Localization of the phosphatidylserine-binding site of glyceraldehyde-3-phosphate dehydrogenase responsible for membrane fusion.
In this study, we demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phosphatidylserine (PS)-binding protein and localized the putative PS-binding site involved in the membrane fusion induced by this enzyme. In an attempt to identify the PS-binding proteins, we raised polyclonal antibodies against a 15-amino-acid synthetic peptide (amino acid residues 390-403 of phosphatidylserine decarboxylase), which was shown to bind specifically to PS. One polyclonal antibody, designated aPSD-2, crossreacted with GAPDH, and its binding to GAPDH was inhibited by PS but not by other phospholipids such as phosphatidylethanolamine and phosphatidylinositol. Kinetic analysis of GAPDH binding to phospholipid membranes by measuring surface plasmon resonance showed that GAPDH associated with the phospholipid membrane containing PS rapidly (k[on] =2.8 X 10(4) M(-1) X s[-1]) and dissociated extremely slowly (k[off]=5.9 X 10(-5) s[-1]), giving a low dissociation constant (KD=2.6nM). GAPDH bound less effectively to membranes without PS with a dissociation constants of 0.2 microM. GAPDH-induced vesicle fusion was also inhibited by aPSD-2, suggesting that this antibody recognizes the putative PS-binding site on GAPDH involved in the enzyme-induced membrane fusion. Chemical fragmentation of GAPDH with cyanogen bromide followed by separation and sequence analysis of the reactive peptide resulted in the identification of a single reactive peptide with the sequence of amino acid residues 45-103 of GAPDH. Analysis of aPSD-2 binding to synthetic peptides derived from the corresponding region localized the antibody-binding site to amino acid residues 70-94 of GAPDH. Both the 25-amino-acid synthetic peptide (amino acid residues 70-94 of GAPDH) and polyclonal antibody raised against this peptide inhibited GAPDH-induced membrane fusion, suggesting that these amino acid residues play a crucial role in this membrane fusion process.
Kaneda
M
M
Department of Inflammation Research, The Tokyo Metropolitan Institute of Medical Science (Rinshoken).
Takeuchi
K
K
Inoue
K
K
Umeda
M
M
D000595:Amino Acid Sequence; D000906:Antibodies; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000276:immunology; D003429:Cross Reactions; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D008561:Membrane Fusion / Q000502:physiology*; D008566:Membranes / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D010718:Phosphatidylserines / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding
D000906:Antibodies; D010446:Peptide Fragments; D010718:Phosphatidylserines; D010743:Phospholipids; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D002262:Carboxy-Lyases; C022353:phosphatidylserine decarboxylase