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

Background: 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. 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. methods. bt18 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. results. anion 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. conclusions. gapdh 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. © 2010 krishnan et al; licensee biomed central ltd.