title: Identification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a binding protein for a 68-kDa Bacillus thuringiensis parasporal protein cytotoxic against leukaemic cells.
abstract: 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.
authors.affiliation: Department of Pharmacy, Faculty of Medicine and Health Sciences, International Medical University, No 126 Jalan 19/155B Bukit Jalil, Kuala Lumpur, 57000 Malaysia.
authors.forename: Kanakeswary
authors.initials: K
authors.lastname: Krishnan
authors.forename: Jeremy Er An
authors.initials: JE
authors.lastname: Ker
authors.forename: Shar Mariam
authors.initials: SM
authors.lastname: Mohammed
authors.forename: Vishna Devi
authors.initials: VD
authors.lastname: Nadarajah
mesh_terms: D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D045744:Cell Line, Tumor / Q000187:drug effects*; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006801:Humans; D007938:Leukemia / Q000378:metabolism*; D011485:Protein Binding; D013171:Spores, Bacterial / Q000737:chemistry / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004731:Endotoxins; C412914:parasporin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases