Investigation of the inhibitory potential of phospholipase A2 inhibitor gamma from Sinonatrix annularis to snake envenomation

Accepted Manuscript Investigation of the inhibitory potential of phospholipase A2 inhibitor gamma from Sinonatrix annularis to snake envenomation Shengwei Xiong, Yunyun Luo, Lipeng Zhong, Huixiang Xiao, Hong Pan, Keren Liao, Mengxue Yang, Chunhong Huang PII:

S0041-0101(17)30230-1

DOI:

10.1016/j.toxicon.2017.07.019

Reference:

TOXCON 5678

To appear in:

Toxicon

Received Date: 9 April 2017 Revised Date:

18 July 2017

Accepted Date: 20 July 2017

Please cite this article as: Xiong, S., Luo, Y., Zhong, L., Xiao, H., Pan, H., Liao, K., Yang, M., Huang, C., Investigation of the inhibitory potential of phospholipase A2 inhibitor gamma from Sinonatrix annularis to snake envenomation, Toxicon (2017), doi: 10.1016/j.toxicon.2017.07.019. 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.

ACCEPTED MANUSCRIPT

Investigation of the inhibitory potential of phospholipase A2 inhibitor

2

gamma from Sinonatrix annularis to snake envenomation

3

Abstract: SaPLIγ is a novel gamma phospholipase A2 inhibitor (PLI) recently

4

isolated from Sinonatrix annularis, a Chinese endemic non-venomous snake. To

5

explore the neutralization effects of saPLIγ in snakebite envenomation, a dose

6

equivalent to LD50 of Deinagkistrodon acutus, Agkistrodon halys and Naja atra

7

venom with/without saPLIγ was inoculated into the gastrocnemius muscle of female

8

Kunming mice. The ability of saPLIγ to inhibit myonecrosis and systemic toxicity

9

were evaluated through investigations of muscle histopathology, and determination of

10

the serum levels of creatine kinase (CK), lactate dehydrogenase isoenzyme1 (LDH1)

11

and aspartate transferase (AST). Edema of the gastrocnemius muscle was evaluated

12

by calculating the width difference between the inoculated limb and the contralateral

13

leg. Desmin loss in the gastrocnemius muscle was determined by Western blot

14

analysis. Co-immunoprecipitation and shotgun LC-MS/MS analyses were performed

15

to identify venom proteins that interact with saPLIγ. All the envenomed mice had

16

significantly elevated serum CK, LDH1 and AST levels, whereas the levels were

17

decreased significantly in the presence of saPLIγ. Histopathological evaluation of

18

gastrocnemius muscle sections showed severe snake venom-induced damage,

19

characterized by leukocyte infiltration and erythrocyte leakage, leading to local edema.

20

Myonecrosis, hemorrhage and desmin loss were significantly attenuated by saPLIγ.

21

SaPLIγ interacted with a wide range of venom proteins, including PLA2s,

22

metalloproteinases and C type lectins, which may contribute to broad anti-venom

23

effects.

24

Keywords: Sinonatrix annularis; PLIγ; Myonecrosis; Anti-venom

25

1. Introduction

SC

M AN U

TE D

EP

AC C

26

RI PT

1

Snakebite envenomation is a brachychronic poisoning event and a common and

27

devastating environmental problem, which constitutes a public health hazard in many

28

regions around the world, particularly in the tropics and rural areas(Warrell, 2010).

29

This issue was underestimated until snake envenomation was added to the World

30

Health Organization’s list of neglected tropical diseases in 2009(Williams et al., 1

ACCEPTED MANUSCRIPT 2010). Anti-snakebite therapy has deteriorated, with rural Africa experiencing a

32

snakebite crisis since the termination of production of Fav-Afrique, which is the most

33

effective anti-venom agent against Africa’s vipers, mambas and cobras(Chippaux et

34

al., 2015). Fortunately, investigations of snakebite epidemiology and the problems

35

associated with envenomation treatment have been reported recently in a number of

36

prestigious international journals, such as Nature(Schiermeier, 2015), The

37

Lancet(Rägo et al., 2015), The British Medical Journal(Williams, 2015), and PLoS

38

One(Feitosa et al., 2015), increasing the focus on research to identify novel attention

39

on anti-snake venom drugs.

RI PT

31

In addition to high rates of mortality, snakebite can result in megalgia and

41

permanent sequelae, which represent a significant challenge to anti-venom therapy.

42

The clinical symptoms of snakebite envenomation are characterized by local effects

43

(edema, hemorrhage, and myonecrosis) and systemic effects (cardiovascular

44

alterations, coagulopathy, and acute kidney injury)(Gutiérrez et al., 2009; Marsh et al.,

45

1997; Peichoto et al., 2004; Pinho et al., 2008). Myonecrosis and hemorrhage are

46

among the most devastating consequences of snakebite. Specifically, dramatic

47

myonecrosis develops in envenomation inflicted by snakes of the Viperidae and

48

Crotalinae families, the venoms of which are rich sources of myotoxic phospholipase

49

A2 enzymes (PLA2s) (Aziz et al., 2016; Denegri et al., 2016; Huancahuire-Vega et al.,

50

2014).

M AN U

TE D

PLA2s (EC.3.1.1.4) specifically catalyze the Ca2+-dependent hydrolysis of the

EP

51

SC

40

sn-2 fatty acids of phospholipids, releasing arachidonic acid (AA) and

53

lysophospholipids. Interestingly, PLA2s are abundant and critical components of the

54

venom of members of the Hydrophiinae, Elapinae, Crotalinae and Viperinae snake

55

families, while these molecules are rare in the venom of low- and non-venomous

56

members of the Colubridae family (Mackessy, 2010). Snake venom PLA2s (svPLA2s)

57

exhibit an amazing variety of toxicologic and pharmacological effects, which cover

58

pre-/post-synaptic neurotoxicity, myotoxicity, hemorrhagic toxicity, cardiotoxicity,

59

inhibition of platelet aggregation and coagulation, as well as the induction of edema,

60

hemolysis and convulsions(Gutiérrez et al., 2013; Pungercar et al., 2007; Santos-Filho

61

et al., 2008). Local and systemic myonecrosis, which are common consequences of

62

snakebite envenomation, are caused largely by the myotoxicity of svPLA2(Gutiérrez

AC C

52

2

ACCEPTED MANUSCRIPT 63

et al., 2003). Envenomation is also associated with the destructive effects of systemic

64

toxicity, such as cardiac and hepatic damage. To protect against auto-intoxication, some species have evolved natural

66

inhibitors present in the circulatory system, including phospholipase A2 inhibitors

67

(PLI), which are among the best characterized(Ohkura et al., 1997). To date, three

68

types of endogenous PLIs have been identified: PLIα, PLIβ and PLIγ, which has been

69

identified as a potential anti-venom. RBaltMIP, a PLIγ isolated from Bothrops

70

alternatus serum, effectively restrained the myotoxicity of Asp49PLA2 (BthTX-II and

71

PrTX-III) and Lys49 PLA2(BthTX-I and PrTX-I) from autologous venom(Santosfilho

72

et al., 2014). Similarly, a protein isolated from P. flavoviridis serum that mediated

73

total suppression of myonecrosis and hemorrhage caused by its own venom was

74

identified as a PLI(Chijiwa et al., 2013). Hence, accumulating evidence indicates that

75

PLI can neutralize the effects of snake venom myotoxicity, thus reducing the damage

76

caused by envenomation.

SC

M AN U

77

RI PT

65

SaPLIγ is a novel PLIγ that we previously isolated from a Chinese endemic non-venous snake, Sinonatrix annularis. Unlike the previously reported PLIs, saPLIγ

79

was shown to be effective in blocking the hemorrhagic toxicities of D. acutus, N. atra

80

and A. halys venom(Le et al., 2015). This endogenous protein is vital for the survival

81

of Chinese ringed water snake species, and represents a potential candidate for the

82

design of a broad range of novel anti-venom drugs. In this study, the inhibitory effects

83

of saPLIγ against the myotoxicity mediated by the three different snake venoms were

84

examined. In addition, we developed a monoclonal antibody against PLIγ based on a

85

saPLIγ epitope, which enabled us to investigate the interactions between PLIγs and

86

svPLA2s and/or other toxic components.

EP

AC C

87

TE D

78

88

2. Materials and methods

89

2.1 Reagents and materials

90

Powdered venom from D. acutus, A. halys and N. atra was purchased from

91

Huangshan snake farm (Huangshan, Anhui Province, China). PLIγ (SaPLIγ) was

92

isolated from Sinonatrix annularis serum by sequential Source Q (GE healthcare) and 3

ACCEPTED MANUSCRIPT UnoQ (Bio-Rad) ion-exchange chromatography as described previously(Chen et al.,

94

2011). Polyacrylamide gel reagents, Clarity™ ECL Western Blotting Substrate

95

(Bio-Rad, Hercules, CA, USA) and assay kits for creatine kinase (CK)(A032), lactate

96

dehydrogenase isoenzyme1 (LDH1)(A020-3) and aspartate transferase (AST)

97

(C010-1) were products of Nanjing Jiancheng Bioengineering Institute (Nanjing,

98

China). sPLA2 IB polyclonal antibody (ab103872), sPLA2II-A monoclonal antibody

99

(mAb; ab24498), anti-desmin mAb(ab32362) were Abcam products (UK). GAPDH

100

antibodies were purchased from Sigma (UK). Anti-PLIγ mAb was raised in mice in

101

our laboratory using KLH conjugated-epitope peptide

102

(CPVLRLSNRTHEANRNDLIKVA) derived from SaPLIγ as the antigen. The PLIγ

103

mAb was used to screen novel PLIγs in the sera of 12 snake species (both venomous

104

and non-venomous). Mouse IgG was purchased from Boster Biotech (Wuhan, China).

105

Protein G Sepharose 4 Fast Flow (GE17-0618-01) was from GE Healthcare. The mass

106

spectrometer (Orbitrap Fusion) and liquid chromatography (EASY-nLCTM 1000)

107

systems were products of Thermo Scientific.

108

2.2 Biological model

SC

M AN U

Female Kunming (KM) mice (20 ± 5 g) were supplied by the Animal Center of

TE D

109

RI PT

93

110

Nanchang University. Mice were acclimatized for one week under standard laboratory

111

conditions of chow, water, and light.

All the mice were randomly divided into seven groups (n = 3 per group). The

113

negative control group was injected with normal saline. Three groups were inoculated

114

with LD50 doses of D. acutus (40 µg), A. halys (20 µg) or Naja atra (14 µg) venom,

115

according to the method described by Liu et al.(Liu et al., 2007). The final three

116

groups were inoculated with the corresponding venoms pre-incubated with 40 µg

117

saPLIγ at 37ºC for 10 min [the dose of saPLIγ was modified based on our previous

118

report (Le et al., 2015)]. All the mixtures were injected into the central gastrocnemius

119

muscle of the left hind limb (50 µL per mouse).

120

2.3 Local and systemic toxic reactions

121 122

AC C

EP

112

The physical state and behavior of mice were observed and recorded at 1, 3, and 6 h after inoculation. Mice were sacrificed at 6 h by CO2 inhalation and blood was 4

ACCEPTED MANUSCRIPT 123

collected by cardiac puncture into heparin-coated tubes. Samples were centrifuged at

124

5,000 ×g for 10 min at 4°C to separate the plasma. Local myonecrosis and systemic

125

toxic reactions were assessed by determination of serum levels creatine kinase (CK),

126

lactate dehydrogenase-1(LDH1) and aspartate transferase (AST) using appropriate

127

assay kits for comparison with the control group. Both the hind legs of each mouse were dissociated at the articulatio coxae and the

RI PT

128

gastrocnemius muscles were exposed by careful dissection of surrounding muscles,

130

such as the rectus femoris, semitendinosus and adductor medial muscles. The width of

131

the gastrocnemius muscle was measured using a Vernier caliper. Local edema

132

(∆width) was estimated based in the difference between the widths of the

133

gastrocnemius muscles of the venom-inoculated limb and that of the contralateral

134

muscle.

135

2.4 Histopathological analysis

M AN U

136

SC

129

After obtaining photographs, the central part of the venom inoculated gastrocnemius was removed and fixed in 10% formaldehyde for at least 24 h at 4ºC.

138

The materials were dehydrated using a graded ethanol series, immersed in xylene, and

139

then embedded in paraffin using conventional protocols(Ramosvara, 2005). Sections

140

(5 µm thick) were prepared using a microtome (Leica RM2235, German), placed on

141

glass slides, deparaffinized in xylene and ethanol, and hydrated in distilled water.

142

Sections were then processed for histological analysis by staining with

143

hematoxylin-eosin prior to examination under a light microscope. The images of

144

sections were captured using an Olympus microscope system (Japan). Muscle damage

145

was evaluated based on the integrity of muscle fibers and the numbers of infiltrating

146

leukocytes and erythrocytes.

147

2.5 Desmin loss in envenomed gastrocnemius muscle

148

The envenomed gastrocnemius muscle was homogenized in RIPA lysis buffer. The

149

supernatant was collected after centrifugation at 10,000 rpm, and the total protein

150

concentration was quantified using the BCA method. Proteins (40 µg/well) were

151

separated by 15% SDS-PAGE and transferred to PVDF membranes according to

152

standard western blot protocols. After blocking, the membranes were incubated with

AC C

EP

TE D

137

5

ACCEPTED MANUSCRIPT rabbit anti-desmin (dilution 1:10,000) and anti-GAPDH primary antibody (1:5,000)

154

overnight at 4ºC. The membranes were then incubated with IgG- radish peroxidase

155

(HRP) conjugated secondary antibody for 1 h at room temperature. Detection was

156

performed with Clarity™ ECL Western Blotting Substrate (Bio-Rad, Hercules, CA,

157

USA). The band intensity was quantified by Image J software (National Institutes of

158

Health), and relative expression (desmin/ GAPDH) was calculated.

159

2.6 Interaction of saPLIγ with snake venom PLA2s

160

Co-immunoprecipitation methods were adapted to explore the interaction of saPLIγ

161

with snake venom PLA2s. Briefly, 2 µL anti-PLIγ monoclonal antibody (mAb) or

162

common mice IgG was mixed with 50 µL of protein G resin in a 1.5 mL EP tube and

163

incubated at 4°C overnight. The supernatant was removed by centrifugation at 500 ×g

164

for 5 min. The sample was prepared by mixing 0.1 mg saPLIγ with 200 µL (1 mg/mL)

165

D. acutus, N. atra or A. halys venom followed by incubation with the “mAb-protein G

166

resin pellets” for 12 h. The new “sPLA2- saPLIγ-mAb-resin” pellets were obtained by

167

centrifugation 5 min at 500 ×g and washed three times using RIPA lysis buffer. The

168

co-immunoprecipitates were suspended in 1× protein loading buffer and boiled for 5

169

min before separation by 12% SDS-PAGE. Electrotransfer to PVDF membranes was

170

carried out at 100 V for 2 h. After blocking in 5% non-fat milk solution for 1 h, the

171

membranes were immune-detected using anti-PLA2 IB (dilution 1:500), sPLA2 IIA

172

(dilution 1:10,000) and anti-saPLIγ (1:1,000)antibodies. Positive controls (venom

173

input) were prepared by mixing 100 µg venom of D. acutus, N. atra and A. halys

174

venom with the corresponding co-immunoprecipitate, and detection using anti-PLA2

175

IB (dilution 1:500), sPLA2 IIA (dilution 1:10,000) antibodies after separation by

176

SDS-PAGE on the same gel.

177

2.7 Shotgun LC-MS/MS of immunoprecipitated venom proteins

178

To identify the proteins that interact with saPLIγ, the coimmunoprecipitate (coIP) of

179

snake venom (D. acutus) was further investigated by shotgun LC-MS/MS. Negative

180

control was simultaneously performed using common mice IgG, venom proteins that

181

pull-downed by mice IgG were excluded from the protein pool immunoprecipitated

182

by saPLIγ. Protein digestion was performed according to the FASP procedure

183

described by Wisniewski and colleagues (Wisniewski et al., 2009). After digestion

AC C

EP

TE D

M AN U

SC

RI PT

153

6

ACCEPTED MANUSCRIPT 184

with 5 µg trypsin (ultra-pure, Promega) at 37ºC for 18 h, the peptide supernatant was

185

obtained by centrifugation and lyophilized using Concentrater plus (Eppendorf).

186

Peptides were separated by reversed-phase high performance liquid chromatography. Peptides were reconstituted in buffer A (HPLC-grade water with

188

0.1% formic acid ) and loaded onto on an analytical column (75 µm × 25 cm, 5 µm,

189

100 Å, C18) and eluted with a gradient of 5%–28% buffer B (0.1% formic acid in

190

acetonitrile) from 0–40 min and 28%–90% buffer B from 40–42 min, after which

191

buffer B was reset to 90%. Peptide fractions were coupled to the input of the mass

192

spectrometer for MS/MS determination. MS data were acquired dynamically choosing

193

the most abundant precursor ions from the survey scan (375–1500 m/z ) for high

194

confidence fragmentation. The target value was determined based on predictive

195

Automatic Gain Control (pAGC), with dynamic exclusion duration of 40 s. Survey

196

scans were acquired at a resolution of 120,000 at m/z 200 and resolution for HCD

197

spectra was set to 50,000 at m/z 200. The acquired MS/MS spectra were searched

198

using the MASCOT search engine (Matrix Science, London, UK; version 2.3)

199

against a combined protein database including the NCBI sequences for D. acutus

200

venom proteins (DA. NCBI 267.fasta, 267 sequences) (Supplemental dataset).

201

Proteins were identified using the following criteria: Peptide mass tolerance, 20 ppm;

202

MS/MS tolerance, 0.1 Da; enzyme, trypsin; missed cleavage, 2. Fixed modifications

203

were as follows: carbamidomethyl (C), variable modification: oxidation (M), false

204

discovery rate (FDR) <0.01 at the peptide and protein level.

205

2.8 Statistical analysis

SC

M AN U

TE D

EP

SPSS software (version 19.0) for Windows was used for statistical analysis. The level of

AC C

206

RI PT

187

207

significance was set at P < 0.05. If P < 0.01 and P < 0.001 was considered to be statistically

208

highly and super significant, respectively.

209

3. Results

210

3.1 Physiological and behavioral signs

211

The mice treated with saline displayed normal behavior in terms of drinking, eating

212

and movement, while the envenomed mice exhibited a variety of signs. Common

213

reactions included torpid movement, head-drooping, protopsis and hematose signs; 7

ACCEPTED MANUSCRIPT some were venom-related, such as somnolence in the N. atra and A. halys groups,

215

subdued behavior in the N. atra and D. acutus groups, hunching (A. halys), and

216

weakening and paralysis in the A. halys and D. acutus groups. Possibly due to the pain

217

caused by the venom, the mice persistently licked the site of envenomation. Unlike

218

the protopsis and hematose signs in envenomed mice, the saPLIγ plus group were

219

exhibited normal, active behavior, except one mouse in the saPLIγ plus N. atra group,

220

which exhibited head-drooping (Figure 1B, left). In addition, one of N. atra venom

221

injected mice died after approximately 4 h, while all the saPLIγ plus mice survived to

222

6 h.

TE D

M AN U

SC

RI PT

214

223 224

Fig. 1. Envenomation signs in mice with/without saPLIγ. A. Control group treated with saline was healthy; B. Mice inoculated with N. atra

226

venom (left, with saPLIγ); C. Mice inoculated with A. halys venom (left, with saPLIγ);

227

D. Mice treated with D. acutus venom (left with saPLIγ). All the mice survived to 6 h

228

except one mouse in the N. atra group, which died after approximately 4 h. Circles

229

indicate mice exhibited head-drooping and arrows indicate protopsis and hematose

230

signs.

231

3.2 Hemorrhage and edema of the gastrocnemius muscle

232

Severe hemorrhage was observed in mice injected with D. acutus and A. halys

233

venoms. The gastrocnemius and surrounding muscles exhibited necrosis, swelling, red

234

or dark red in color, and erosion of the muscular fasciae (Figure 2A). The

AC C

EP

225

8

ACCEPTED MANUSCRIPT corresponding saPLIγ plus mice were nearly normal in color and size of the

236

gastrocnemius when compared to the non-injected leg (right limb of the same

237

individual) and/or the saline group. Hemorrhaging was less marked in the mice

238

inoculated with neurotoxic N. atra venom, although obvious swelling was also

239

observed in the injected gastrocnemius. The edema index was significantly higher in

240

all three of the venom groups compared with that in the corresponding saPLIγ plus

241

groups (Figure 2C). These results indicated that saPLIγ was effective in attenuating

242

the hemorrhagic and edematous toxicities of the different types of snake venom.

243

TE D

M AN U

SC

RI PT

235

Fig. 2. Hemorrhage and edema in the gastrocnemius muscle. A. Gastrocnemius

245

muscles injected with D. acutus and A. halys venom exhibited necrosis, swelling, red

246

or dark red in color, and erosion of the muscular fasciae (indicated by the arrow). The

247

muscles in the N. atra venom group were less hemorrhagic but similarly edematous.

248

All the mice in the saPLIγ plus groups appeared normal, both in color and size. B.

249

Control mice injected with an equivalent volume of saline; C. Suppressive effect of

250

saPLIγ on edema induced in the gastrocnemius muscles of mice inoculated with snake

251

venom. ∆ mm represents the difference in the width of the left limb gastrocnemius

252

compared to the contralateral muscle. Data represent means ± SD (n = 3). *P < 0.05,

253

**P < 0.01, ***P < 0.001.

254

3.3 Histopathological analysis

AC C

EP

244

9

ACCEPTED MANUSCRIPT Histopathological damage to the gastrocnemius muscle fibers were evaluated by

256

hematoxylin and eosin (HE) staining of tissue sections. Evaluation of lateral and

257

longitudinal sections of tissue samples from the control group revealed normal

258

histological architecture, including intact fascicles, clear perimysium and

259

endomysium (Figure 3 A1 and A2). The gastrocnemius muscle fibers subjected to N.

260

atra venom were severely damaged, devoid of structural organization, and exhibited

261

characteristic features such as amorphous cytoplasm containing clumps of contractile

262

myofilaments. There was also significant neutrophil infiltration of the muscle fibers,

263

indicating the existence of inflammation (Fig. 3 B1). The myocytes, perimysium and

264

endomysium in the gastrocnemius muscle of mice inoculated with A. halys venom

265

were swollen and amorphous. The damaged muscle fibers consisted of little more

266

than occasional clumps of cellular debris, erythrocytes and inflammatory infiltration

267

by neutrophils (Fig. 3 C1). Inoculation with D. acutus venom caused even greater

268

hemorrhaging, and severe myonecrosis. The architectural organization of the

269

myofibers was completely lost, with massive erythrocyte infiltration (Fig. 3 D1).

270

Although the necrosis was severe, all these signs of damage were inhibited by the

271

addition of SaPLIγ. As shown in Figure 3 (B2–D2), the gastrocnemius muscle

272

microstructure in all three of the groups was visually normal. The myocytes and

273

endomysium were almost intact with only occasional myonecrosis, and occurrence of

274

hemorrhage and neutrophil infiltration.

AC C

EP

TE D

M AN U

SC

RI PT

255

275

10

SC

RI PT

ACCEPTED MANUSCRIPT

276

Fig 3. Microscopic architecture of the gastrocnemius muscles subjected to snake

278

venom. The mice were intramuscularly injected with A: saline, B: N. atra venom, C:

279

A. halys venom, D: D. acutus venom, with synchronous saPLIγ inoculation in the

280

saPLIγ plus groups. Muscle sections were examined at the magnification of 200×.

281

Venom-injected muscle was architecturally amorphous, containing damaged

282

myocytes and cellular debris, as well as characteristic features erythrocyte and

283

neutrophil infiltration. Abbreviations: Er, erythrocyte; Nc, necrosis; Ne, neutrophil.

284

3.4 Biochemical assays of serous CK, LDH1 and AST

285

The damage to skeletal muscle induced by snake venom inoculation was evaluated by

286

determination of serous CK activity. The basal level of CK was approximately 180

287

U/L, whereas the levels increased sharply (6.5–17.7 fold) in the snake venom

288

inoculated groups. In the presence of saPLIγ, the serum CK values were significantly

289

decreased (Fig 4.A). These data were consistent with the suppression of muscle tissue

290

degeneration and necrosis observed in the histological analysis.

TE D

EP

AC C

291

M AN U

277

Serum AST and LDH1 were also assayed for the assessment of systemic

292

toxicities of the three venoms in the viscera, with particular reference to damage to

293

liver and heart. As shown in Figure 4B and C, the AST levels in the N. atra and A.

294

halys groups were increased significantly (P < 0.05 and P < 0.01, respectively)

295

compared with those in the control groups. In addition, highly significant increases in

296

the AST activity in the D. acutus group (P = 0.057), indicated a high level of damage 11

ACCEPTED MANUSCRIPT to the liver. The LDH1 was also remarkably elevated in the snake venom inoculated

298

groups, especially in the D. acutus and N. atra venom groups. Thus, local

299

envenomation in the muscle induced a systemic toxic response, for example

300

cardiotoxicity. Both the AST and LDH1 levels were attenuated remarkably by the

301

addition of saPLIγ (Fig. 4 B, C). In conclusion, these observations indicated that

302

saPLIγ effectively reduced the toxicity of various types of snake venom.

M AN U

SC

RI PT

297

304

EP

TE D

303

Fig. 4. Alterations in serum CK, LDH1 and AST levels following snake

306

envenomation. Serum CK, LDH1 and AST levels were all increased dramatically in

307

the venom groups versus the controls. However, this effect was significantly relieved

308

by the addition of saPLIγ. * Venom vs. saPLIγ plus; *P < 0.05, **P < 0.01, ***P <

309

0.001. # Venom group vs. control

310

3.5 Desmin loss in envenomed gastrocnemius muscle

AC C

305

311

Desmin is a vital protein responsible for regulation of sarcomere architecture in

312

muscle. Desmin was degraded significantly in all the venom treated groups, with 12

ACCEPTED MANUSCRIPT higher loss in the D. acutus and A. halys groups (predominantly hemorrhagic) than in

314

the N. atra group (predominantly neurotoxic). SaPLIγ showed significant protective

315

effects against desmin degradation, especially in the N.atra and D. acutus venom

316

groups (P < 0.05) (Fig. 5).

M AN U

SC

RI PT

313

317

Fig.5. Desmin loss in gastrocnemius muscle exposed to different venoms. Desmin

319

was partially degraded in envenomed gastrocnemius muscles. Nevertheless, the

320

degradation was significantly relieved in the presence of saPLIγ. # Venom group vs.

321

control; *Venom group with vs. without saPLIγ.

322

3.6 Interaction of saPLIγ and venom sPLA2s

323

The interactions of saPLIγ with the three types of venom were evaluated by coIP and

324

verified by Western blot analysis. As shown in Figure 6, saPLIγ showed positive

325

binding to venom PLA2s of D. acutus, A. halys and N. atra, and these svPLA2s were

326

identified as IB/IIA subtypes.

AC C

EP

TE D

318

13

SC

RI PT

ACCEPTED MANUSCRIPT

327

Fig 6. Interaction of saPLIγ with snake venom PLA2s. IP represents

329

co-immunoprecipitates pulled down by common mouse IgG or saPLIγ mAb. IB

330

represents the immuno-binding of anti-PLA2 or anti-saPLIγ antibody with IP products.

331

Venom input comprised a mixture of D. acutus, A. halys or N. atra venom with their

332

corresponding IP products. A: Interaction of saPLIγ with IB type PLA2 of D. acutus,

333

A. halys or N. atra venom; B: Interaction of saPLIγ with IIA type PLA2 of the three

334

venoms.

335

3.7 MS/MS identification of venom proteins in coIP products

336

Using shotgun LC-MS/MS, the coIP complex of saPLIγ and D. acutus venom was

337

analyzed and identified by searching for D. acutus venom protein sequences in the

338

NCBI database. Interestingly, in addition to the expected svPLA2s (Lys49 subtype,

339

basic), the most targeted proteins were metalloproteinases (accession numbers

340

7340956, 283825330 and 82221933). The rest were snake C type lectins (agglucetin)

341

(Table 1). Searches of an extended database of Agkistrodon genus proteins identified

342

four more svPLA2s (1041577407, 46015749, 1160578115 and 1041577421, basic)

343

from A. piscivorus and A. Contortrix, respectively.

344

Table 1.

345

4. Discussion

AC C

EP

TE D

M AN U

328

14

ACCEPTED MANUSCRIPT PLIγs are known to mediate inhibitory effects on all classes (I, II and III) of venom

347

PLA2s(Lizano et al.,2003), exerting broad anti-venom effects. Our previous study

348

revealed that saPLIγ was an effective inhibitor of the hemorrhagic activity of D.

349

acutus, and A. halys venoms(Le et al., 2015). As a potent inhibitor of svPLA2, saPLIγ

350

exerted strong suppression of the myonecrosis induced by N. atra, A. halys and D.

351

acutus venoms, leading a significant decrease in serum CK activity. Features of

352

severe membrane destabilization were observed, including erosion of the muscular

353

fasciae in D. acutus and A. halys venom inoculated mice (Figure 2A) as well as

354

impaired perimysium and endomysium in the gastrocnemius muscles of all the three

355

venom groups (architectural amorphous myofibers, Figure 3 B1–D1). Skeletal muscle

356

necrosis is among one of the most dramatic consequences of envenomation and the

357

endogenous resistance conferred by proteins (saPLIγ) to myotoxins is an evolutionary

358

benefit to the survival of S. annularis. Furthermore, saPLIγ mediated significant

359

inhibition of the increase in serum AST and LDH1 levels caused by envenomation in

360

all three venom groups. Generally, increases in AST and LDH1 levels are

361

biochemical indicators of hepatic and cardiac injury, which are common in snakebite

362

victims(Rahmy et al., 2000; Marsh et al., 1997). These findings indicate that saPLIγ is

363

also effective in preventing systemic damage to the heart and liver.

SC

M AN U

TE D

364

RI PT

346

Desmin is a vital muscle-specific, type II intermediate filament that integrates the sarcolemma, Z-disk, and nuclear membrane in sarcomeres. It links the myofibrils

366

laterally by connecting the Z-disks. Through its connection to the sarcomere, desmin

367

links the contractile apparatus to the cell nucleus, mitochondria, and post-synaptic

368

areas of motor endplates(Asad et al., 2014). Harris et al. reported degradation of 50%

369

of the desmin and titin content of skeletal muscles in the early stages of degeneration

370

caused by Notechis scutatus venom(Harris et al., 2003). Similar breakdown of desmin

371

was also observed in our study, with 22%, 39% and 42% loss caused by inoculation

372

with N. atra, A. halys and D.acutus venoms, respectively. Despite attempts to detect

373

titin loss by western blot analysis, we failed due to the extremely large size of this

374

molecule. Nevertheless, the relaxed myofibers in the gastrocnemius and paralysis of

375

the venom inoculated limb clearly indicated muscle contraction dysfunction.

376 377

AC C

EP

365

In this study, we investigated the interactions of saPLIγ with three different types of snake venom. We revealed that saPLIγ binds with IB and IIA PLA2s of the three 15

ACCEPTED MANUSCRIPT venoms. However, the existence of IB PLA2 in Viperid and IIA subtype in Elapid

379

venoms that showed by western blot is opposite to general knowledge of svPLA2s’

380

distribution. This was possibly resulted from the low specifity of IB and IIA

381

antibodies which direct against mammal sPLA2s. What’s more, the antigen for PLA2

382

IB antibody generation was C terminal amino acids 110-140 of human PLA2g1b, a

383

peptide with high identity (70-80%) to a variety of snake species, including Viperidae

384

and Elapidae. Interestingly, the binding targets in these snake venoms were not

385

limited to PLA2s, but also zinc metalloproteinases, such as aculysin 1 and SVMP MD,

386

and snake C type lectins (agglucetin). In other words, saPLIγ is involved in the

387

interaction with three venom protein families, thus exerting a variety of anti-venom

388

effects. However, the mechanism by which these components bind remains obscure,

389

although PLA2 was shown to exert synergistic effects with SVMPs in damaging

390

skeletal muscle (C2C12) cells (Bustillo et al., 2012) and detaching endothelial cells

391

(Bustillo et al., 2015).

SC

M AN U

392

RI PT

378

The induction of myonecrosis by svPLA2, either by catalytic (Asp49-PLA2) or non-catalytic (Lys49-PLA2) mechanisms, is characterized by disruption of the

394

sarcolemma membrane integrity(Asad et al., 2014; Lomonte et al., 2011). The soleus

395

muscle (weighing 60–100 mg) of an adult rat can be entirely destroyed by as little as

396

2.0 µg of the myotoxic phospholipases A2 notexin or notechis (Harris et al., 1978).

397

As a svPLA2 inhibitor, saPLIγ was found to be effective in preventing skeletal

TE D

393

muscle damage initiated by hemorrhagic venom (D. acutus), neurotoxic venom

399

venom (N. atra) and a mixed activity venom (A. halys). Hence, saPLIγ represents a

400

potential broad-spectrum first-line therapeutic agent for snakebite envenomation.

401

5. Conclusion

AC C

402

EP

398

SaPLIγ was an effective antagonist of svPLA2s. The hemorrhage and myonecrosis

403

initiated by D. acuts, A. halys and N. atra could be highly inhibited by saPLIγ. Thus saPLIγ is

404

potential for wide spectrum anti-venom drug development.

405

Ethical statement

406 407

The experiments were carried out in accordance with the guidelines issued by the Ethical Committee of Nanchang University.

408

Funding 16

ACCEPTED MANUSCRIPT 409 410 411 412 413 414

We are grateful for the support of the National Natural Science Foundation of China (NO.31260209) and (NO.31460227); Natural Science Foundation of Jiangxi Province ( 20171BAB204015), Cultivating Foundation of Young Scientists of Jiangxi Province (20171BCB23018).

Conflict of interest statement The authors declare no conflict of interest. Reference

416 417 418 419 420 421 422 423 424 425

Asad, M.H., Murtaza, G., Ubaid, M., DurreSabih, Sajjad, A., Mehmood, R., Mahmood, Q., Ansari, M.M., Karim, S., Mehmood, Z., 2014. Naja naja karachiensis envenomation: biochemical parameters for cardiac, liver, and renal damage along with their neutralization by medicinal plants. Biomed Res. Int. 2014, 970540-970553. Aziz, S.A., Mehta, R., 2016. Technical Aspects of Toxicological Immunohistochemistry. Springer New York. Bustillo, S., Gay, C.C., García Denegri, M.E., Ponce-Soto, L.A., Bal de Kier Joffé, E., Acosta, O., Leiva, L.C., 2012. Synergism between baltergin metalloproteinase and Ba SPII RP4 PLA2 from Bothrops alternatus venom on skeletal muscle (C2C12) cells. Toxicon 59, 338-343.

426 427 428 429

Bustillo, S., García-Denegri, M.E., Gay, C., Velde, A.C.V.D., Acosta, O., Angulo, Y., Lomonte, B., Gutiérrez, J.M., Leiva, L., 2015. Phospholipase A 2 enhances the endothelial cell detachment effect of a snake venom metalloproteinase in the absence of catalysis. Chem.Biol. Interact. 240, 30-36.

430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453

Chen, K., Zhong, L.P., Chen, L.Z., Li, X., Xu, X., Huang, C.H., 2011. Investigation and purification of snake venom secretory phospholipase A 2 inhibitors from sera of some common snake species in Jiangxi province. Pharm.l Biotech. 18, 220-223. Chijiwa, T., So, S., Hattori, S., Yoshida, A., Odaueda, N., Ohno, M., 2013. Suppression of severe lesions, myonecrosis and hemorrhage, caused by Protobothrops flavoviridis venom with its serum proteins. Toxicon 76, 197-205. Chippaux, J.P., Massougbodji, A., Diouf, A., Baldé, C.M., Boyer, L.V., 2015. Snake bites and antivenom shortage in Africa. Lancet 386, 2252. Denegri, M.E.G., Teibler, G.P., Maruñak, S.L., Hernández, D., Acosta, O.C., Leiva, L.C., 2016. Efficient muscle regeneration after highly haemorrhagic bothrops alternatus venom injection. Toxicon 122, 167-175. Feitosa, E.L., Sampaio, V.S., Salinas, J.L., Queiroz, A.M., da Silva, I.M., Gomes, A.A., Sachett, J., Siqueira, A.M., Ferreira, L.C., Dos Santos, M.C., Lacerda, M., Monteiro, W., 2015. Older age and time to medical assistance are associated with severity and mortality of snakebites in the Brazilian amazon: a case-control study. PLoS One 10, e0132237. Gutiérrez, J.M., Lomonte, B., 2013. Phospholipases A2: unveiling the secrets of a functionally versatile group of snake venom toxins. Toxicon 62, 27-39. Gutiérrez, J.M., Rucavado, A., Chaves, F., Díaz, C., Escalante, T., 2009. Experimental pathology of local tissue damage induced by Bothrops asper snake venom. Toxicon 54, 958-975. Gutiérrez, J.M.A., Ownby, C.L., 2003. Skeletal muscle degeneration induced by venom phospholipases A2: insights into the mechanisms of local and systemic myotoxicity. Toxicon 42, 915-931.

AC C

EP

TE D

M AN U

SC

RI PT

415

17

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

Harris, J.B., Johnson, M.A., 1978. Further observations on the pathological responses of rat skeletal muscle to toxins isolated from the venom of the Australian tiger snake, Notechis scutatus scutatus. Clin. Exp. Pharmacol. Physiol. 5, 587. Harris, J.B., Vater, R., Wilson, M., Cullen, M.J., 2003. Muscle fibre breakdown in venom-induced muscle degeneration. J. Anat. 202, 363-372. Huancahuire-Vega, S., Ponce-Soto, L.A., Marangoni, S., 2014. PhTX-II a basic myotoxic phospholipase A₂ from Porthidium hyoprora snake venom, pharmacological characterization and amino acid sequence by mass spectrometry. Toxins 6, 3077-3097. Wiśniewski J.R, Zougman A, Nagaraj N, Mann M., 2009. Universal sample preparation method for proteome analysis. Nat. Methods 6(5), 359-362. Le, Z., Li, X., Yuan, P., Liu, P., Huang, C., 2015. Orthogonal optimization of prokaryotic expression of a natural snake venom phospholipase A2 inhibitor from Sinonatrix annularis. Toxicon 108, 264-271. liu, D., Jiang, K., Shu, P., 2007. Snakes and snake toxins, in: liu, D., et al. (Eds.), Biotoxin development and utilization. Chemical Industry Press, Beijing, pp. 44-45. Lizano, S., Domont, G., Perales, J., 2003. Natural phospholipase A2 myotoxin inhibitor proteins from snakes, mammals and plants. Toxicon 42, 963-977. Lomonte, B., Gutiérrez, J.M., 2011. Phospholipases A2 from viperidae snake venoms: how do they induce skeletal muscle damage? Acta Chim. Slov. 58, 647-659. Mackessy, S.P., 2010. The Field of Reptile Toxinology--Snakes, Lizards, and Their Venoms, in: Mackessy, S.P. (Ed.), Handbook of Venoms & Toxins of Reptiles. CRC Press, London pp. 8-10. Marsh, N., Gattullo, D., Pagliaro, P., Losano, G., 1997. The Gaboon viper, Bitis gabonica : Hemorrhagic, metabolic, cardiovascular and clinical effects of the venom. Life Sci. 61, 763-769. Ohkura, N., Okuhara, H., Inoue, S., Ikeda, K., Hayashi, K., 1997. Purification and characterization of three distinct types of phospholipase A2 inhibitors from the blood plasma of the Chinese mamushi, Agkistrodon blomhoffii siniticus. Biochem. J. 325 ( Pt 2), 527-531. Peichoto, M.E., Acosta, O., Leiva, L., Teibler, P., Maruñak, S., Ruíz, R., 2004. Muscle and skin necrotizing and edema-forming activities of Duvernoy's gland secretion of the xenodontine colubrid snake Philodryas patagoniensis from the north-east of Argentina. Toxicon 44, 589-596. Pinho, F.M., Yu, L., Burdmann, E.A., 2008. Snakebite-induced acute kidney injury in Latin America. Semin. Nephrol. 28, 354-362. Pungercar, J., Krizaj, I., 2007. Understanding the molecular mechanism underlying the presynaptic toxicity of secreted phospholipases A2. Toxicon 50, 871. Rägo, L., Marroquin, A.M., Nübling, C.M., Sawyer, J., 2015. Treating snake bites--a call for partnership. Lancet 386, 2252. Rahmy, T.R., Hemmaid, K.Z., 2000. Histological and histochemical alterations in the liver following intramuscular injection with a sublethal dose of the Egyptian cobra venom. J. Nat. Toxins 9, 21-32. Ramosvara, J.A., 2005. Technical aspects of immunohistochemistry. Vet. Pathol. 42, 405-426. Santos-Filho, N.A., Boldrini-Franca, J., Santos-Silva, L.K., Menaldo, D.L., Henrique-Silva, F., Sousa, T.S., Cintra, A.C., Mamede, C.C., Oliveira, F., Arantes, E.C., Antunes, L.M., Cilli, E.M., Sampaio, S.V., 2014. Heterologous expression and biochemical and functional characterization of a recombinant alpha-type myotoxin inhibitor from Bothrops alternatus snake. Biochimie 105, 119-128. Santos-Filho, N.A., Silveira, L.B., Oliveira, C.Z., Bernardes, C.P., Menaldo, D.L., Fuly, A.L., Arantes, E.C., Sampaio, S.V., Mamede, C.C.N., Beletti, M.E., 2008. A new acidic

AC C

454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505

18

ACCEPTED MANUSCRIPT myotoxic, anti-platelet and prostaglandin I 2 inductor phospholipase A 2 isolated from Bothrops moojeni snake venom. Toxicon 52, 908-917. Schiermeier, Q., 2015. Africa braced for snakebite crisis. Nature 525, 299. Warrell, D.A., 2010. Snake bite. Lancet 375, 77-88. Williams, D., Gutierrez, J.M., Harrison, R., Warrell, D.A., White, J., Winkel, K.D., Gopalakrishnakone, P., Global Snake Bite Initiative Working, G., International Society on, T., 2010. The Global Snake Bite Initiative: an antidote for snake bite. Lancet 375, 89-91. Williams, D.J., 2015. Snake bite: a global failure to act costs thousands of lives each year. BMJ 351, h5378.

RI PT

506 507 508 509 510 511 512 513 514 515

AC C

EP

TE D

M AN U

SC

516

19

ACCEPTED MANUSCRIPT

Table 1. Venom proteins of D. acutus co-immunopreciptated by saPLIγ Description

Coverage

Peptides

agglucetin-beta 1 subunit

QSKTWADAEK

7340956

metalloproteinase MD1

402810593 283825330 17224439 82221933

snake venom serine protease Da-36 acutusin 1 Lys49-phospholipase A2 Zinc metalloproteinase/disintegrin; SVMP MD2

23321265 23200502

agglucetin-beta 2 subunit Lys49-Phospholipase A2

97180272 1041577407* 46015749* 1160578115*

Basic phospholipase A2 DAV-N6 Phospholipase A2 (A. piscivorus)

Asp49-svPLA2 (A. piscivorus) Phospholipase A2 li(A. piscivorus)

MW [kDa]

calc. pI

146

16.7

6.51

ISNSEAHAVFK;YMEIVIVVDHSMVKK

2

1

466

52.4

5.6

GLNIYLGMHNQSIQFDDEQR KASQLIVSTEFQRYMEIVIVVDHSMYTK ENLDTYNK; NYGLYGCNCGVGGR KASQLIVTPEHQRYMEIVIVVDHSMYTK; YMEIVIVVDHSMYTKYNGDSDKIK; GETYLIEPMKISNSEAHAVYK AWAKTSDCLIGK NYGLYGCNCGVGGR; SLFELGK; ENLDTYNK; GEPLDATDR TGVIICGEGTPCEK; AAAVCLGENLR

1 1 2 3

1 1 2 3

260 413 138 466

29 46.6 15.8 52.5

6.51 5.73 8.46 5.68

1 4

1 4

149 122

17.2 14.1

7.9 8.46

2

2

138

15.8

8.25

KQICECDRAAAICFR SLLELGK;ENLDTYNK VTGCNPK; VTGCNPKMDIYTYSVDNGNIVCGGTNPCK AAAICFGDNLKTYDSK; VTGCNPK

1 2

1 2

139 121

15.7 14

8.12 8.54

2

2

123

14

8.24

2

2

139

15.7

8.12

M AN U

TE D

AC C

1041577421*

AAs

1

EP

Lys49-Phospholipase A2(A. Contortrix) Basic phospholipase A2

Unique Peptides

1

SC

23321263

RI PT

Accession

* Identified by an extended search in NCBI protein database of Agkistrodon genus.

ACCEPTED MANUSCRIPT Highlights 1. SaPLIγ has a wide protection effect to local myonecrosis and systemic toxic reaction against venom of D. acutus, N. atra and A. halys. 2. Snake envenomation also leads to severe leukocyte infiltration and erythrocyte leakage. 3. Desmin loss in damaged myofiber is relieved in the presence of saPLIγ.

AC C

EP

TE D

M AN U

SC

RI PT

4. SaPLIγ interacts with a number of venom proteins, including PLA2s, metalloproteinases, C type lectins.

ACCEPTED MANUSCRIPT Address: No. 461 Bayi street, NanChang,

Ethical statement

RI PT

Jiangxi province, China TEL: 86-791-83969075 web: www.ncu.edu.cn

The animal experiments were carried out in accordance with the guidelines issued

AC C

EP

TE D

M AN U

SC

by the Ethical Committee of Nanchang University.