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: Kanakeswary Krishnan, Jeremy Er An Ker, Shar Mariam Mohammed, Vishna Devi Nadarajah
• Journal: Journal of biomedical science (J Biomed Sci) Vol. 17 Pg. 86 (Nov 13 2010) ISSN: 1423-0127 [Electronic] England
• PMID: 21073742 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Bacterial Proteins
• Endotoxins
• parasporin
• Glyceraldehyde-3-Phosphate Dehydrogenases

Topics

• Bacillus thuringiensis (genetics, metabolism)
• Bacterial Proteins (genetics, metabolism, pharmacology)
• Cell Line, Tumor (drug effects)
• Endotoxins (genetics, metabolism, pharmacology)
• Glyceraldehyde-3-Phosphate Dehydrogenases (metabolism)
• Humans
• Leukemia (metabolism)
• Protein Binding
• Spores, Bacterial (chemistry, metabolism)
• T-Lymphocytes (cytology, drug effects, metabolism)