Localization of the Phosphatidylserine-Binding Site of Glyceraldehyde-3-Phosphate Dehydrogenase Responsible for Membrane Fusion
Abstract
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 residue…
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GAPDH binds to the nuclear membrane via a conserved PS-binding domain located between amino acids 70-94.
“GAPDH protein has been reported to bind to the nuclear membrane via a conserved PS-binding domain, located between amino acids 70–94 (Supplementary Fig. 2c)39.”
From GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain (2021) · cites this paper as [39]
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- Authors
- Kaneda, M., Takeuchi, K.-I., Inoue, K., & Umeda, M.
- Affiliations
- Tokyo Metropolitan Institute of Medical Science (JP)
- The University of Tokyo (JP)
- ORCID
- Umeda, M.
- Venue
- Journal of Biochemistry
- Publisher
- Oxford University Press (OUP)
- Published
- 1997-12-01
- Volume
- 122
- Issue
- 6
- Pages
- 1233-1240
- ISSN
- 0021-924X (ISSN-L)
- OpenAlex
- W2029165599
- Cited by
- 31
- References
- 0
- Citations by year
- 2026: 1 · 2024: 1 · 2021: 3 · 2020: 2 · 2019: 1 · 2018: 1 · 2017: 4 · 2016: 1 · 2015: 1 · 2014: 2 · 2012: 3
- FWCI
- 0.36
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- The Journal of Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Lipid Membrane Structure and Behavior
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Sphingolipid Metabolism and Signaling
- Keywords
- glyceraldehyde-3-phosphate dehydrogenase, phosphatidylserine binding, membrane fusion, phosphatidylserine decarboxylase, polyclonal antibodies, binding site localization, vesicle fusion, synthetic peptides
- MeSH terms
- Amino Acid Sequence, Antibodies, Binding Sites, Carboxy-Lyases, Cross Reactions, Glyceraldehyde-3-Phosphate Dehydrogenases, Kinetics, Membrane Fusion, Membranes, Molecular Sequence Data, Peptide Fragments, Phosphatidylserines, Phospholipids, Protein Binding
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