title: Cloning, expression and characterization of a mucin-binding GAPDH from Lactobacillus acidophilus.
abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous enzyme involved in glycolysis. It is also referred to as a moonlighting protein as it has many diverse functions like regulation of apoptosis, iron homeostasis, cell-matrix interactions, adherence to human colon etc. apart from its principal role in glycolysis. Lactobacilli are lactic acid bacteria which colonize the human gut and confer various health benefits to humans. In the present study, we have cloned, expressed and purified the GAPDH from Lactobacillus acidophilus to get a recombinant product (r-LaGAPDH) and characterized it. Size exclusion chromatography shows that r-LaGAPDH exists as a tetramer in solution and have a mucin binding and hemagglutination activity indicating carbohydrate like binding adhesion mechanism. Fluorescence spectroscopy studies showed an interaction of r-LaGAPDH with mannose, galactose, N-acetylgalactosamine and N-acetylglucosamine with a Kd of 3.6±0.7×10(-3)M, 4.34±0.09×10(-3)M, 4±0.87×10(-3)M and 3.7±0.28×10(-3)M respectively. We hope that this preliminary data will generate more interest in further elucidation of the roles of GAPDH in the adhesion processes of the bacteria.
authors.affiliation: Department of Bioinformatics and Structural Biology, Indian Institute of Advanced Research, Koba, Gandhinagar 382007, Gujarat, India.
authors.forename: Dhaval K
authors.initials: DK
authors.lastname: Patel
authors.affiliation: Department of Bioinformatics and Structural Biology, Indian Institute of Advanced Research, Koba, Gandhinagar 382007, Gujarat, India.
authors.forename: Kunal R
authors.initials: KR
authors.lastname: Shah
authors.affiliation: Department of Bioinformatics and Structural Biology, Indian Institute of Advanced Research, Koba, Gandhinagar 382007, Gujarat, India.
authors.forename: Anju
authors.initials: A
authors.lastname: Pappachan
authors.affiliation: Department of Biochemistry, The Maharaja Sayajirao University of Baroda, Vadodara 390002, Gujarat, India.
authors.forename: Sarita
authors.initials: S
authors.lastname: Gupta
authors.affiliation: Department of Bioinformatics and Structural Biology, Indian Institute of Advanced Research, Koba, Gandhinagar 382007, Gujarat, India. Electronic address: ddsingh@iiar.res.in.
authors.forename: Desh Deepak
authors.initials: DD
authors.lastname: Singh
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D015870:Gene Expression; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007779:Lactobacillus acidophilus / Q000201:enzymology / Q000235:genetics; D009077:Mucins / Q000737:chemistry*; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D009077:Mucins; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)
keywords: Fluorescence spectroscopy; GAPDH; Lactobacillus; Moonlighting; Protein-carbohydrate interaction