title: Phosphoproteomic analysis of differentiating Leishmania parasites reveals a unique stage-specific phosphorylation motif.
abstract: Protists of the genus Leishmania are obligatory intracellular parasites that cause a wide range of cutaneous, mucocutaneous, and visceral diseases in humans. They cycle between phagolysosomes of mammalian macrophages and the sand fly midgut, proliferating as intracellular amastigotes and extracellular promastigotes, respectively. Exposure to a lysosomal environment, i.e. acidic pH and body temperature, signals promastigotes to differentiate into amastigotes. Time course analyses indicated that Leishmania differentiation is a highly regulated and coordinated process. However, the role of posttranslational events such as protein phosphorylation in this process is still unknown. Herein, we analyzed and compared the phosphoproteomes of L. donovani amastigotes and promastigotes using an axenic host-free system that simulates parasite differentiation. Shotgun phosphopeptide analysis revealed 1614 phosphorylation residues (p-sites) corresponding to 627 proteins. The analysis indicated that the majority of the p-sites are stage-specific. Serine phosphorylation in a previously identified trypanosomatid-specific "SF" motif was significantly enriched in amastigotes. We identified a few phosophotyrosines (pY), mostly in proteins known to participate in signal transduction pathways. The analysis indicated that Leishmania contains proteins with multiple p-sites that are phosphorylated at distinct stages of the life cycle. For over half of the phosphorylation events, changes in phosphoprotein abundance did not positively correlate with changes in protein abundance, suggesting functional regulation. This study compares, for the first time, the phosphoproteins of L. donovani axenic promastigotes and amastigotes and provides the largest data set of the Leishmania phosphoproteome to date.
authors.affiliation: Technion-Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: Polina
authors.initials: P
authors.lastname: Tsigankov
authors.forename: Pier Federico
authors.initials: PF
authors.lastname: Gherardini
authors.forename: Manuela
authors.initials: M
authors.lastname: Helmer-Citterich
authors.forename: Gerald F
authors.initials: GF
authors.lastname: Späth
authors.forename: Dan
authors.initials: D
authors.lastname: Zilberstein
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics*; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D006801:Humans; D007891:Leishmania / Q000254:growth & development / Q000378:metabolism*; D008018:Life Cycle Stages; D010271:Parasites / Q000737:chemistry / Q000378:metabolism; D010588:Phagosomes / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000302:isolation & purification; D010766:Phosphorylation; D040901:Proteomics
chemical_list: D000596:Amino Acids; D010750:Phosphoproteins