The Giardia duodenalis 14-3-3 protein is post-translationally modified by phosphorylation and polyglycylation of the C-terminal tail.
The flagellated protozoan Giardia duodenalis (syn. lamblia or intestinalis) has been chosen as a model parasite to further investigate the multifunctional 14-3-3s, a family of highly conserved eukaryotic proteins involved in many cellular processes, such as cell cycle, differentiation, apoptosis, and signal transduction pathways. We confirmed the presence of a single 14-3-3 homolog gene (g14-3-3) by an in silico screening of the complete genome of Giardia, and we demonstrated its constitutive transcription throughout the life stages of the parasite. We cloned and expressed the g14-3-3 in bacteria, and by protein-protein interaction assays we demonstrated that it is a functional 14-3-3. Using an anti-peptide antibody raised against a unique 18-amino acid sequence at the N terminus, we observed variations both in the intracellular localization and in the molecular size of the native g14-3-3 during the conversion of Giardia from trophozoites to the cyst stage. An affinity chromatography, based on the 14-3-3 binding to the polypeptide difopein, was set to purify the native g14-3-3. By matrix-assisted laser desorption ionization mass spectroscopy analysis, we showed that polyglycylation, an unusual post-translational modification described only for tubulin, occurred at the extreme C terminus of the native g14-3-3 on Glu246, Glu247, or both and that the Thr214, located in the loop between helices 8 and 9, is phosphorylated. We propose that the addition of the polyglycine chain can promote the binding of g14-3-3 to alternative ligands and that the differential rate of polyglycylation/deglycylation during the encystation process can act as a novel mechanism to regulate the intracellular localization of g14-3-3.
Lalle
Marco
M
Department of Infectious, Parasitic and Immunomediated Diseases and Department of Environment and Primary Prevention, Istituto Superiore di Sanità, 00161 Rome, Italy.
Salzano
Anna Maria
AM
Crescenzi
Marco
M
Pozio
Edoardo
E
D048948:14-3-3 Proteins / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D015152:Blotting, Northern; D015153:Blotting, Western; D002453:Cell Cycle; D002454:Cell Differentiation; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D003720:Densitometry; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D005872:Giardia / Q000378:metabolism*; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014404:Tubulin / Q000737:chemistry
D048948:14-3-3 Proteins; D010455:Peptides; D011994:Recombinant Proteins; D014404:Tubulin; C011080:polyglycine