Proteomics

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Plasmodium spp. Glyceraldehyde 3-phosphate dehydrogenase: Diverse subcellular localization and multiple post-translational modifications


ABSTRACT: Glyceraldehyde 3-phosphate dehydrogenase (Gapdh), a glycolytic enzyme is known to be associated with multiple non-glycolytic functions. To examine the possibility of Plasmodium spp Gapdh having moonlighting functions, PfGapdh was cloned, overexpressed, purified to homogeneity and characterized. Polyclonal antibodies were raised in mice and used for immuno detection. Western analysis of P. yoelii whole cell extract, soluble and particulate fractions showed three major species with molecular mass ~27, 37 and 51 kDa. All three forms were equally abundant in the soluble fraction. Particulate fraction that accounted for ~10-15% of total PyGapdh did not show ~51 kDa form. Immunological evidence suggests~51 kDa form to arise by ubiquitination of native 37kDa form. For determining subcellular distribution, P. yoelii cells were subjected to differential detergent fractionation and analyzed using two-dimensional gel electrophoresis. Various subcellular fractions showed multiple species of Gapdh, indicating multiple post-translational modifications. For the determination of the nature of PTMs and residues modified, ‘Top-Down’ and ‘Bottom-Up’ approaches were used. Spots in a 2D gel that showed high intensity in western were excised and analyzed for identification of protein as well as PTMs. For more comprehensive analysis of PTM, P. yoelii whole cell extract was run on a 1D gel and bands that showed reactivity in western were cut out and analyzed by LC-MS/MS. In all, eleven residues were identified that underwent modifications. Modifications consisted of four phosphorylations, five ubiquitinations, two acetylations, two methylations and one nitrosylation. Physiological relevance of these modifications is not yet understood

INSTRUMENT(S): 6520 Quadrupole Time-of-Flight LC/MS

ORGANISM(S): Plasmodium Yoelii Yoelii

DISEASE(S): Plasmodium Falciparum Malaria

SUBMITTER: Nitin Jindal  

LAB HEAD: Gotam K Jarori

PROVIDER: PXD002313 | Pride | 2018-01-08

REPOSITORIES: Pride

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