Identification of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a binding protein for a 68-kDa Bacillus thuringiensis parasporal protein cytotoxic against leukaemic cells

Kanakeswary Krishnan,Shar Mariam Mohammed,Jeremy Er An Ker,Vishna Devi Nadarajah

doi:10.1186/1423-0127-17-86

Kanakeswary Krishnan, Shar Mariam Mohammed + Show 2 more

Open Access

https://doi.org/10.1186/1423-0127-17-86

Copy DOI

Abstract

BackgroundBacillus thuringiensis (Bt), an ubiquitous gram-positive spore-forming bacterium forms parasporal proteins during the stationary phase of its growth. Recent findings of selective human cancer cell-killing activity in non-insecticidal Bt isolates resulted in a new category of Bt parasporal protein called parasporin. However, little is known about the receptor molecules that bind parasporins and the mechanism of anti-cancer activity. A Malaysian Bt isolate, designated Bt18 produces parasporal protein that exhibit preferential cytotoxic activity for human leukaemic T cells (CEM-SS) but is non-cytotoxic to normal T cells or other cancer cell lines such as human cervical cancer (HeLa), human breast cancer (MCF-7) and colon cancer (HT-29) suggesting properties similar to parasporin. In this study we aim to identify the binding protein for Bt18 in human leukaemic T cells.MethodsBt18 parasporal protein was separated using Mono Q anion exchange column attached to a HPLC system and antibody was raised against the purified 68-kDa parasporal protein. Receptor binding assay was used to detect the binding protein for Bt18 parasporal protein in CEM-SS cells and the identified protein was sent for N-terminal sequencing. NCBI protein BLAST was used to analyse the protein sequence. Double immunofluorescence staining techniques was applied to localise Bt18 and binding protein on CEM-SS cell.ResultsAnion exchange separation of Bt18 parasporal protein yielded a 68-kDa parasporal protein with specific cytotoxic activity. Polyclonal IgG (anti-Bt18) for the 68-kDa parasporal protein was successfully raised and purified. Receptor binding assay showed that Bt18 parasporal protein bound to a 36-kDa protein from the CEM-SS cells lysate. N-terminal amino acid sequence of the 36-kDa protein was GKVKVGVNGFGRIGG. NCBI protein BLAST revealed that the binding protein was Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Double immunofluorescence staining showed co-localisation of Bt18 and GAPDH on the plasma membrane of the CEM-SS cells.ConclusionsGAPDH has been well known as a glycolytic enzyme, but recently GAPDH was discovered to have roles in apoptosis and carcinogenesis. Pre-incubation of anti-GAPDH antibody with CEM-SS cells decreases binding of Bt18 to the susceptible cells. Based on a qualitative analysis of the immunoblot and immunofluorescence results, GAPDH was identified as a binding protein on the plasma membrane of CEM-SS cells for Bt18 parasporal protein.

Highlights

Bacillus thuringiensis (Bt), an ubiquitous gram-positive spore-forming bacterium forms parasporal proteins during the stationary phase of its growth
It was reported that selective human cancer cell-killing activity is associated with some non-insecticidal Bt isolates resulting in a new category of Bt parasporal protein called parasporin
Immunoblot assay to detect the binding of anti-Bt18 antibody to Bt18 parasporal protein Solubilised and activated Bt18 parasporal protein was separated by SDS-PAGE and transferred to a nitrocellulose membrane

Summary

Introduction

Bacillus thuringiensis (Bt), an ubiquitous gram-positive spore-forming bacterium forms parasporal proteins during the stationary phase of its growth. Recent findings of selective human cancer cell-killing activity in non-insecticidal Bt isolates resulted in a new category of Bt parasporal protein called parasporin. It was reported that selective human cancer cell-killing activity is associated with some non-insecticidal Bt isolates resulting in a new category of Bt parasporal protein called parasporin. Mizuki et al, (2000) obtained the first parasporin by expressing the cry gene encoding the Cry31Aa protein ( known as parasporin-1), which exhibits strong cytotoxicity against human leukemic T cells (MOLT-4), but did not exhibit insecticidal or hemolytic activities [4]. A Malaysian Bt isolate, designated Bt18 produces parasporal protein that exhibit cytotoxic activity preferentially for human leukaemic T cells (CEM-SS) but is non-cytotoxic to normal T cells or other cancer cell lines such as HeLa, MCF-7 and HT-29 [9]. Insecticidal Bt parasporal proteins are known to bind receptors on the insect brush border membrane and it is suggested that these receptors play a role in the specificity of insecticidal activity [10,11]

Objectives

Methods

Results

Conclusion