Title : O-GlcNAcylation disrupts
glyceraldehyde-3-phosphate dehydrogenase homo-tetramer formation and mediates its nuclear translocation
Abstract :
- Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) is a typical glycolytic enzyme comprised of four identical 37 kDa subunits
- In addition to its glycolytic function, GAPDH has a number of biological functions which are related to its subcellular localization
- Generally, protein O-linked N-acetylglucosamine modification (O-GlcNAcylation) is considered, among other effects, to mediate the nuclear transportation of cytosolic proteins
- To elucidate the effect of O-GlcNAcylation on GAPDH , we determined the location of the O-GlcNAcylation site by tandem mass spectrometry, and subsequently examined the biological significance of this derivatization
- The site involved was identified to be Thr227 by beta-elimination and Michael addition
- Transient transfection assays demonstrated that the T227A mutation induced the cytoplasmic accumulation of GAPDH , whereas the wild type was present in the cytoplasm and nuclei
- Structural modeling, mutagenesis of Thr227 to Lys and Arg , and gel filtration chromatography of mutated and wild type GAPDH , together suggested that O-GlcNAcylation at Thr227 interrupts the hydrophobic interfaces formed between GAPDH monomers in its tetrameric state
- The present study identified Thr227 as the major GAPDH O-GlcNAcylation site , which suggests that this modification mediates the nuclear translocation of GAPDH , presumably by disrupting the conformation of tetrameric GAPDH