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Transglutaminase 2-dependent Deamidation of Glyceraldehyde-3-phosphate Dehydrogenase Promotes Trophoblastic Cell Fusion
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a 36-kDa protein with pleiotropic functions as well as the classical role in glycolysis. We found that GAPDH in the plasma membrane of BeWo choriocarcinoma cells displayed a striking acidic shift, which was represented by several spots in two-dimensional electrophoresis, after cell-cell fusion induction by forskolin. The acidic isoforms of GAPDH were separated by an OFFGEL Fractionator and subjected to structural characterization. Liquid chromatography-nanoelectrospray ionization mass spectrometry (LC-nanoESI MS) suggested an increase by 1 Da each for the acidic shift of the molecule. Tandem MS of tryptic peptides identified conversion of Glu for Gln at residues 78 and 204, and the same post-translational modification was suggested for all Gln residues to various extent. The Gln deamidation is catalyzed by transglutaminase (TG) family proteins, among which TG2 was expressed in BeWo cells. Finally, TG inhibitors, knockdown of the TG2 gene by short hairpin RNA, site-directed mutagenesis of GAPDH and microscopic observations revealed that the Gln/Glu deamidated GAPDH accumulated in the plasma membrane binds F-actin via an electrostatic interaction and thereby participates in the cytoskeletal remodeling required for cell fusion. This study revealed a novel function of GAPDH in cellular fusion. [K. Iwai et al., J. Biol. Chem. 289, 4989-4999 (2014)]