Characterization of the First Secreted Sorting Nexin Identified in the Leishmania Protists.

Proteins of the sorting nexin (SNX) family present a modular structural architecture with a phox homology (PX) phosphoinositide (PI)-binding domain and additional PX structural domains, conferring to them a wide variety of vital eukaryotic cell's functions, from signal transduction to membrane deformation and cargo binding. Although SNXs are well studied in human and yeasts, they are poorly investigated in protists. Herein, is presented the characterization of the first SNX identified in Leishmania protozoan parasites encoded by the Ld BPK_352470 gene. In silico secondary and tertiary structure prediction revealed a PX domain on the N-terminal half and a Bin/amphiphysin/Rvs (BAR) domain on the C-terminal half of this protein, with these features classifying it in the SNX-BAR subfamily of SNXs. We named the Ld BPK_352470.1 gene product Ld SNXi, as it is the first SNX identified in Leishmania ( L. ) donovani. Its expression was confirmed in L. donovani promastigotes under different cell cycle phases, and it was shown to be secreted in the extracellular medium. Using an in vitro lipid binding assay, it was demonstrated that recombinant (r) Ld SNXi (rGST- Ld SNXi) tagged with glutathione-S-transferase (GST) binds to the PtdIns3 P and PtdIns4 P PIs. Using a specific a- Ld SNXi antibody and immunofluorescence confocal microscopy, the intracellular localization of endogenous Ld SNXi was analyzed in L. donovani promastigotes and axenic amastigotes. Additionally, r Ld SNXi tagged with enhanced green fluorescent protein (r Ld SNXi-EGFP) was heterologously expressed in transfected HeLa cells and its localization was examined. All observed localizations suggest functions compatible with the postulated SNX identity of Ld SNXi. Sequence, structure, and evolutionary analysis revealed high homology between Ld SNXi and the human SNX2, while the investigation of protein-protein interactions based on STRING (v.11.5) predicted putative molecular partners of Ld SNXi in Leishmania .