicrobial susceptibility was assayed using the disk diffusion

(Oxyuranus scutulatus), Q9PTC0 (Pseudonaja textilis), and

technique (K-B method). Staphylococcus aureus ATCC-259232

Q90358 (Crotalus durissus terrificus). Signal-peptides were

was supplied by the centers for disease control and prevention in

removed from all the sequences. We use maximum likelihood

China (CDC). S. aureus used in the disk diffusion was grown to

inference

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exponential phase at a concentration of 5 × 107 CFU/ml. Disks of

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6 mm diameters were made using saline as a negative control and

phylogenetic tree was built using the amino acid sequences and

SVPLA2/PLI mixtures with varied concentrations as the

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Q9I8P7 (Python reticulatus PLI), Q9PU34 (Notechis scutatus),

as

implemented

in

(http://www.megasoftware.net/mega4/mega.html).

multicintus ACCEPTED MANUSCRIPT

serum (E); Macropisthodon rudis

experimental group. For each treatment, three replicates were

serum (F); Sinonatrix annularis serum (G); Naja prepared. For inhibition studies, SVPLA2 (5 µg) was preincubated with or without different concentration of PLI (2.5, 5,

naja atra.serum. 3.2.

Purification

of

PLI

protein

from

the

serum

of

Macropisthodon rudis

37°C for 20 h.

PLI from the serum of Macropisthodon rudis was purified by

3. Results

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or 10 µg). Bacterial growth was determined by nutrient agar at

affinity chromatography using a CNBr-activated Sepharose 4B-

3.1. Inhibitory activity of different snakes’ serum to SVPLA2

conjugated with SVPLA2 (Figure 2A), and the purity of the

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The crude serum of six species of snakes (Elaphe carinata,

isolated protein was analyzed by RP-HPLC (Figure 2B). SDSBungarus multicintus, Natrix annularis, Macropisthodon rudis,

PAGE (12%) showed only a single band of 25.4 kDa, as

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Naja naja atra, and Sinonatrix annularis) were mixed with equal

analyzed by Quantity One software (Figure 2C). However, using

volume of SVPLA2 (1 mg/ml) to a total volume of 40µl. The

the Superdex 200HR 10/30 size exclusion column, the molecular

mixtures were added to each sampling well in the agarose gel

mass of PLI was determined to be 160 kDa, (Figure 4D). This

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made with yolk lecithin, and incubated for 8 h at 37°C. Of the six

indicates that PLI is an oligomeric protein likely composed of six

species tested, only serum from Macropisthodon rudis and

subunits of 25.4 kDa. PLI was determined to have a pI value of

Sinonatrix annularis showed significant inhibitory activity for

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SVPLA2 (Figure 1).

5.8, as estimated by IEF. Western blot analysis confirmed that PLI belongs to the γ-type phospholipase A2 inhibitor family (data not shown). Inhibition of PLA2 enzymatic activity was approximately 80% (Figure 2E) at the molar ratios of 1.2:1 (PLI: PLA2). Native (nondenaturing) PAGE analysis identified the formation of a PLI-

Figure 1. Screening serum inhibitors of SVPLA2 from the six species of snake. Each well contained 20 µl SVPLA2 and 20 µl of the following: (A); saline negative control (B); Elaphe carinata serum (C); Natrix annularis serum (D); Bungarus

PLA2 complex, indicating that PLI and PLA2 form a noncovalent interaction (Figure 2F).

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A

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E

F

Figure 2. Purification and SDS-PAGE of anti(PLI)

protein

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PLA2

Macropisthodon

from

rudis.

the

serum

(A)

of

Affinity

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chromatography of CNBr-activated Sepharose 4B conjugated with SVPLA2. (B) HPLC of PLI from

B

Macropisthodon rudis. (C) SDS-PAGE of PLI from Macropisthodon rudis. The molecular mass

calculated from the single subunit was 25.4 kDa.

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C

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(D) The molecular weight of PLI was determined

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by gel filtration on a Superdex 200HR 10/30 column. The elution coefficient (Kav) was determined by the equation: Kav = (Ve－Vo)/ (Vt－Vo), where Ve = elution volume, Vt = column volume, and Vo = void volume. (E) PLI was incubated at 37°C for 8 h with a fixed concentration of SVPLA2 at different molar ratios to PLI. Phospholipase activity was calculated by measuring transparent circle. (F) The formation of a non-covalent PLI-PLA2 complex was measured by native (non-denaturing) PAGE (20% gel). Lane 1, PLI; lane 2, PLA2 + PLI; lane 3, PLA2. 3.3 cDNA cloning and sequence analysis

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sequence alignment and the variability of their primary structure.

Macropisthodon rudis by RT-PCR. Amplification products were

Non-venomous snakes and venomous snakes were separated. The

of the expected size of 550 bps. The amino acid sequence was

phylogenetic tree analysis shows that PLIγ of Macropisthodon

aligned with the following proteins: Sinonatrix annularis PLIγ,

rudis was closely related to the PLIγ of Naja kaouthia (Figure

E.climacophora PLIγ (Shirai et al., 2009), E. quadrivirgata

3B). The similarity in PLI sequence between Macropisthodon

(Okumura et al., 1999), Agkistrodon blomhoffii siniticus PLIγA

rudis and Naja kaouthia is 74.4%.

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cDNA coding for PLIγ was isolated from the liver tissue of

al., 1989), Bothrops erythromelas PLIγA (Estevao-Costa et al.,

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2008), Naja kaouthia PLIγ (Doley and Mukherjee, 2003), Python

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(Ohkura et al., 1997), Trimeresurus elegans PLIγA (Kogaki et

reticulatus PLI (Thwin et al., 2002), Notechis scutatus PLI

(Hains et al., 2001), Notechis ater (Hains et al., 2000), Bothrops

jararaca (Estevao-Costa et al., 2008), Oxyuranus scutulatus, Pseudonaja textilis, and Crotalus durissus terrificus (Sekuloski

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A

S., 1999). The highest similarity to purified PLI based on amino

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acid sequence alignment was Notechis scutatus PLI (ID: Q9PU33), which was 79% similar. The similarity of PLI purified

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from Macropisthodon rudis with Sinonatrix annularis PLIγ and Python reticulatus PLI were 73.8% and 55.9% respectively B

(Figure 3A). PLI displays a potential site for N-glycosylation on the asparagine residues in the sequence

157

Asn-Arg-Thr159

Figure 3. Amino acid sequence alignment and phylogenetic tree. (A) Amino acid sequence was compared with other known PLI sequences using

(Signal-peptides were removed from all the sequences). The phylogenetic tree of PLIγ was constructed from the amino

Genedoc. The strictly conserved residues are highlighted

in

bold.

(B) The phylogenetic

relationship of PLIγ from Macropisthodon rudis acid sequences by a neighbor-joining method based on their and other known PLIγ, was analyzed using MEGA

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5.1. The phylogenetic tree represented ACCEPTED the nearest gene with respect to reference gene (Expasy ID:

SVPLA2 + 5 µg PLI; E: 5 µg SVPLA2 + 10 µg PLI.

Q7LZI1).

4. Discussion 3.4 Inhibition of SVPLA2 in vitro In screening the inhibitory activity of different snake serums In vitro, anti-PLA2 (PLI) protein from Macropisthodon rudis significantly inhibited the SVPLA2 activity as measured by the annularis

showed

decreasing amount of arachidonic acid (AA) produced in RAW

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to SVPLA2, the serum of Macropisthodon rudis and Sinonatrix the

greatest

inhibitory

capacity.

This work shows the successful purification and characterization 264.7 cells (Figure 4A). The antibacterial effects of SVPLA2

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of a novel PLIγ from the serum of Macropisthodon rudis. PLI were inhibited by PLI in a dose-dependent manner (Figure 4B).

shows an antagonistic effect of the pro-inflammation and

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antibacterial activities of SVPLA2, but the mechanism of PLISVPLA2 interaction is not clear. There is a strong correlation

between PLA2 hydrolysis and antibacterial activities (Nair et al.,

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2007; Samy et al., 2012; Sudharshan and Dhananjaya, 2015). In

(AA)

concentration

suggested using PLI as supplement to anti-venom serum therapy;

was

measured via ELISA in the supernatant of RAW 264.7 cells. Control (saline), SVPLA2 (20 µg), SVPLA2

SVPLA2 binds to a three-finger motif in PLIγ of other species

sequence was not found. In addition, numerous authors have

Figure 4. Inhibition of SVPLA2 in vitro. (A) acid

inhibited by PLI in a dose-dependent manner.

(Thwin et al., 2002), yet, conservation of this amino acid

B

Arachidonic

this research, we find that antibacterial effects of SVPLA2 can be

PLI (20 µg SVPLA2, 20 µg PLI) are

however, as an exogenous protein, there are concerns about immunogenicity.

Therefore,

further research

on PLI of

Macropisthodon rudis will be needed to determine ways to

shown. (B) Bactericidal activity of SVPLA2 against Staphylococcus aureus. A: saline; B: 5 µg

prevent or reduce potential adverse side effects in snakebites treatment.

Hains, P.G., Nield, B., Sekuloski, S., Dunn, R., Broady, K., 2001. ACCEPTED MANUSCRIPT

Acknowledgments

We are grateful for the support of the National Natural Science

Sequencing and two-dimensional structure prediction of a phospholipase A(2) inhibitor from the serum of the common tiger

Foundation of China (NO. 31460227), and Jiangxi Science and

snake (Notechis scutatus). Journal of molecular biology 312, 875-884.

Technology -Development Funds (NO. 20122BBG70091).

Hains, P.G., Sung, K.L., Tseng, A., Broady, K.W., 2000.

Author Contributions

Functional characteristics of a phospholipase A(2) inhibitor from

Lipeng Zhong devised the experiment and analyzed data.

Notechis ater serum. The Journal of biological chemistry 275, 983-991. Kihara, H., 1976. Studies on phospholipase A in Trimeresurus

manuscript.

flaoviridis venom. III. Purification and some properties of phospholipase

A

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Lipeng Zhong and Chunhong Huang contributed to writing the

inhibitor in

Habu

serum.

Journal

of

biochemistry 80, 341-349.

Conflicts of Interest

Kogaki, H., Inoue, S., Ikeda, K., Samejima, Y., Omori-Satoh, T.,

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Hamaguchi, K., 1989. Isolation and fundamental properties of a

The authors declare no conflict of interest.

phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis. Journal of biochemistry 106, 966-971.

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ACCEPTED MANUSCRIPT ► Isolation of a novel gamma-type PLA2 inhibitory protein from Macropisthodon rudis snake serum. ►The amino acid sequences of the cDNAs were analyzed with other related inhibitors by multiple sequence alignment. ►Phylogenetic trees were constructed to study their evolutionary relationship.

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►PLIγ neutralizes the enzymatic, inflammatory, and antibacterial activities of a PLA2 isolated from Agkistrodon acutus.

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The authors declare that they have no conflict of interest. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional

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and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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This article does not contain any studies with animals performed by any of the authors. Informed consent was obtained from all

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individual participants included in the study.