Proteomic analysis of Moroccan cobra Naja haje legionis venom using tandem mass spectrometry

J O U RN A L OF P ROTE O M IC S 9 6 ( 2 01 4 ) 2 4 0 –25 2

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Proteomic analysis of Moroccan cobra Naja haje legionis venom using tandem mass spectrometry Ibtissam Maliha,b,c,⁎, Muhamad Rusdi Ahmad rusmilib , Ting Yee Teeb , Rachid Sailec , Noreddine Ghalima , Iekhsan Othmanb a

Venom and Toxins Laboratory, Pasteur Institute of Morocco, Casablanca, Morocco Department of Biomedical Sciences, Jeffrey Cheah School of Medicine and Health Sciences, Monash University Sunway Campus, Malaysia c Laboratory of Biology and Health, URAC 34, Hassan II University Mohammedia — Casablanca, Faculty of Science Ben M'sik, Morocco b

AR TIC LE I N FO

ABS TR ACT

Article history:

The proteome of the venom of Naja haje legionis, the only medically important elapid species

Received 3 September 2013

in Morocco, has been elucidated by using a combination of proteomic techniques that

Accepted 12 November 2013

includes size exclusion chromatography, reverse-phase HPLC, Tricine/SDS-Page, tryptic digestion, Q-TOF tandem mass spectrometry and database search. The sequence analysis of venom fractions revealed a highly complex venom proteome which counts a total of 76

Keywords:

proteins identified from database that can be assigned into 9 proteins families. We report

Venom proteome

the identification of: cobra venom factor (CVF), L-amino-acid oxidases (LAAO), acetylcholin-

Naja haje legionis

esterase (AChE), snake venom metalloproteinases (SVMP), cysteine rich secretory proteins

Moroccan cobra

(CRISP), venom nerve growth factor (vNGF), phospholipases A2 (PLA2), vespryns, kunitz-type

Tandem mass spectrometry

inhibitor, short neurotoxins, long neurotoxins, weak neurotoxins, neurotoxin like proteins, muscarinic toxins, cardiotoxins and cytotoxins. Comparison of these proteins showed high sequence homology with proteins from other African and Asian cobras. Further works are needed to assess the contribution of individual toxins in venom toxicity. Biological significance Naja haje legionis is one of the medically important snakes implicated in the pathogenesis of snake bite in Morocco. The absence of information about venom composition and clinical manifestations of envenomation by this cobra represents an obstacle for the management of this environmental disease in the country. The elucidation of Moroccan cobra venom composition will provide a reasonable guidance for clinician to understand the pathophysiological conditions associated with cobra envenomation and the elaboration of better management strategies. © 2013 Elsevier B.V. All rights reserved.

1.

Introduction

In Morocco, snake envenomation is considered a serious public health problem. A recent retrospective study reported 1478 envenomation by snake bites between 1992 and 2008, occurring

mainly in the rural populations, with 62.1% admitted with snake envenomation symptoms and 8.4% of the cases that were lethal [1]. However, it is reckoned that the total number of envenomation cases in Morocco may be underestimated as there is no reliable data and systematic snake bite reporting [1,2].

⁎ Corresponding author at: Laboratory of Biology and health, URAC 34, Hassan II University Mohammedia — Casablanca, Faculty of Science Ben M'sik, Avenue Idriss El Harti, Casablanca, Morocco. Tel.: + 212 610907222. E-mail address: [email protected] (I. Malih). 1874-3919/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jprot.2013.11.012

J O U RN A L OF P ROT EO M IC S 9 6 ( 2 01 4 ) 2 4 0 –2 52

Naja haje is the only medically important terrestrial elapid found in North Africa [2,3]. This species can be found from Morocco to Egypt with the exception of coastal Algeria [4,5]. Envenomation by N. haje caused prominent neurotoxic symptoms with local swelling and pain at bite site [6,7]. Besides neurotoxicity, N. haje was found to cause cardiovascular disturbance, myotoxicity, cytotoxicity and nephrotoxicity in animals and in-vitro studies [8–10]. The neurotoxic effects of N. haje venom are attributed to the presence of highly potent alpha neurotoxins while myotoxicity and cytotoxicity are caused by phospholipase A2 enzymes and cytotoxins [10–13]. Naja haje legionis is a geographically isolated subspecies among the N. haje complex which can be found only in Morocco and was considered as separate subspecies [14]. Latest molecular and morphological analysis in N. haje complex revealed that N. h. legionis is synonymous with N. haje from Egypt and West Africa [4,15]. In Morocco, this snake is distributed over a large geographical area that goes from Oued Assag through Laayoune, Agadir, Ouarzazate with an extension eastwards to Figuig [2]. This snake can be easily be recognized by its relatively large size (150–200 cm), thick black body, large head and display wide expandable neck upon provocation [14]. Recent insights in proteomic studies provided convenient methods for the characterization of the toxic content and immunological profiling of snake venoms [16,17]. The obtained information from these studies will help for the production of specific and more effective antivenoms and the identifications of proteins that can be used in therapeutics [18]. With recent development and expansion of proteomics and genomics databases, venom protein profiling has improved tremendously with increasing numbers of snake venom proteomes that have been reported [16]. Previously, venom proteome profiling of venom from genus Naja have been conducted by using Egyptian cobra (N. haje), monocled cobra (Naja kaouthia) and Chinese cobra (Naja atra) [17,19]. However, due to limitation in the database depository at that time, not all proteins were fully elucidated and confirmed by this approach [17,19]. In this study, we have conducted the proteomic profiling of N. h. legionis venom by using chromatographic fractionation and proteomics techniques to determine the major proteins composition of the venom. This information provides new insight into the types of toxins presents and a scientific base for the elaboration of adequate management strategies for envenomation by N. h. legionis and other N. haje subspecies.

2.

Methods and materials

2.1.

Venom and chemicals

N. h. legionis venom was milked from forty adult snakes collected from four regions of Morocco (Marrakech, Ouarzazat, Taroudant, and Errachidia) and kept at the serpentarium of Pasteur Institute of Morocco (Casablanca, Morocco). Pooled venoms were centrifuged, lyophilized and stored at − 20 °C until use. All used chemicals were of analytical grade and all chromatography solvents were of HPLC or mass spectrometry grades. Milli-Q water was used for the preparation of all buffers.

241

Fig. 1 – Gel filtration chromatography of crude Naja haje legionis venom on Sephadex G-75 (two columns of 1.6 × 100 cm, ammonium acetate 0.05 M pH 7.4).

2.2.

Gel filtration chromatography

N. h. legionis venom (400 mg) was fractionated by gel filtration by using two columns (1.6 × 100 cm) of Sephadex G75 mounted in series and pre-equilibrated with 0.05 M ammonium acetate pH 7.4. Elution of proteins venom was conducted with the same equilibration buffer at a flow rate of 24 ml/h. Protein elution was monitored at 280 nm and fractions of 1.2 ml/tube were collected. Fractions of individual peaks based on obtained chromatogram were pooled, dialyzed against distilled water and lyophilized.

2.3.

Protein estimation by bicinchoninic acid (BCA)

Protein content of each peak was determined using Pierce BCA assay Kit. Serial dilutions of bovine serum albumin (BSA)

Fig. 2 – Tricine SDS Page of gel filtration fractions on a 10% gel (49.5%T, 3%C). Lane 1 and 10 correspond to molecular standards (from 200 to 6.5 kDa). Lanes 2–5 are respectively venom fractions (F1, F2, F3, and F4) under reducing conditions. Lanes 6–9 are venom fractions under non reducing conditions. Comments on the figure correspond to the different proteins identified by LC MS/MS analysis.

242

J O U RN A L OF P ROTE O M IC S 9 6 ( 2 01 4 ) 2 4 0 –25 2

(A) Fractions A1- A17

(B)

Fig. 3 – (A) Reverse-phase HPLC separation of fraction F1 purified using a C18 column (4.6 × 250 mm, 4 μm particle size, 300 Å pore size) and Agilent LC 1200 chemstation. (B) Tricine SDS Page of F1 derived RP-HPLC fractions (A1–A17) on a 10% gel (49.5%T, 3%C) under reducing (upper panel) and non-reducing conditions (lower panel). Peaks labeled with a question mark correspond to unidentified fractions. SVMP: snake venom metalloproteinases, LAAO: L-amino acid oxydase, AChE: acetycholinesterase, CVF: cobra venom factor.

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ranging from 60 to 2000 μg/ml were used as standards. 25 μl of venom fractions, standards and Blank (distilled water) were deposited into a 96 well microplate. The working reagent (WR) was prepared by mixing 50 part of BCA Reagent A with 1 part of BCA reagent B. 200 μl of WR was added to each well at a ratio of 1/8 (v/v) and the mixture was incubated at 37 °C for 30 min. After cooling the samples, absorbance was measured at 562 nm using a Microplate spectrophotometer system (Benchmark plus, Bio-Rad).

2.4.

243

Reverse phase HPLC

Fractions (~ 1 mg) from previous fractionation step were further separated on reverse phase Jones semi-preparative C18 column (4.6 × 250 mm, 4 μm particle size, 300 Å pore size) using an Agilent 1200 series LC system (Agilent Technologies, Santa Clara, USA). Elution buffers were 0.1% TFA in water (solution A) and 90% acetonitrile with 0.1% TFA (solution B). The column was equilibrated with 0.5% solution B and elution

(A) Fractions B1- B14

(B)

Fig. 4 – (A) Reverse-phase HPLC separation of fraction F2 purified using a C18 column (4.6 × 250 mm, 4 μm particle size, 300 Å pore size) and Agilent LC 1200 chemstation. (B) Tricine SDS Page of F2 RP-HPLC fractions (B1–B14) on a 10% gel (49.5%T, 3%C) under reducing (upper panel) and non-reducing conditions (lower panel). Peaks labeled with a question mark correspond to unidentified fractions. SVMP: snake venom metalloproteinases.

244

J O U RN A L OF P ROTE O M IC S 9 6 ( 2 01 4 ) 2 4 0 –25 2

was carried out at a flow rate of 1 ml/min. An optimized linear gradient was used for the fractionation of each gel filtration peak. For peak F1, the run gradient was modified with a 5%B for 10 min, followed by 5–40% solution B for 20 min and 40–60% solution B for 30 min. For peak F2, the gradient started with 5% solution B followed by 5–40% solution B

for 10 min and 40–70% solution B for 30 min. For peak F3, elution was conducted with 5% solution B for 10 min, 5–30% solution B for 20 min and 30–50% solution B for 60 min. For peak F4, the used gradient was 5% solution B for 10 min, 5–25% solution B for 20 min, 25–40% solution B for 120 min. Protein detection was carried out at 214 nm with a reference

(A) Fractions C1- C13

(A)

Fig. 5 – (A) Reverse-phase HPLC separation of fraction F3 purified using a C18 column (4.6 × 250 mm, 4 μm particle size, 300 Å pore size) and Agilent LC 1200 chemstation. (B) Tricine SDS Page of F3 derived RP-HPLC fractions (C1-C13) on a 16% gel (49.5%T, 6%C) under reducing (upper panel) and non-reducing conditions (lower panel). SVMP: snake venom metalloproteinases, NTX: neurotoxins, vNGF: venom nerve growth factor, PLA2: phospholipase A2, CRISP: cysteine-rich secretory protein.

J O U RN A L OF P ROT EO M IC S 9 6 ( 2 01 4 ) 2 4 0 –2 52

245

(A) Fractions D1- D20

(B)

Fig. 6 – (A) Reverse-phase HPLC separation of fraction F4 purified using a C18 column (4.6 × 250 mm, 4 μm particle size, 300 Å pore size) and Agilent LC 1200 chemstation. (B) Tricine SDS Page of F1 derived RP-HPLC fractions (D1–D20) on a 16% gel (49.5%T, 6%C) under reducing (upper panel) and non-reducing conditions (lower panel). Short NTX: short neurotoxins, long NTX: long neurotoxins, WTX: weak neurotoxins, MTLP: muscarinic like toxin proteins, CTX: cytotoxins.

246 Table 1 – Assignment of reverse-phase HPLC fractions isolated from the venom of Naja haje legionis (Moroccan cobra) to protein families identified by Q-TOF MS/MS analysis of selected peptide ions from in gel and in solution digested proteins. Apparent molecular masses were determined by SDS-PAGE (■ non reducing/♦ reducing conditions). Cysteine residues determined in MS/MS analysis are carbamidomethylated. ND: not determined, CVF: cobra venom factor, LAAO: L-amino acid oxidase, AChE: acetylcholinesterase, SVMP: snake venom metalloproteinase, CRISP: cysteine rich secretory protein, vNGF: venom nerve growth factor, PLA2: phospholipase A2, MTLP: muscarinic toxin like protein, NTX: neurotoxins. HPLC fraction

Molecular No. of AA mass matched % (kDa) peptides – ~60■/♦

A12

66■/♦

A12, 14 A11, 13, 14 A11–14

A14 A15

66■/♦

A16–17, B3–5, 14, C13 – B1, 2, 6–9, 11–13 A7, B3, 5, 10 B3–5, 10 31–66■/♦ B14 60■/♦ C2 ~12■ C3, 4, 6 C4 ~14■/♦

C7 C2–7

~12■/♦

C8

~27■/♦

z

4 1 1 1 4 1

– 2 3 2 2 2 2 2 2 2 3 2 2 2 2 2 – 14.5 3 1.5 2 1.6 2 5.1 2 34.7 3 25.2 5

1 1 3 6 1 2 3 1 7 1 1 1

1 2 1 4 2 1 4 2 7 2 4 9 2 40 10

Matched MH + error SPI (%) MH+ (ppm)

851.4 570.7 446.7 486.1 410.5 773.5

25.4 5

776.7

3863.7

4.9

69.7

3.0 23.2 51.7 4.9 13.0 20.1 9.2 48.3 21.7 6.4 10.5

481.7 1130.9 613.3 682.3 475.2 1276.1 1211.4 771.3 597.8 512.6 905.9

962.4 2260.9 1838.0 1363.6 949.4 2551.2 2421.9 1541.7 1194.6 1535.7 1810.8

3.9 0.8 4.8 1.3 4.1 0.7 1.1 0.0 1.7 1.6 0.3

72.9 95.1 94.3 98.4 90.8 100 72.1 100 70.8 71.1 95.9

2 2 3 2 2 2 2 2 2 3 2

Swiss-Prot ID

Protein family/species

m/z – 1028.5 1386.7 907.4 1133.5 886.5 1003.5 650.3 1010.5 2477.1 1193.5 1344.6 1183.5 1609.7 1504.7 1197.6 – 2552.2 1140.4 892.5 971.3 1229.6 3879.7

1.5 4.0 1.5 6.3 3.7 1.9 4.8 3.2 17.8 3.0 7.2 20.4 1.7 45.2 20.5

MS/MS derived sequence

514.7 462.9 454.2 567.2 443.7 502.2 325.6 505.7 1239.0 398.5 672.8 592.2 805.4 752.8 599.3

2.3 2.4 3.9 4.8 2.6 2.1 3.3 1.6 0.1 4.2 4.0 2.1 0.2 0.0 3.1

91.1 84.0 91.1 100 100 68.5 85.0 82.2 100 94.9 96.1 95.5 87.0 98.8 95.9

−0.7 −0.1 3.7 4.1 1.5 3.8

97.2 70.8 83.4 66.2 95.6 64.1

– (K)IPCAPEDVK(C) (K)VTLLEASERVGGR(V) (R)VTYQTPAK(N) (K)YPVKPSEEGK(S) (K)VIEELKR(T) (K)VTVLEASER(A) (K)KFDLK(L) (K)IFLTCTKK(F) (K)LNEFFQENENAWYYINNIR(K) (R)RSPLEECFR(E) (R)KFWEADGIHGGK(S) (K)VYAYLFDHR(A) (K)GICVAEPYEITVMK(D) (K)DLTEEPNSQGISSK(T) (K)KSVAVVQDHSK(S) – (R)TAPAFQFSSCSIRDYQEYLLR(D) (K)DSCFQENLK(G) (K)VTLDLFGK(W) (R)FVCDCDR(T) (R)GGKGTPVDDLDR(C) (–)NLYQFKNMIHCTVPNRSWWHFA NYGCYCGR(G) (–)NLYQFKNMIHCTVPSRPWWHF ADYGCYCGR(G) (K)QYFFETK(C) (R)GIDSSHWNSYCTETDTFIK(A) (R)FIRIETACVCVITKK(K) (R)ALTMEGNQASWR(F) (K)GLCTNPCK(R) (K)YLYVCQYCPAGNIIGSIATPYK(S) (K)SGPPCADCPSACVNGLCTNPCK(H) (K)HHNVFSNCQSLAK(Q) (K)EIVDKHNALR(R) (K)YGTQREWAVGLAGK(S) (K)TVENVGVSQVAPDNPER(F)

– Q2UXR0 Q4JHE3 A6MFL0 Q90W54 Q4JHE2 P0DI84 O93364 Q6TGQ9 A8QL58 A8QL51 A8QL52 Q92035 Q01833 Q91132 Q10749 P82942 Q7LZ61 Q4VM08 Q6SLM2 P00599 P00601

ND SVMP (Echis ocellatus) LAAO (Oxyuranus scutellatus) LAAO (Demansia vestigiata) LAAO (Agkistrodon halys blomhoffi) LAAO (Notechis scutatus scutatus) LAAO (Vipera ammodytes ammodytes) LAAO (Crotalus adamenteus) LAAO (Bothrops jararacussu) LAAO (Naja atra) LAAO (Bungarus multicinctus) LAAO (Bungarus fasciatus) AChE (Bungarus fasciatus) Complement C3 (Naja naja) CVF (Naja kaouthia) SVMP (Naja mossambica) ND SVMP (Naja kaouthia) SVMP (Daboia russelli) SVMP (Macrovipera lebetina) PLA2 (Bungarus caeruleus) PLA2 (Naja melanoleuca) PLA2 (Naja melanoleuca)

P00602

PLA2 (Naja mossambica)

Q2XXL6 Q5YF89 P61898 Q3HXY7 A6MFK9 Q8JI38 Q3SB03 Q7ZZN8 P81991 Q27J48 P82885

vNGF (Azemiops feae) vNGF (Naja sputatrix) vNGF (Naja atra) vNGF (Notechis scutatus scutatus) CRISP (Demansia vestigiata) CRISP (Laticauda semifasciata) CRISP (Hoplocephalus stephensii) CRISP (Naja atra) CRSIP (Austrelaps superbus) Vespryn (Lachesis muta muta) Vespryn (Naja kaouthia)

–

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A1–6, 8–10 A7 A11–14

Peptide ion

21–31■/♦

C10–11 C10–12

21–31■/♦ 21–31■/♦

D1, 2

7–9■/♦

D3 D4

– 7–9■/♦

D5 D6, 7 D8–10

7–9■/♦ 6–7■/♦ 6–9■/♦

D11, 15 D12

7■/♦

D13

7■/♦

18.3 46.5 46.5 8.5 98.6 13.8 46.0 7.0 24.4 80.0 9.3 96.7 16.0 15.6 22.8 64.6

2 2 2 2 2 2 2 2 3 2 – 2 2 3 2 3 2 2 2 3 3 2 3 3 2 2 3

696.3 688.7 719.7 492.2 948.7 520.7 529.7 460.6 491.9 784.7 498.7 1005.4 518.9 610.2 811.8 1091.8

D14–16 D14–18 D14–19 D14, 15, 17–19

3 2 3 2 1 1 3 1 2 1

29.3 30.8 86.6 30.0 26.6 11.6 65.0 23.3 30.0 17.5

2 2 2 2 2 3 5 2 2 2

552.7 987.4 989.4 742.8 973.9 404.2 419.6 781.3 443.7 685.8

D14–17, 19 D14, 17–19 D15, 16 D16–18 D16–20 D18 D19

2 1 1 1 6 1 1

38.3 26.6 11.6 16.6 92.0 23.3 12.3

3 703.3 2 973.4 2 400.7 2 667.2 2 831.4 2 787.9 2 667.7

C3–7, D8–18 D13–19 D13, 14, 17–19 D14

~ 6♦

~6♦ ~6♦

2 1 1 3 5 1 2 4 1 1

10.6 37.9 48.0 20.5 28.0 5.2 12.6 77.0 24.5 7.3

1 2 2 1 4 1 2 1 2 3 1 4 1 1 1 2

645.8 663.3 726.8 916.4 1007.4 652.8 1204.4 655.7 585.6 344.7

1290.6 1325.6 1452.7 1831.9 2013.9 1304.6 2407.9 1310.5 1754.8 688.3 – 1391.6 1376.5 2157.1 983.4 2844.3 1040.5 1058.4 920.375 1473.7 2352.1 996.4 3014.2 1554.7 1219.5 1622.7 3273.4

3.2 1.4 0.0 −0.1 0.4 2.3 −1.2 −0.6 0.9 3.9

88.3 90.9 79.5 100 98.6 81.8 98.0 90.5 91.5 76.5

2.3 −1.4 1.1 2.3 0.8 2.9 1.9 −0.9 3.3 4.1 4.6 1.7 −2.7 −3.2 3.8 −3.3

87.2 92.0 100 75.1 100 91.5 88.1 81.7 89.2 98.4 84.6 94.4 67.2 86.7 92.5 80.7

1104.5 1973.8 1977.8 1484.7 1946.8 1210.7 2094.0 1561.7 886.519 1370.6

1.0 0.9 0.5 4.8 −3.6 2.6 3.1 3.9 0.0 3.2

80.6 100 88.3 91.2 69.0 88.1 93.2 93.7 94.5 68.4

(R)RSVSPTASNMLK(M) Q2XXQ1 (–)DVDFNSESTRR(K) P84807 (–)NVDFNSESTRRK(K) P84806 (R)AWTEIIQLWHDEYK(N) Q7ZT98 (R)VLEGIQCGESIYMSSNAR(T) Q7T1K6 (R)RSVSPTATNMLK(M) Q2XXQ0 (K)SGPPCGDCPSACVNGLCTNPCK(H) P0CB15 (K)GIEINCCTTDK(C) P68417 (–)LECHNQQSSQPPTTK(S) P01425 (R)GTIIER(G) P80958 – – (R)VDLGCAATCPTVK(T) P01391 (K)MWCDNFCGMR(G) P25674 (K)RVDLGCAATCPTVKPGVDIK(C) P34074 (R)GQNLCYTK(T) P01384 (–)IRCFITPDVTSQACPDGQNICYTK(T) P01389 (R)FYEGNLLGK(R) P01400 (–)LECYQMSK(V) P25678 (K)YVYCCR(R) A8N286 (R)RGCAATCPEAKPR(E) Q802B2 (–)LTCLICPEKYCNKVHTCR(N) P01401 (R)DGEKICFK(K) O42256 (–)FTCFTTPSDTSETCPDGQNICYEKR(W) P01415 (K)MFMMSDLTVPVKR(G) Q98956 (K)FKTIEECHR(T) B6RLX2 (R)FKTIDECHRTCVG(–) P00986 (K)FLFSETTETCPDGQNVCFNQAHLIY P82462 PGK(Y) (K)RGCIDVCPK(S) Q9W6W6 (K)NSALVKYVCCNTDRCN(–) Q9DGH9 (K)NSALVKYMCCNTDKCN(–) P01463 (K)MYMVSSSTVPVKR(G) P01457 (K)NSALVKYVCCSTDRCN(–) P01452 (K)GITRLPWVIR(G) Q9W717 (–)LECNKLVPIAHKTCPEGK(N) P01454 (R)GCIDVCPKDSALVK(Y) P01456 (K)LVPPFWK(T) P01462 (K)TCPEGKDLCYK(M) P19003

2108.0 1945.8 800.499 1333.4 1661.8 1574.8 1334.4

0.4 0.5 2.3 2.9 0.2 3.9 −3.4

75.7 97.7 81.2 91.3 97.4 70.8 61.5

(–)LECNQLIPIAHKTCPEGK(N) (K)NSALVKYVCCNTDKCN(–) (K)STIPVKR(G) (K)YVCCNTDKCN(–) (–)LKCHNTQLPFIYK(T) (R)GCIDACPKNSLLVK(Y) (K)YVCCNTDRCS(–)

P01453 P01459 P01448 P01441 P62394 P01446 P49123

CRISP (Leioheterodon madagascariensis) CRISP (Naja haje haje) CRISP (Naja haje haje) CRSIP (Ophiophagus hannah) CRISP (Naja atra) CRISP (Liophis poecilogyrus) CRISP (Trimeresurus flavoviridis) 3FTX–short NTX (Naja annulifera) 3FTX–short NTX (Hemachatus haemachatus) 3FTX–short NTX (Naja atra) ND 3FTX–long NTX (Naja kaouthia) 3FTX–Long NTX (Naja haje haje) 3FTX–long NTX (Boulengerina annulata) 3FTX–long NTX (Notechis scutatus) 3FTX — long NTX (Naja haje anchietae) 3FTX — weak NTX (Naja melanoleuca) 3FTX — weak NTX (Naja annulifera) 3FTX — MTLP (Ophiophagus hannah) 3FTX — weak NTX (Naja sputatrix) 3FTX — weak NTX (Naja haje haje) 3FTX — Weak NTX (Naja sputatrix) 3FTX — weak NTX (Naja haje haje) 3FTX — cardiotoxin (Naja atra) BPTI/Kunitz (Ophiophagus hannah) BPTI/Kunitz (Naja nivea) 3FTX — MTLP (Naja kaouthia) 3FTX — cytotoxin 10 (Naja atra) 3FTX — cytotoxin 2 (Naja kaouthia) 3FTX — cytotoxin 2 (Naja nivea) 3FTX — cytotoxin 5 (Naja haje haje) 3FTX — cytotoxin-4 (Naja mossambica) 3FTX — NTX-like protein (Naja atra) 3FTX — cytotoxin 9 (Naja annulifera) 3FTX — cytotoxin-1 (Naja nivea) 3FTX — cytotoxin 2 (Naja annulifera) 3FTX — cytotoxin S3C2 (Aspidelaps scutatus) 3FTX — cytotoxin-10 (Naja annulifera) 3FTX — cytotoxin-3 (Naja annulifera) 3FTX — cytotoxin-1 (Naja melanoleuca) 3FTX — cytotoxin 2 (Naja oxiana) 3FTX — cytotoxin 11 (Naja haje haje) 3FTX — cytotoxin 3 (Naja kaouthia) 3FTX — cytotoxin-8 (Naja atra)

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C8–12 C9–12

247

248

J O U RN A L OF P ROTE O M IC S 9 6 ( 2 01 4 ) 2 4 0 –25 2

wavelength of 360 nm. Fractions of eluted peaks were collected automatically.

2.5.

Tricine SDS-PAGE

Venom fractions purified from gel filtration and RP-HPLC were subjected to Tricine SDS-PAGE according to Schagger protocol (2006) [20]. This protocol uses a continuous pH buffers system with the cathode buffer at pH 8.25, gel buffer at pH 8.45 and anode buffer at pH 8.9. Gels of 0.075 × 10 × 10,5 cm were prepared using two acrylamide/bisacrylamide mix solutions, AB3 stock solution (49.5% T, 3% C) and AB6 (49.5% T, 6% C). High molecular weight proteins (> 45 kDa) were resolved on a 10% running gel (using AB3) while low molecular weight fractions (< 45 kDa) were run through a 16% gel (using AB6) overlayed with a 10% spacer gel. A 4% stacking gel was also prepared using AB3 stock solution. Samples (40 μg of protein) were mixed with equal volume of 2 × denaturing sample buffer (50 mM Tris–HCL, 2% SDS, 0,1% bromophenol blue, 10% glycerol) in reducing (with 100 mM of DTT) and non-reducing conditions (without DTT). Prestained molecular standards from 6.5 to 200 kDa (Biorad161-0317) were diluted with reducing sample buffer at a ratio of 1/20. Samples and standards were simultaneously heated at 95 °C for 5–10 min prior to loading on the gel. Electrophoresis was performed using a Hoefer SE 260 device. Efficiency of separation was visualized by staining the gels with PhastGel Blue R dye (GE, Healthcare). After overnight staining, gels were destained using a 30% methanol, 10% acetic acid in water solution. Gel images were captured with ChemiDoc system (Bio-Rad, Hercules, CA, USA).

2.6.

Tryptic digestion

For in gel digestion, SDS-PAGE gels were carefully rinsed with double distilled water. Bands of interest were manually excised and destained using 200 mM ammonium bicarbonate in 40% acetonitrile and incubated at 37 °C for 30 min. Proteins were reduced with 10 mM DTT and incubated at 56 °C for 1 h and alkylated by 55 mM iodoacetamide in the dark at room temperature for 30 min. Gel pieces were subsequently washed with 50 mM ammonium bicarbonate in 50% acetonitrile at room temperature for 15 min, before been dehydrated with pure HPLC grade acetonitrile at 37 °C for 15 min. Proteins were digested in 20 μl of trypsin solution (20 ng/μl in 1 mM hydrochloric acid) and incubated at room temperature for 5 min. Later 50 μl of 40 mM ammonium bicarbonate in 9% acetonitrile was added into the tubes and incubated overnight at 37 °C. After overnight incubation, supernatant was collected and peptides were extracted from gel pieces in three steps using 5% formic acid only, 5% formic acid in 50% acetonitrile and pure acetonitrile with subsequent incubation times at 37 °C for 15 min. Supernatant was collected each time and gel pieces were discarded. Extracted peptides were dried overnight on a vacuum concentrator and 10 μl of 0.1% formic acid in water was added prior to analysis by tandem mass spectrometry. For in solution digestion, approximately 0.5 mg of proteins was mixed with 25 μl of 100 mM ammonium bicarbonate, 25 μl of trifluoroethanol and 1 μl of 200 mM DTT, vortexed

and heated at 60 °C for 1 h. After cooling samples at room temperature, proteins were alkylated with 4 μl of 200 mM iodoacetamide in the dark at room temperature for 1 h. Excess iodoacetamide was blocked by addition of 1 μl DTT and incubation for 1 h at room temperature. Samples were diluted with double distilled water and ammonium bicarbonate to adjust pH (7–9). Digestion was performed by using trypsin solution at a ratio of 1/20 (enzyme: substrate) followed by incubation overnight at 37 °C. Trypsin activity was later stopped with 1 μl of formic acid. The samples were later concentrated by vacuum concentrator and kept at −20 °C until use. The samples were dissolved in 0.1% formic acid prior to injection into the LCMS/MS system.

2.7. Nano ESI-liquid chromatography and tandem mass spectrometry (nano-ESI-LCMS/MS) Digested peptides from in gel and in solution tryptic digestions were subjected to nano-electrospray ionization (ESI) MS/MS experiments using an Agilent 1200 HPLC-Chip/MS Interface, coupled with Agilent 6520 Accurate-Mass Q-TOF LC/MS system. Samples were loaded in a large capacity chip 300 Å, C18, 160 nL enrichment column and 75 μm × 150 mm analytical column (Agilent part N° G4240-62010) with a flow rate of 3 μl/min from a capillary pump and 0.3 μl/min from a Nano pump of Agilent 1200 series. Injection volume was adjusted to 1 μl per sample and the mobile phases were 0.1% formic acid in water (solution A) and 90% acetonitrile in water with 0.1% formic acid (solution B). A gradient of 47 min was applied using Agilent 1200 series nano-flow LC pump: 3–50% solution B for 30 min, 50–95% solution B for 2 min, and 95% solution B for 5 min. Ion polarity was set to positive ionization mode. Drying gas flow rate was 5 l/min and drying gas temperature was 325 °C. Fragmentor voltage was 175 V and the capillary voltage was set to 1850– 2000 V. Spectra were acquired in a MS/MS mode with a MS scan range of 110–3000 m/z and MS/MS scan range of 50–3000 m/z. Precursor charge selection was set as doubly, triply or up to triply charged state with the exclusion of precursors 922.0098 m/z (z = 1) and 121.0509 (z =1) set as reference ions. Data was extracted with MH+ mass range between 600 and 4000 Da and processed with Agilent Spectrum Mill MS Proteomics Workbench software packages (Version Rev A.03.03.084 SR4). Carbamidomethylation of cysteine was set as a single modification. The search was performed against SwissProt database (Version. March 2012). Protein identifications validated with the following filters: protein score >11, peptides score >6 and scored peak intensity (SPI) >80%.

3.

Results and discussions

3.1.

The venom proteome of N. h. legionis

Venom composition of N. haje or its Moroccan subspecies, N. h. legionis has never been thoroughly profiled by using proteomics techniques. Early works on N. haje venom have shown that the venom has differences in the chromatography and electrophoretic profile compared to other elapids and viperids [17]. In this study, gel filtration of the N. h. legionis venom yielded 4 different peaks, namely, fraction 1 (F1), fraction 2 (F2), fraction 3 (F3) and

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fraction 4 (F4) (Fig. 1). Tricine SDS-PAGE of these fractions showed that the protein components have molecular weights in the range from 6.5 kDa to 200 kDa (Figs. 1 and 2). Proteins with molecular weight of 45 kDa–200 kDa were detected in F1 and F2

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while proteins with lower molecular weight (6.5–31 kDa) were found in F3 and F4 (Fig. 2). Further fractionation of gel filtration fractions by reverse phase liquid chromatography yielded 17 fractions from F1, 14 fractions from F2, 13 fractions from F3 and

(A)

(B)

Fig. 7 – Relative abundance of protein families identified by LC MS/MS analysis (A) from gel filtration fractions and their corresponding RP-HPLC peaks, (B) within whole venom of Moroccan cobra Naja haje legionis. ND: not determined, CVF: cobra venom factor, LAAO: L-amino acid oxidase, AChE: acetylcholinesterase, SVMP: snake venom metalloproteinase, CRISP: cysteine rich secretory protein, vNGF: venom nerve growth factor, PLA2: phospholipase A2, MTLP: muscarinic toxin like protein, NTX: neurotoxins.

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20 fractions from F4 (Figs. 3–6). The results showed that reverse phase HPLC was able to further fractionate the proteins into partially purified and purified proteins. Analysis on Tricine SDS-PAGE and LCMS/MS showed that many of these fractions were not homogeneous and require further purification. This could be due to the presence of structural isoforms and venom proteins with closely similar molecular weights and charge. Analysis of venom fractions tryptic digests by LCMS/MS revealed the presence of 76 proteins matched from public available database that can be assigned into 9 proteins families (Table 1). The three finger toxins (3FTx) constitute the major components of crude venom and 58% of fraction F4 (Table 1 and Fig. 7). 3FTx is a family of toxins that have nearly similar structural arrangement that resembles three protruding fingers with conserved cysteine residues in their primary structures [21,22]. Toxins of 3FTx family have been reported to have diverse effects such as neurotoxicity, cardiotoxicity, cytotoxicity, and platelet aggregating activities [22,23]. In N. h. legionis, the 3TFx that were detected were short and long neurotoxins, weak neurotoxins, muscarinic toxins, cardiotoxins and cytotoxins (Table 1). α-Neurotoxins from N. h. legionis and other cobra venoms are perhaps among the most important venom component. The detected α-neurotoxins were found to be homologous to both Asian and African cobra neurotoxins. Cytotoxins are other common components in Naja venoms [13,19]. These toxins were found to constitute 54% of all 3FTx detected in the N. h. legionis venoms. Although cytotoxins are one of the major components in the venom, necrosis is not one of major symptoms in N. haje envenomation [7]. In addition, cytotoxins from N. haje were found to be 10–20 times less toxic than cytotoxins isolated from Naja oxiana and N. kaouthia [13]. The presence of these types of 3TFx is in favor of a neurotoxic and cytotoxic venom profile. The second largest non-enzymatic toxin families in the venom are cysteine-rich secretory proteins (CRISPs). These toxins have been found to constitute 49% of fraction F3 and ~10% of total venom proteins (Fig. 7, Table 1). Although constitute an important part of cobra venoms, activities of many of these proteins are not fully characterized and fully understood [24,25]. LCMS/MS analysis detected several peptides with homology with different CRISPs found in viperid, elapid and colubrid species (Table 1). Homology with CRISPs from various species may indicate that the CRISPs content in N. h. legionis are heterogeneous and may exert various effects. Venom nerve growth factors (vNGF) were also found to constitute an important part of fraction F3 (25%) either 5% of total venom proteins. vNGFs are ubiquitous proteins that have been detected in all snake families [26]. Several homologous peptides of vNGF related to elapidae venoms have been detected in N. h. legionis venom. Besides non-enzymatic toxic protein families, there are also several families of enzymatic toxins that were detected in N. h. legionis venom, such as snake venom metalloproteinase (SVMP), acetylcholinesterase (AChE), L-amino acid oxidase (LAAO) and phospholipase A2 (PLA2). These enzymatic proteins were mainly distributed among fractions F1 and F2 except for PLA2 which was detected in F3 (Fig. 7). Interestingly, SVMPs were found to constitute an important part of N. h. legionis venom (~9%) and the major part of fraction F2 (76%). While these toxins have demonstrated an important role in the pathogenesis of envenomation by colibridae and viperidae,

the role of SVMPs in cobra venoms remains unclear in absence of hemorrhagic symptoms in envenomation by elapids [27,28]. LAAOs are among well characterized venom enzymatic toxins that showed high degree of conservation among the different snake families [29]. Several isoforms related to elapidae and viperidae venoms have been detected in N. h. legionis venom. The role of these enzymes in cobra envenomation is not fully understood [30]. LAAO has been suggested to be involved in immobilizing prey by inducing hypotension and platelet aggregation [30–32]. PLA2 play an important role in the pathogenesis of cobra envenomations [33]. Due to their ability to form multimeric complexes with other venom components, PLA2 enzymes trigger a wide range of physiopathological activities [34]. In this venom, several isoforms of PLA2 were detected and were found to be homologous with PLA2 from Bungarus and Naja venoms (Table 1). The presence of these toxins may indicate that neurotoxicity observed upon envenomation could be partly induced by presynaptic neurotoxic PLA2 in addition to postsynaptic neurotoxin activity.

3.2.

Unique peptides from N. h. legionis venom

We have also detected peptides that exhibited sequence homology with muscarinic-like toxin proteins (MLTP). This is the first report of the presence of MLTP in N. haje crude venom. Previously discovered MLTP from other cobra species showed that these proteins are not toxic and have low affinity toward muscarinic acetylcholine receptors [35]. Several peptides with homology with acetylcholinesterase from Bungarus fasciatus were also detected in N. h. legionis venom. AChE from Naja species has only been reported from N. oxiana but never from an African cobra [36]. In addition, peptides with homology with Kunitz type inhibitors from elapid venoms were also detected (Table 1). Previously, protease inhibitors have only been found in Asian cobra species and one African cobra species, Naja nivea [37,38]. The presence of complement factor and cobra venom factor (CVF) is one of the characteristic of cobra venoms [39]. Several peptides of complement factors from N. kaouthia and Naja naja were detected in this venom (Table 1). This is quite interesting as the complements depleting factors has never been isolated from African cobra venoms and were only detected from venom gland transcripts. Other less known and unique polypeptides that have been detected in N. h. legionis venoms are vespryns. Thaicobrin from N. kaouthia and Ohanin-like protein from Lachesis muta are two isoforms of vespryn that have been detected. These toxins have never been described from African cobra venoms and were only deduced from venom gland transcripts [40]. There are also a number of high molecular weight proteins that were not determined from fractions F1 and F2 (Figs. 3 and 4). These proteins couldn't be identified and matched to known snake venom proteins from databases. The unknown proteins may be novel proteins that are not listed in databases and which may require farther characterization and sequencing.

4.

Conclusion

By using a simple proteomic approach, we have successfully elucidated the venom proteome of the Moroccan cobra, N. h. legionis which is a geographically isolated subspecies among

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the N. haje complex. The present proteomic data confirm the particularity and complexity of N. h. legionis venom. The revealed venom composition will help clinicians to understand the clinical manifestations of envenomation by N. h. legionis. It will also provide a reasonable guidance for better snake bite management and the production of effective antivenoms. More works on venom toxicity, pharmacological activities and neutralization studies are necessary to assess the potency of venom toxins and cross neutralization properties of commercially available antivenoms against N. h. legionis venom.

[12]

[13]

[14] [15]

Acknowledgment [16]

We thank the International Division of Pasteur Institute for supporting Ibtissam Malih with Calmette Program Traineeship Grant (Ref: RIIP/EC/MAN/N°16/11). This work was partly supported by Monash University Sunway Campus Major Research Grant Scheme.

[17]

[18]

Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jprot.2013.11.012.

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