Elsevier

Toxicon

Volume 52, Issue 2, 1 August 2008, Pages 348-353
Toxicon

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

https://doi.org/10.1016/j.toxicon.2008.06.003Get rights and content

Abstract

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.

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 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 (Jäger 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 this communication, we report the purification, characterization and N-terminal sequencing of a serine proteinase-like protein from the venom of the Buthus martensii Karsch, which binds to the cancer cell membrane.

Section snippets

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.

Gel filtration of B. martensii Karsch venom

B. martensii Karsch venom (100 mg) was dissolved in 1 ml of 50 mM NH4HCO3

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

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

Acknowledgement

The work was supported by grant R-398-000-030-305 from A*STAR SERC (Singapore).

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