Purification and N-terminal sequence of a serine proteinase-like protein (BMK-CBP) from the venom of the Chinese scorpion (Buthus martensii Karsch)

Toxicon 52 (2008) 348–353

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Purification and N-terminal sequence of a serine proteinase-like protein (BMK-CBP) from the venom of the Chinese scorpion (Buthus martensii Karsch) Rong Gao a, Yong Zhang a, b, *, Ponnampalam Gopalakrishnakone c a

Nanoscience and Nanotechnology Initiative, National University of Singapore, Singapore 117576, Singapore Division of Bioengineering, Faculty of Engineering, National University of Singapore, Singapore 117576, Singapore c Department of Anatomy, Faculty of Medicine, National University of Singapore, Singapore 117597, Singapore b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 January 2008 Received in revised form 4 June 2008 Accepted 5 June 2008 Available online 14 June 2008

A serine proteinase-like protein was isolated from the venom of Chinese red scorpion (Buthus martensii Karsch) by combination of gel filtration, ion-exchange and reveres-phase chromatography and named BMK-CBP. The apparent molecular weight of BMK-CBP was identified as 33 kDa by SDS-PAGE under non-reducing condition. The sequence of N-terminal 40 amino acids was obtained by Edman degradation. The sequence shows highest similarity to proteinase from insect source. When tested with commonly used substrates of proteinase, no significant hydrolytic activity was observed for BMK-CBP. The purified BMK-CBP was found to bind to the cancer cell line MCF-7 and the cell binding ability was dose-dependent. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Scorpion venom Serine proteinase Purification Cell binding

1. Introduction Animal venoms are rich source of protein and peptide toxins, which can reach over 90% of the dry weight of the snake venom (Mebs, 1969). After injection, these proteins or peptides could target to different receptors on cells or molecules and induce various kinds of physiological changes. These include blood coagulation disorder (Kornalik, 1991), cytotoxic effect (Dufton and Hider, 1991), blockage of neuro-muscle transmission (Endo and Tamiya, 1991) and ion-channel blockage (Harvey and Anderson, 1991), etc. Many of these venom proteins or peptides have been isolated and well characterized. The high specificity and strong potency made them the good choice as the targeting molecules of drug delivering and the model for

* Corresponding author. Division of Bioengineering, Faculty of Engineering, Block EA-03-12, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore. Tel.: þ65 65164871; fax: þ65 68723069. E-mail address: [email protected] (Y. Zhang). 0041-0101/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2008.06.003

the drug design (Lewis and Garcia, 2003; Blumenthal and Seibert, 2003; Armishaw and Alewood, 2005). Anticancer therapy is one of the major applications for the using of venom proteins and peptides. Some isolated proteins or peptides specifically bind to the cancer cell membrane and affect the cancer cell migration and proliferation. Based on their mechanisms, few families of venom proteins and peptides have been identified: [1] ion-channel toxins from scorpion, which affect the cancer cell physiology through blockage of the specific ion-channel (Ja¨ger et al., 2004); [2] ligand of specific target on the membrane of cancer cell, such as chlorotoxin from scorpion venom (DeBin et al., 1993; Deshane et al., 2003), which binds to the metalloproteinase on gliomas and kill the cancer cells (Nabors, 2004; Mamelak and Jacoby, 2007); and [3] disintegrins or disintegrin-containing proteins from snake venoms, which affect the cancer cell through binding to integrin on cell surface (McLane et al., 2004). To find the new proteins or peptides which could bind to the membrane receptor of cancer cell, we screen several snake and scorpion venom from Asia–Pacific region. In

R. Gao et al. / Toxicon 52 (2008) 348–353

this communication, we report the purification, characterization and N-terminal sequencing of a serine proteinaselike protein from the venom of the Buthus martensii Karsch, which binds to the cancer cell membrane. 2. Materials and methods 2.1. Materials Superdex 75 (Hiload 16/60) and Sephasil C8 columns were from GE Healthcare Life Sciences, while UNO Q1 column was from Bio-Rad (USA). Chinese red scorpion (B. martensii Karsch) venom was bought from the scorpion farm of China and other snake venoms were purchased from the Venom Supplier (Australia). The chromogenic substrates of proteinase were bought from Chromogenix (Molndal, Sweden). All other chemicals were analytic scale. 2.2. Purification of BMK-CBP

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2.5. Reduction and pyridylethylation of BMK-CBP BMK-CBP was dissolved in 100 ml of the denaturant buffer (6.0 M guanidine hydrochloride, 0.13 M Tris, 1 mM EDTA, pH 8.0) containing 0.07 M b-mercaptoethanol. The mixture was incubated at 37  C for 2 h under nitrogen. Subsequently, 1.5-fold molar excess (over sulfhydryl groups) of 4-vinylpyridine was added and incubated at 37  C for 2 h under nitrogen (Chow et al., 1998). The sample was then desalted on a Sephasil C8 column. 2.6. Amino acid sequencing Amino terminal sequencing of PE-BMK-CBP was carried out by automated Edman degradation using an Applied Biosystems 494 pulsed-liquid-phase sequencer. Phenylthiohydantoin (PTH) amino acids were identified using on-line reverse-phase HPLC on a PTH-C18 column on an Applied Biosystems 140C analyzer. 2.7. Cell culture

2.2.1. Gel filtration of B. martensii Karsch venom B. martensii Karsch venom (100 mg) was dissolved in 1 ml of 50 mM NH4HCO3 buffer (pH 8.9) and load onto the Superdex 75 column (Hiload 16/60). The proteins were then eluted with same buffer at a flow rate of 1 ml/ min. Totally 10 peaks were obtained and pooled as indicated (Fig. 2). The cell binding ability of each peak was tested separately and the protein peak (peak 3) with highest cell binding ability was then further purified with UNO Q1 column. 2.2.2. Ion-exchange chromatography of cell binding protein from Superdex 75 column Pooled peak 3 from Superdex 75 column was loaded on UNO Q1 column (pre-equilibrated with 50 mM NH4HCO3, pH 8.9 buffer) and the protein was then eluted with linear gradient of NaCl at a flow rate of 1 ml/min. Totally three protein peaks were obtained and the cell binding activity was found to locate in peak 2 (Fig. 4). 2.2.3. Reverse-phase chromatography of cell binding protein from UNO Q1 column To obtain the ultra-pure sample, peak 2 from UNO S1 was loaded on a Sephasil C8 column (pre-equilibrated with 0.1% TFA) and the protein was then eluted with linear gradient of solution B (80% acetonitrile, containing 0.1% TFA) at a flow rate of 1 ml/min. A single protein peak was obtained (Fig. 5) and named as BMK-CBP. 2.3. SDS-PAGE analysis SDS-PAGE analysis was carried out on polyacrylamide gel containing 1% SDS in Tris–glycine buffer as described by Laemmli (1970).

MCF-7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with L-glutamine (2 mM), heat-inactivated fetal bovine serum (10%), penicillin (50 IU/ ml) and streptomycin (50 mg/ml). Cells were cultured at 37  C in a gas phase of air with 5% CO2. 2.8. Cell adhesion assay The experiment was done following the method described by Marcinkiewicz et al. (2000) with modification: Testing proteins were dissolved in PBS buffer and applied to the wells overnight at 4  C. Excess proteins were washed away by PBS. Cell suspension (100 ml, 7.5  105) was seeded in each well and allowed to adhere for 1 h at 37  C. The attached cells were then stained for 10 min with 0.5% crystal violet in 25% methanol, rinsed with distilled water to remove excess stain not absorbed by cells and air-dried overnight. The crystal violet was then extracted with 100 ml of 0.1 M sodium citrate in 50% ethanol. The absorbance was measured at 585 nm using Genova Life Science Analyzer (Jenway, Felsted, England) spectrophotometer. 2.8.1. Caseinolytic activity This was tested using azo-casein as substrate, according to Rowan and Buttle (1994) modification. Enzyme was incubated with 0.1 ml of 2% azo-casein solution for 2 h at 37  C. Then, 0.1 ml of 20% trichloroacetic acid was added and sample left at room temperature for 30 min. The tubes were centrifuged and the absorbance of the clear supernatant was determined by spectrophotometric measurement at 405 nm. The control solution was applied, in which the enzyme solution was omitted. The caseinolytic activity, expressed as units/ mg, where 1 U was defined as the amount of enzyme that in 20 min at 37  C caused an increase of 0.001 in absorbance (at 405 nm) of trichloroacetic acid supernatant.

2.4. Mass spectrometry Determination of the molecular mass was carried out by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF, ABI 4700 linear mode) mass spectrometry.

2.8.2. Amidolytic activity The activity was measured according to Zhang et al. (1998) using chromogenic substrate S-2238 (D-Phe(pipecolyl)-Arg-p-nitroaniline hydrochloride), S-2222

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(benzoyl-Ile-Glu-(g-OR)-Gly-Arg-p-nitroaniline hydrochlo ride), S-2251 (H-D-Val-Leu-Lys-p-nitroaniline dihydrochloride) and S-2366 (L-pyro-Glu-Pro-Arg-p-nitroanilinehydrochloride). Assays were performed in 50 mM Tris–HCl, pH 7.8, 0.01% Tween-80 in a total volume of 500 ml. The reaction was initiated by the addition of the testing sample and the formation of p-nitroanilide (pNA) was monitored continuously at 405 nm. The amount of substrate hydrolyzed was calculated from the absorbance at 405 nm by using a molar extinction coefficient of 10,000 M1 cm1 for free pNA. 2.8.3. Fibrinogenolytic activity (Gao et al., 1998) Fibrinogenolytic activity was measured as follows: test samples and human fibrinogen (5 mg/ml) were dissolved in 50 mM Tris–HCl buffer, pH 7.4, 0.1 M NaCl (containing 10 mM CaCl2) and incubated at 37  C. At various time intervals, 20 ml of the incubation mixture was pipetted into a small test tube and analyzed by SDS-PAGE under reducing condition.

3. Results 3.1. Screening of cell binding ability of snake and scorpion venoms To identify the venom components, which could bind to cancer cell, we used breast cancer cell line MCF-7 as the host. Several snake venoms (including Elapid and Viperidae families) and scorpion venom from Asia–Pacific region were screened. As shown in Fig. 1, significant changes were observed for the venom of Agkistrodon halys, Russell’s viper and B. martensii Karsch. Since the venoms of A. halys and Russell’s viper are rich sources of disintegrin and disintegrin-containing proteins, these cell binding could be attributed to the presence of the

3.2. Purification of cell binding protein from Chinese red scorpion venom Venom of B. martensii Karsch was first dissolved in 50 mM NH4HCO3 buffer (pH 8.9) and loaded onto the Superdex 75 column and the proteins were eluted with same buffer. As shown in Fig. 2, totally 10 peaks were obtained and pooled as indicated. The protein components of each peak were first checked with SDS-PAGE (Fig. 3). Peaks 1–4 represented the proteins with the molecular weight high than 20 kDa while the peaks 5–10 represented the components with the molecular weight lower than 20 kDa. Since the SDS-PAGE could not show the accurate molecular weight of small peptides, the peaks 5–10 were then applied to MALDI-TOF to check the molecular weight distribution and the results indicated that peaks 5–10 contained only the small peptides with the molecular between 3 and 8 kDa (data not shown). The cell binding ability of each peaks was also tested separately. To our interest, the cell binding activity was mainly associated with peaks 2–4, but no significant activity was found for peaks 5–10, which contained the channel toxins from scorpion venom. The peak 3 from Superdex 75 column was then further purified with UNO Q1 column. Totally three protein peaks were obtained and the cell binding activity was found to locate in peak 2 (Fig. 4). To obtain the ultra-pure sample, peak 2 was pooled and further purified with reverse-phase C8 column. A single protein peak (with cell binding activity) was obtained (Fig. 5). The final preparation of cell binding protein (from reverse-phase C8 column) was named as

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Fig. 1. Screening of cell binding activity. The experiment was done as described in Section 2. Venom concentration used was 0.1 mg/ml while the PBS alone was used as the negative control. After experiment, the crystal violet was then extracted with 100 ml of 0.1 M sodium citrate in 50% ethanol. The absorbance was measured at 585 nm.

Absorbance at 280 nm

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proteins mentioned above. In case of scorpion venom, numerous ion-channel toxins have been isolated, which target to the different ion-channel on the cell membrane (see reviews by Rodrı´guez de la Vega and Possani, 2004; Goudet et al., 2002). In addition, peptide toxin which specially bind to the target of cancer cell and induces anticancer effect was also been reported (McLane et al., 2004). These encouraged us to continue the investigation of B. martensii Karsch venom.

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Elution volume (ml) Fig. 2. Gel filtration of Chinese red scorpion venom. The crude venom (100 mg) was dissolved in NH4HCO3 buffer (50 mM, pH 8.9). After filtered through 0.22 mm membrane, the venom solution was loaded onto Superdex 75 column (16/60) and run under same buffer (1 ml/min). The elute was monitored under 280 nm and collected (1 ml/tube). Totally 10 protein peaks were obtained and pooled.

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Fig. 3. SDS-PAGE of pooled protein peaks from Superdex 75 column. Crude venom (Line 2) and protein peaks 1–10 (Lines 3–12) from Superdex 75 column were run on 15% SDS-PAGE under non-reducing condition. Line 1 shows the protein molecular marker: (a) 100 kDa, (b) 93 kDa, (c) 52 kDa, (d) 37 kDa, (e) 28 kDa (f) 19 kDa.

BMK-CBP and was used for all later works. The apparent molecular weight of BMK-CBP was estimated as 33 kDa by SDS-PAGE (Fig. 5 inset). 3.3. Cancer cell binding activity of purified protein The cell binding activity of BMK-CBP was also tested by MCF-7 cell. A clear dose-dependent curve was observed for the purified protein (Fig. 6). The cell binding activity could be observed starting from 1 mg of protein coated and increased following the increasing of protein coated. The curve formed a platform when the protein amount reached around 5–7 mg. 3.4. N-terminal sequence The N-terminal 40 amino acid of BMK-CBP was obtained by Edman degradation and sent for homology searching by BLAST. The searching result indicated that BMK-CBP was a serine proteinase-like protein. The sequence was also

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3.5. Enzymatic activity As the purified protein shows the sequence of trypsinlike serine proteinase, we also tested its enzymatic activity using different substrates. Table 1 shows the proteinase activity assay for B. martensii Karsch venom and its fractions. The results showed that the crude venom could degrade the purified fibrinogen among the substrates tested. This fibrinolytic activity was further confirmed by peaks 1 and 2 from gel filtration. But the BMK-CBP did not show any activity to the substrates tested.

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Elution Volume Fig. 4. Ion-exchange chromatography of cell binding protein from Superdex 75 column. Peak 3 of Superdex 75 column was pooled and loaded onto UNO Q1 column (pre-equilibrated with 50 mM NH4HCO3 buffer, pH 8.9). The binding protein was then eluted with linear gradient of NaCl. The cell binding activity was mainly associated with peak 2.

Absorbsance at 585 nm

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compared with the selected serine proteinase from different sources (Fig. 7). Lower sequence identity was observed between the BMK-CBP and snake venom serine proteinases and trypsin (around 30%), while higher sequence identity was found when compared with the serine proteinases from insect source.

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Fig. 5. Reverse-phase chromatography of cell binding protein. Peak 2 from UNO Q column was pooled and further loaded onto Sephasil C8 column (pre-equilibrated with 0.1% TFA). The binding protein was then eluted with linear gradient of solution B (80% acetonitrile, containing 0.1% TFA). One single protein peak was obtained and named as BMK-CBP. Inset: SDSPAGE analysis of BMK-CBP. Crude venom (Line 2) and BMK-CBP (Line 3) were run on 15% SDS-PAGE under non-reducing condition. Line 1 shows the protein molecular marker: (a) 100 kDa, (b) 93 kDa, (c) 52 kDa, (d) 37 kDa, (e) 28 kDa (f) 19 kDa.

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Protein Amount (µg) Fig. 6. Cell binding activity of purified BMK-CBP. The experiment was done as described in Section 2. Different amount of purified BMK-CBP was applied and the PBS was used as control.

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Fig. 7. Sequence comparison of serine proteinase from different sources; (1) serine proteinase from Manduca sexta (Jiang et al., 2005); (2) accession: XP_001122037; (3) serine proteinase from Deinagkistrodon acutus venom (Wang et al., 2001); (4) serine proteinase from Crotalus durissus durissus venom, accession: Q2QA04; (5) bovine trypsin (Roach et al., 1997).

4. Discussion In this communication, we report the screening of cancer cell binding ability of several snake and scorpion venoms. In case of B. martensii Karsch venom, the venom was fractionated into 10 peaks by gel filtration: [1] high molecular proteins (including peaks 1–4, 19 kDa) and [2] small peptides (including peaks 5–10, 3–8 kDa). The small peptide fractions of scorpion venom are rich source of ion-channel toxins, which could bind to different channels on cell membrane and affect their function (see reviews by Rodrı´guez de la Vega and Possani, 2004; Goudet et al., 2002). But in our assay system, no significant cell attachment was observed for these fractions. The high molecular weight fractions of the scorpion venom were neglected because of the high abundance of bioactive peptides in scorpion venom. These bioactive peptides include channel toxins and antibacterial peptides. Only few papers were published concerning proteins from these fractions, which include hyaluronidase (Morey et al., 2006), PLA2 (Conde et al., 1999), C-type lectin (Khoang et al., 2001) and serine proteinase (Almeida et al., 2002) from the venom of different scorpion species. To our interest, the cell binding activity of the crude venom was found from high molecular protein fractions. After three steps of purification, a serine proteinase-like protein was obtained, which was responsible for the cell binding activity of the crude venom. This forms the first serine proteinase-like protein isolated from scorpion venom. Cellular receptors for serine proteinase are widely reported in various kind of the cell. They anchor the proteinase on the cell membrane and trigger various kind of physiological events, such as tissue factor on the cell (Rao and Pendurthi, 1998) which form complex with Factor VII and trigger the blood coagulation cascade; uPAR (urokinase-type plasminogen Table 1 Proteinase activity assay of Buthus martensii Karsch venom

Acknowledgement

Casein Fibrinogen S-2238 S-2222 S-2251 S-2261 Crude Venom Gel filtration P-1 Gel filtration P-2 Gel filtration P-3 Gel filtration P-4 Gel filtration P-5 Gel filtration P-6 Gel filtration P-7 Gel filtration P-8 Gel filtration P-9 Gel filtration P-10 Purified BMK-CBP

           

þ þþ þþ         

þ þþþ þþ         

           

           

activator receptor) which involved in the tumor associated proteolysis (Laufs et al., 2006). This membrane receptor could enrich the uPA (urokinase-type plasminogen activator) on cell surface and catalyze the conversion of plasminogen to plasmin. The generated plasmin will degrade the extracellular matrix proteins and thus enhance the cancer cell invasion (Danø et al., 1985). In addition, binding of uPA with its receptor uPAR could activate downstream signaling molecules and lead to cell proliferation, migration, and invasion (Ma et al., 2001; Aguirre-Ghiso et al., 2003). Venom metalloproteinases were also been found to bind to the cell membrane receptor and induce various physiological changes for the cells, such as inhibition of platelet aggregation and collagen adhesion (Moura-da-Silva et al., 2001; Zigrino et al., 2002), cancer cell adhesion and migration (Cox and Huttenlocher, 1998). The key factor in these activities was the disintegrin-like domain of venom metalloproteinase, through which the metalloproteinase binds to the a2b1 on cell surface and works through either enzymatic activity or no-enzymatic mechanism. In experiment, we first test the cell binding ability of BMK-CBP. A dose-dependant binding was obtained and the curve formed a platform when the amount of BMKCBP reached 5–7 mg. The saturation of the binding capacity of 96-well plate could be the main reason as we used extra amount of the cell in the experiment. The result indicated that BMK-CBP could recognize and bind to the membrane on MCF-7 cell. In addition, we have also test the hydrolytic activity of BMK-CBP on commonly used proteinase substrates. Even through, fibrinolytic activity was recorded for the crude venom and gel filtration fractions, but no significant hydrolyzing activity was observed for BMP-CBP. In conclusion, we reported here the isolation and characterization of the first serine proteinase-like protein which could bind to the MCF-7 cell with dose-dependant manner.

           

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