Identification of Leishmania-specific protein phosphorylation sites by LC-ESI-MS/MS and comparative genomics analyses.
Human pathogenic protozoa of the genus Leishmania undergo various developmental transitions during the infectious cycle that are triggered by changes in the host environment. How these parasites sense, transduce, and respond to these signals is only poorly understood. Here we used phosphoproteomic approaches to monitor signaling events in L. donovani axenic amastigotes, which may be important for intracellular parasite survival. LC-ESI-MS/MS analysis of IMAC-enriched phosphoprotein extracts identified 445 putative phosphoproteins in two independent biological experiments. Functional enrichment analysis allowed us to gain insight into parasite pathways that are regulated by protein phosphorylation and revealed significant enrichment in our data set of proteins whose biological functions are associated with protein turn-over, stress response, and signal transduction. LC-ESI-MS/MS analysis of TiO(2)-enriched phosphopeptides confirmed these results and identified 157 unique phosphopeptides covering 181 unique phosphorylation sites in 126 distinct proteins. Investigation of phosphorylation site conservation across related trypanosomatids and higher eukaryotes by multiple sequence alignment and cluster analysis revealed L. donovani-specific phosphoresidues in highly conserved proteins that share significant sequence homology to orthologs of the human host. These unique phosphorylation sites reveal important differences between host and parasite biology and post-translational protein regulation, which may be exploited for the design of novel anti-parasitic interventions.
Institut Pasteur, Plate-forme de Protéomique and CNRS URA 2185, Department of Structural Biology and Chemistry, Pasteur-Genopole Ile-de-France, Paris, France.
Sonia
S
Hem
Pier Federico
PF
Gherardini
José
J
Osorio y Fortéa
Veronique
V
Hourdel
Miguel A
MA
Morales
Reiko
R
Watanabe
Pascale
P
Pescher
Michael A
MA
Kuzyk
Derek
D
Smith
Christoph H
CH
Borchers
Dan
D
Zilberstein
Manuela
M
Helmer-Citterich
Abdelkader
A
Namane
Gerald F
GF
Späth
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods*; D016000:Cluster Analysis; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional; D005656:Fungal Proteins; D006801:Humans; D007891:Leishmania / Q000737:chemistry* / Q000378:metabolism; D008018:Life Cycle Stages; D051379:Mice; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
D005656:Fungal Proteins; D010750:Phosphoproteins