Identification of snake venom allergens by two-dimensional electrophoresis followed by immunoblotting

Toxicon 125 (2017) 13e18

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Identification of snake venom allergens by two-dimensional electrophoresis followed by immunoblotting Yujing Hu a, b, e, 1, Liming Yang c, 1, Haiwei Yang d, 1, Shaoheng He a, *, Ji-Fu Wei b, ** a

Allergy and Clinical Immunology Research Centre, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, Liaoning 121001, China Research Division of Clinical Pharmacology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China c Jiangsu Collaborative Innovation Center of Regional Modern Agriculture and Environment Protection, Jiangsu Key Laboratory for Eco-Agriculture Biotechnology around Hongze Lake, Huaiyin Normal University, Huai'an, 223001, China d Department of Urology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China e Department of Clinical Laboratory, The Changzhou No 2 People's Hospital, Changzhou, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 October 2016 Received in revised form 10 November 2016 Accepted 16 November 2016 Available online 17 November 2016

This allergic reaction to snake venom was described to occur in patients after recurrent exposure through bites in amateur and professional snake handlers, which might be underestimated and contribute to fatal snakebites in victim, independently from the toxicity of the venom itself. Few allergens were identified from snake venoms by normal SDS-PAGE, which cannot separate the snake venom completely. In the present study, we identified nine potential allergens by two-dimensional (2D) electrophoresis followed by immunoblotting (named as allergenomics) in Protobothrops mucrosquamatus venom. By multidimensional liquid chromatography-ion trap mass spectrometry (MDLC-ESI-LTQ-MS/MS) analysis, six allergens showed sequence similarity to snake venom serine proteinases. Other allergens showed sequence similarity to snake venom metalloproteinase. These allergic reactions to snake venom allergens might contribute to fatal snakebites in victim, independently. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Snake venom Allergen Allergenomics Serine proteinase Metalloproteinase

1. Introduction Envenomation by snakebite is a common and generally harmful, environmentally and occupationally neglected tropical disease and highly relevant public health problem, estimating to cause around 50,000 deaths annually around the world (Cruz et al., 2009). Snake venoms are mixtures of proteins, toxins and enzymes that may cause coagulopathy, neurotoxicity, myotoxicity, hypotension and n et al., 2009). In addition to their direct tissue necrosis (Alape-Giro toxic effects, snake venoms also activate the victims' normal tissues and cells to release some harmful components to cause secondary injury. For example, many snake venom components could activate

* Corresponding author. Allergy and Clinical Immunology Research Centre, The First Affiliated Hospital of Jingzhou Medical University, No. 2, Section 5, Renmin Street, Guta District, Jinzhou, Liaoning, 121001, China. ** Corresponding author. Research Division of Clinical Pharmacology, The First Affiliated Hospital, Nanjing Medical University, 300 Guangzhou Road, 210029, Nanjing, China. E-mail addresses: [email protected] (S. He), [email protected] (J.-F. Wei). 1 Contributed to this paper equally. http://dx.doi.org/10.1016/j.toxicon.2016.11.251 0041-0101/© 2016 Elsevier Ltd. All rights reserved.

n et al., mast cells directly and cause inflammatory cascade (Leo 2011; Stone et al., 2013; Wei et al., 2009a, 2009b, 2006a, 2006b). Besides the direct and secondary injury caused by snake venom, there are some reports about allergic sensitization to snake venom (Wadee and Rabson, 1987; Reimers et al., 2000; Prescott and Potter, 2005; de Medeiros et al., 2008; Madero et al., 2009; de Pontes et al., 2016). The allergic reaction to snake venom was described to occur in patients after recurrent exposure through bites in amateur and professional snake handlers (Wadee and Rabson, 1987; de Medeiros et al., 2008; Madero et al., 2009). The snake venom allergy might be underestimated as it contributes to fatal snakebites in victim, independently from the toxicity of venom itself (Wadee and Rabson, 1987; Reimers et al., 2000; Prescott and Potter, 2005; de Medeiros et al., 2008; Madero et al., 2009; de Pontes et al., 2016). Madero et al. identified 4 IgE-binding bands of about 60, 28, 14 and 7 kDa in the Bothrops extract by SDS-PAGE followed by immuno-blotting methods (Madero et al., 2009). Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) analysis demonstrates that 14-kDa protein has similarity with snake venom phospholipase A2 and the 60- and 28-kDa proteins shows significant similarity with snake venom metalloproteinases (Madero et al., 2009). Recently, de Pontes et al. identified crotoxin as an

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Y. Hu et al. / Toxicon 125 (2017) 13e18

allergen to cause occupational anaphylaxis in one patient who worked with Crotalus durissus terrificus venom for 4 years (de Pontes et al., 2016). All these evidences suggested that snake venom contained allergens which could cause serious allergic reactions in sensitive patients who had the experience to meet the snake venom. Snake venoms comprise of complex mixtures of toxins, which largely belong to a few major protein families (Fry et al., 2009, 2008). These snake venom families have similar molecular weight, which cannot be separated from each other by normal SDSPAGE. Indeed, two-dimensional (2D) electrophoretic analysis of snake venom provides a more realistic view of venom complexity (Calvete, 2014; Calvete et al., 2007). On the other hand, allergentargeted proteomics based on 2D electrophoresis followed by immunoblotting (named as allergenomics), have been used for comprehensive identification and/or quantification of potential allergens that bind to IgE (Di Girolamo et al., 2015; Nakamura and Teshima, 2013). The aim of the present study is to identify the potential allergen in snake venom (Protobothrops mucrosquamatus) in occupational snake venom handler by allergenomics method. 2. Materials and methods 2.1. Chemicals Chemicals for electrophoresis, including acrylamide, bisacrylamide, SDS and TriseHCl (pH 8.8) were purchased from GE Healthcare Life Sciences China (Shanghai, China). Dithiothreitol (DTT) and iodacetamide (IAM) were purchased from Promega (Beijing) Biotech Co (Beijing, China). Water from a Millipore MilliRO4 reverse osmosis system was used for making all solutions. 2.2. Patients' sera and preparation of Protobothrops mucrosquamatus venom Sera from Protobothrops mucrosquamatus snake venom handlers who had worked or studied in the department of animal toxicology, Kunming Institute of Zoology, Chinese Academy of Sciences were collected and stored in aliquots at
70  C. The allergic response to snake venom was noticed by their clinical histories. Sera from nonallergic individuals were used as controls. The study protocol was approved by the ethical committee of the First Affiliated Hospital of Nanjing Medical University. Written informed consent for the use of blood samples were obtained from all participants before study entry according to the declaration of Helsinki.

4  C at 1 mA/cm2 for 1.5 h. After electro transfer step, all transferred protein spots on PVDF membranes were stained temporarily with Ponceau S solution and then scanned. These Ponceau S-stained images served as reference gel images to match spots to Coomassiestained protein spots. The transferred PVDF membranes were blocked for overnight at 4  C with blocking solution [3% BSA in TBST (Tris-buffered Saline with Tween 20)], and incubated with sera from Protobothrops mucrosquamatus allergic persons respectively for 5 h at 37  C at a dilution of 1:5 in TBST. After washing four times in TBST, the membranes were incubated with monoclonal anti-human IgE Ab (Promega, 1:2500 dilution) for 2 h at 37  C. The protein spots were then detected with an enhanced chemiluminescence kit (SuperSignalTM West Pico substrate; Pierce Biotechnology) for 5 min before scanning. Image analysis software PDQuest (Bio-Rad) was used to match Coomassie-stained spots in gels to Ponceau Sstained spots in PVDF membrane or Ponceau S-stained protein spots to immunoblotting spots in the same PVDF membranes. Since no mass spectra could be obtained from proteins blotted onto PVDF membranes, protein spot identities were assigned by matching the chemiluminescence images with Coommassie-stained gels run in parallel. 2.5. Peptide analysis by multidimensional liquid chromatographyion trap mass spectrometry (MDLC-ESI-LTQ-MS/MS) The stained protein spots matching the immunoblotting signals were cut out of the gels. In-gel digestion of protein spots followed the procedures described previously (Yang et al., 2010). Mass spectra analysis was performed by Shanghai applied protein technology company (Shanghai, China). We used SEQUEST to search the theoretical sequence databases and identify the best matches to the spectra. Then we compared these peptides with the results that SEQUEST has identified from the UniProt database (Eng et al., 1994). 3. Results 3.1. 2D gel electrophoresis of Protobothrops mucrosquamatus Protobothrops mucrosquamatus venom was subjected to 2D gel electrophoresis. Fig. 1 shows the 2D gel profile of the Protobothrops mucrosquamatus snake venom, in which 83 distinct protein spots

2.3. 2D gel electrophoresis For total protein separation, the immobilized pH gradient (IPG) strips (pH 3e10, linear, 13 cm, Bio-Rad) were rehydrated passively for 13 h. The voltage settings for isoelectric focusing (IEF) in the Protean system (GE EttanIPGphor 3) were 2 h at 250 V, 1 h at 1000 V, 3 h at 8000 V, and then keep at 8000 V until a total of 50000 Vh was reached. After IEF and equilibration, the second dimensional SDS-PAGE gels of 12.5% were run at 2 W/gel for 2 h and 12 W/gel for 1.5 h using Multiphor system (GE Ettan DALT six). The gels were visualized by colloidal Coomassie brilliant blue staining. 2.4. Western blotting For each sample, duplicate 2D gels were run under the same conditions. One gel was subjected to colloidal Coomassie staining to visualize the protein spots and analyze the spots using mass spectrometry. The other gel was transferred onto PVDF membrane (Bio-Rad, Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell) at

Fig. 1. 2D gel profile of the Protobothrops mucrosquamatus snake venom. The arabic number represents serial number of the spots.

Y. Hu et al. / Toxicon 125 (2017) 13e18

15

Table 1 Identification of protein spots from 2-D gels by MDLC-ESI-LTQ-MS/MS. Spot no.

Internal peptide sequence

Charge

MHþ

Diff MHþ

XC

DeltaCn

1

K.DM*EIYLGVHSK.K K.DSCVGDSGGPLICNGQFQGIVSWGGDPCAQPR.E K.EKFFCDSSK.T K.FFCDSSK.T K.FFCDSSKTYTK.W K.GIIAGNTDVTCPL.K.KVPNKDVQR.R K.SAHIAPLSLPSSPPSVGSVCR.I K.VPNKDVQR.R R.AAYAGLPATSR.T R.EPGVYTNVFDHLDWIK.G R.KSAHIAPLSLPSSPPSVGSVCR.I R.TLCAGILEGGK.D R.TLCAGILEGGKDSCVGDSGGPLICNGQFQGIVSWGGDPCAQPR.E R.VM*GWGTITSPQETYPDVPHCANINLLDYEVCR.A K.DSCVGDSGGPLICNGQFQGIVSWGGDPCAQPR.E K.EKFFCDSSK.T K.FFCDSSK.T K.FFCDSSKTYTK.W K.GIIAGNTDVTCPL.K.KVPNKDVQR.R K.SAHIAPLSLPSSPPSVGSVCR.I K.VPNKDVQR.R K.WNKDIMLIR.L R.AAYAGLPATSR.T R.EPGVYTNVFDHLDWIK.G R.KSAHIAPLSLPSSPPSVGSVCR.I R.TLCAGILEGGK.D R.TLCAGILEGGKDSCVGDSGGPLICNGQFQGIVSWGGDPCAQPR.E R.VM*GWGTITSPQETYPDVPHCANINLLDYEVCR.A K.GIIAGNTDVTCPL.K.SAHIAPLSLPSSPPSVGSVCR.I K.VTLPDVPR.C R.CANINLLDYEVCR.A R.IM*GWGTISPTK.V R.TLCAGILEGGK.D K.DIMLIR.L K.EKFICPNK.K K.EKFICPNKK.N K.ILNEDEQTR.D K.ILNEDEQTRDPK.E K.KILNEDEQTR.D K.KILNEDEQTRDPK.E K.NFQMLFGVHSK.K K.GIIAGNTDVTCPL.K.SAHIAPLSLPSSPPSVGSVCR.I K.VTLPDVPR.C K.WNKDIMLIR.L R.CANINLLDYEVCR.A R.IM*GWGTISPTK.V R.TLCAGILEGGK.D K.DIMLIR.L K.GIIAGNTDVTCPL.K.SAHIAPLSLPSSPPSVGSVCR.I K.VTLPDVPR.C R.CANINLLDYEVCR.A R.IM*GWGTISPTK.V R.KSAHIAPLSLPSSPPSVGSVCR.I R.TLCAGILEGGK.D K.LFSDCSK.K K.LFSDCSKNDYQTFLTK.Y K.NDYQTFLTK.Y K.YNPQCILNAP.K.CEACIM*SDVISDKPSK.L K.CKCEACIMSDVISDKPSK.L K.LFSDCSK.K K.LFSDCSKNDYQTFLTK.Y K.NDYQTFLTK.Y K.NDYQTFLTKYNPQCILNAP.K.YNPQCILNAP.K.LAIVVDYR.M K.PLNVAITLSLLR.I K.VTLGSFGDWR.K

2 3 2 2 2 2 2 2 2 2 2 3 2 3 3 3 2 1 2 2 2 2 2 2 2 2 3 2 3 3 2 2 1 2 2 1 1 2 3 2 2 2 2 2 2 2 1 2 2 2 2 1 2 2 1 2 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

1308.4858 3393.6034 1148.2409 890.9534 1384.5091 1331.4909 1084.2545 2120.3887 956.0816 1078.2036 1934.1398 2248.5616 1119.2873 4493.868 3754.1226 3393.6034 1148.2409 890.9534 1384.5091 1331.4909 1084.2545 2120.3887 956.0816 1189.4557 1078.2036 1934.1398 2248.5616 1119.2873 4493.868 3754.1226 1331.4909 2120.3887 897.054 1640.8068 1207.4246 1119.2873 760.9688 1036.2 1164.3729 1118.1797 1458.5562 1246.3526 1586.7291 1308.5339 1331.4909 2120.3887 897.054 1189.4557 1640.8068 1191.4252 1119.2873 760.9688 1331.4909 2120.3887 897.054 1640.8068 1191.4252 2248.5616 1119.2873 856.937 1968.1464 1130.2321 1190.3241 1841.0613 2129.3994 856.937 1968.1464 1130.2321 2301.5335 1190.3241 949.1289 1310.6109 1138.2575


1.4962
1.9416 0.2559
0.6526 0.5321 0.2059 0.0275 0.6847
0.9214 0.3426
1.6332
0.1744 1.2553
0.618 2.1296
1.4016
1.5361 0.4854 0.3611 0.6589 0.1655 0.1767
0.7824 2.1167
2.5194
1.5532
0.2864 0.2403 0.049 1.0286 1.0959 0.7277
1.631
2.5822
1.6034
1.3767 0.4168 2.001
1.0741
0.1443
0.4608 1.0866 0.5011 0.2909
0.4321
0.3663
1.556 0.1647
1.7632
1.5378 0.2683 1.3938 0.8029
0.3633 0.389
1.8422 2.0482
2.5304
1.5407
2.001
0.2766
1.6039
1.2389
1.3307
1.9006
1.774 0.6644 0.9321
0.0105
0.6499 0.7319 0.1519 1.2535

3.4748 4.8425 2.3971 2.4065 2.5908 3.7922 2.9833 4.9453 2.287 2.5761 3.0991 4.653 3.0641 5.2244 4.0323 4.682 2.5618 2.0269 3.4681 3.59 2.6611 4.0642 2.5534 2.5972 2.5498 3.6183 3.9561 2.797 4.6036 3.9902 2.768 3.5822 1.969 4.6512 3.1133 2.0143 2.0358 2.2666 3.7897 2.9956 3.3663 3.2587 3.8356 2.5949 3.5893 3.8897 2.0413 3.0089 4.7739 3.6852 2.7138 2.0083 3.5932 3.8319 1.9064 4.3614 2.9196 4.3452 2.8364 2.2424 4.7278 3.1132 3.2781 2.3023 2.366 2.4357 4.6831 2.9536 2.304 2.843 2.4895 2.302 2.432

0.1939 0.6484 0.5099 0.4534 0.3367 0.6284 0.5166 0.7324 0.4777 0.6025 0.5177 0.3764 0.2714 0.5661 0.1753 0.5969 0.5416 0.5565 0.3602 0.6814 0.4948 0.6356 0.5137 0.5811 0.4129 0.6091 0.4023 0.3378 0.6251 0.1004 0.6092 0.6647 0.5082 0.5858 0.2024 0.2528 0.3141 0.111 0.1489 0.1366 0.4303 0.3416 0.52 0.325 0.6931 0.6801 0.5192 0.1407 0.7258 0.1974 0.3015 0.3807 0.6667 0.6853 0.616 0.6601 0.111 0.408 0.3902 0.3074 0.549 0.5775 0.5622 0.2506 0.5013 0.37 0.7174 0.4941 0.6263 0.5789 0.488 0.598 0.7375

2

3

4

5

6

7

8

9

(continued on next page)

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Y. Hu et al. / Toxicon 125 (2017) 13e18

Table 1 (continued ) Spot no.

Internal peptide sequence

Charge

MHþ

Diff MHþ

XC

DeltaCn

R.DLITVQSDSK.V R.DLITVQSDSKVTLGSFGDWR.K R.DLITVQSDSKVTLGSFGDWRK.T

2 2 2

1106.2088 2225.4436 2353.6165


1.8792 0.7146 0.1665

2.6339 5.0349 2.8102

0.5849 0.7055 0.6996

MHþ: The experimental mass of the peptide (M þ H) determined by MDLC-ESI-LTQ-MS/MS. Diff(MHþ): The delta mass between the experimental mass and the candidate sequence identified by the SEQUEST search. Diff(MHþ) is computed as Mass(candidate) - Mass(experimental). Xcorr: The cross correlation value computed from cross correlation of the experimental MS/MS spectrum vs. the candidate peptides in the database. The candidate peptide producing the highest Xcorr value is chosen as the #1 hit by SEQUEST. DeltaCn: The delta correlation value between the #1 hit and the #2 hit in the search. Cn is the normalized correlation (normalized to 1.0). DeltaCn is the delta Cn between the top 2 hits in the search. This is an indication of the similarity of scoring of the top hits.

sensitized to one or more allergen proteins, and 9 IgE-binding spots were detected. Each patient showed an individual IgE-binding pattern with 4e9 different allergen spots (Fig. 2AeG). The relative Mr and the pI of total proteins were between 30 and 40 kDa/ 5.20e7.90 (Table 2). Among them, 6 with a molecular mass of 39.4 kDa and a pI of 5.22, 8 with a molecular mass of 30.3 kDa and a pI of 7.84, and 9 with a molecular mass of 30.5 kDa and a pI of 7.4. The most reactive IgE-binding spots were numbers 6, 8 and 9, with 100% reaction rate. Other IgE-binding spots showed 42.85e85.71% reaction rate to the sera from Protobothrops mucrosquamatus allergy patients. No positive spots were detected when sera from nonallergic individuals were applied (Fig. 2H). 3.3. Mass spectrometry and database search To identify specific proteins following 2D-PAGE, spots were manually picked from colloidal Coomassie blue-stained gels and analyzed by MS. A total of 9 spots, consisting of both abundant and less abundant proteins, were selected and analyzed to check their quality. MDLC-ESI-LTQ-MS/MS analyses showed that all of the excised spots led to good quality spectra. Data listed in Tables 1 and 2 include an assigned protein spot number, protein identity, percentage sequence coverage, expect value, and NCBI database accession number of the best match and databases that yielded concurrent identification. 4. Discussion

Fig. 2. Identification of allergens from Protobothrops mucrosquamatus snake venom by 2D electrophoresis followed by immunoblotting. Protobothrops mucrosquamatus snake venom proteins were separated by 2D electrophoresis and immunostained with sera from patients with snake venom allergy, and alkaline phosphataseelabeled antihuman IgE antibody. The arabic number represents serial number of the patients. AeG: gel was blotted with serum from Protobothrops mucrosquamatus snake venom sensitive patient. H: gel was blotted with serum from healthy control subject.

were detectable by Coomassie blue staining. The relative molecular mass (Mr) of approximately 90% total proteins were among 15e45 kDa. The isoelectric points (pI) of approximately 72.29% of total proteins were among 4.0e7.0 (Table 1). 3.2. Identification of allergens from Protobothrops mucrosquamatus venom Protobothrops mucrosquamatus allergens were identified by immunoblotting with the sera from Protobothrops mucrosquamatus allergy patients following 2D electrophoresis. All patients were

Protobothrops mucrosquamatus is a venomous pit viper species endemic to Asia. In China, it is distributed mainly in south of China (from north Yunnan to west Gansu, and east Zhejiang province). Clinical effects of Protobothrops mucrosquamatus envenoming ranged from local signs including tenderness, edema, and erythema to systemic complications, including thrombocytopenia, coagulopathy, rhabdomyolysis, acute renal failure, and spontaneous systemic bleeding (Chen et al., 2009). Snake venoms comprise complex mixtures of toxins, which largely belong to a few major protein families. By 2DePAGE, 83 distinct protein spots were detected in Protobothrops mucrosquamatus snake venom. In fact, many components including phospholipase A2s (Wei et al., 2006c, 2010), serine proteinases (Wei et al., 2002a), C-type lectin-like proteins (Wei et al., 2002b), Lamino acid oxidase (Wei et al., 2003), and metalloproteinase (Chou et al., 2013) in Protobothrops mucrosquamatus venom had been purified and identified. Obviously, these proteins cannot be separated from each other by only 1DePAGE. The allergic reactions to snake venom were mostly found in patients by inhalation of dry venom and by handling (Wadee and Rabson, 1987; de Medeiros et al., 2008; Madero et al., 2009; de Pontes et al., 2016). Using 1DePAGE followed by immunoblotting methods, four allergens corresponding to snake venom phospholipase A2 and metalloproteinases (de Medeiros et al., 2008) were identified, and one allergen, which is a heterodimeric neurotoxin containing a nontoxic fraction named crotapotin and a

Y. Hu et al. / Toxicon 125 (2017) 13e18

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Table 2 Immunoblotting detection of proteins identified by MDLC-ESI-LTQ-MS/MS. Spot no.

Exp. MW(Da)/pI

NCBI accession no.

Identified name

Species

Coverage(%)

1 2 3 4 5 6 7 8 9

35200/5.89 35900/5.24 39300/5.85 37900/5.65 39500/5.56 39400/5.22 31000/7.11 30300/7.84 30500/7.40

CAA58223.1 CAA58223.1 AAG27253.1 Q9YGJ8.2 AAG27253.1 Q9DG84.1 1510259A 1510259A ADV71357.1

mucofirase 3 mucofirase 3 serpentokallikrein-2 Plasminogen activator Haly-PA serpentokallikrein-2 Serpentokallikrein-2 H2 proteinase H2 proteinase metalloproteinase PMMP-3

Protobothrops mucrosquamatus Protobothrops mucrosquamatus Protobothrops mucrosquamatus Gloydius blomhoffi brevicaudus Protobothrops mucrosquamatus Protobothrops mucrosquamatus Trimeresurus flavoviridis Trimeresurus flavoviridis Protobothrops mucrosquamatus

66.15 65.37 29.96 15.12 33.46 32.68 12.94 21.89 9.98

toxic fraction, belongs to the PLA2 family (Madero et al., 2009). In the present study, we identified more allergens in snake venom than previous studies, which only identified one or two allergens in each study. At least 9 different allergens with molecular masses of 30e40 kDa and pI values ranging from 5.2 to 7.9 were confirmed by allergenomic method. Among these allergens, three were 100% positive response to Protobothrops mucrosquamatus allergic sera. Others showed 42.85e85.71% positive response to Protobothrops mucrosquamatus allergic sera. By MDLC-ESI-LTQ-MS/MS analysis, 6 allergens showed sequence similarity to snake venom serine proteinases (No 1, 2 to mucofirase, No 3, 5, 6 to serpentokallikrein-2 and No 4 to plasminogen activator Haly-PA). Other allergens showed sequence similarity to snake venom metalloproteinase (No 7, 8 to H2 proteinse and No 9 to metalloproteinase PMMP-3). In a given snake venom, serine proteinases and metalloproteinases consist of many different components originated from gene duplication due to accelerated evolution. These serine proteinases and metalloproteinases showed similar amino acid sequences, but distinct functions. The identified allergens may be different isoforms of snake venom serine proteinases and metalloproteinase. While snake venom metalloproteinases have been identified as allergens in Bothrops venom (de Medeiros et al., 2008), it is the first time that snake venom serine proteinases are identified as allergens. The studies that Serine proteinases from mite (Stewart et al., 1992), dog (Mattsson et al., 2009), bee venom (Winningham et al., 2004) and wasp venom (Winningham et al., 2004) possess allergen properties may supports our current observation. Although metalloproteinases and serine proteinases seem to be the most allergenic components in this venom, other components may play a role in allergenicity in other venoms. In conclusion, 9 different allergens had been are identified from Protobothrops mucrosquamatus snake venom by using allergenomics method. These allergens belong to snake venom serine proteinases and metalloproteinase. Snake venom allergens are likely to contribute to fatal snake biting injury in sensitive patients, independently from the toxicity of the venom. Ethical statement All experiments involving human subjects were performed in accordance with relevant guidelines and regulations of the Institutional Ethics Committee of the First Affiliated Hospital of Nanjing Medical University, and all examination were performed after obtained informed consents. Animal studies were conducted according to the Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Nanjing Medical University. Acknowledgments This project was sponsored by the grants from Special Fund for

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