(A) Gel stained with Flamingo for total protein visualization and (B,C) ECLs of a Western blot probed with the PC-specific antibody TEPC15

(A) Gel stained with Flamingo for total protein visualization and (B,C) ECLs of a Western blot probed with the PC-specific antibody TEPC15. AZD1152-HQPA (Barasertib) identified as eEF1. We could demonstrate that this binding of eEF1 to one of its downstream effectors is dependent on its PC-modification. In this study we provide evidence that in this parasite the modification of eEF1 with PC may be essential for its function as an important virulence factor. and [9]. Open in a separate window Physique 1 Structure of the phosphocholine functionality with R being an [25], [26,27], and [27], indicating that arthro-series glycosphingolipids carrying, in part, PC substituents represent highly conserved glycolipid markers within the nematode phylum. A biosynthetic route homologous to glycosphingolipids was also confirmed for the free-living nematode [23,28,29]. Analogous analyses of the AZD1152-HQPA (Barasertib) PC-substituted glycoprotein ES-62, an excretory/secretory (ES) product of and confirmed a high conservation of such PC-substituted hybrid-type bi- and triantennary [37]. Fourteen putative proteins carrying the PC modification were identified, among them, proteins that are located on the surface of the parasite, or are involved in metabolism. Erythrocyte membrane protein 1 (EMP1, a member of the gene family), together with the heat shock protein 70 (HSP-70), was detected in every stage of the erythrocyte pathway. In genes encode adhesive proteins that are transported to the surface of infected erythrocytes, thereby acting as major virulence determinants for immune evasion [38,39]. There is increasing evidence that HSP-70 could play an important role in the life cycle of both as a chaperone and as immunogen [40]. In the merezoite-stage, only one surface protein (EMP1, P154varH), was detected. The eukaryotic elongation factor-1 (eEF1) is an enzyme that catalyzes the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein synthesis and is involved in the capture of deacylated tRNA [41,42]. Furthermore, eEF1 was found to serve as a central hub in protein networks with hundreds of interacting partners [43,44]. eEF1 was found to bind and activate the Src-homology 2 domain name containing protein tyrosine phosphatase-1 (SHP-1), a protein known to be involved in the macrophage inactivation pathogenesis of leishmaniasis [41]. Additionally, eEF1 was found in exosomes and identified as an important factor for immunosuppression AZD1152-HQPA (Barasertib) and priming host cells for invasion [41,45]. In this study, we identified eEF1 as the only PC-positive protein found in MON-1 by a 2D-gel proteomic approach. Furthermore, we confirmed the presence of PC modifications by quantitative determination of its choline content. Additionally, we localized the PC epitopes within procyclic and stationary phase promastigotes by confocal microscopy. Finally, we were able to demonstrate that this conversation of EF1 and human SHP-1 is dependent on the PC modification of EF1. 2. Materials and Methods 2.1. Cultivation of Leishmania infantum Promastigotes zymodeme MON-1 promastigotes were cultured in TobieCEvans altered medium at 24 C [46]. Promastigotes at day 3 or 10 of culture were used. The liquid phases were centrifuged at 1000 for 5 ATF3 min at 4 C. Supernatant was then discarded and the pellet was resuspended in phosphate buffered saline (PBS) pH 7.2, and washed 3 times by centrifugation at 1000 for 10 min. 2.2. Immunofluorescence promastigotes were washed twice in PBS before fixation in 200 L of 1% formaldehyde in PBS for 30 min at room heat (RT). After a PBS wash, the cells were permeabilized by resuspension in 200 L of 0.1% Triton X-100 in PBS for 10 min. Following an additional PBS wash, the cells were resuspended in 200 L of 0.1 M glycine in PBS and incubated for a further 10 min at RT before being washed in PBS. Glass slides were washed with 70% ethanol and coated with a 0.01% solution of poly-l-lysine (0.1% stock; Sigma Alrich, Taufkirchen, Germany), and the fixed, permeabilized cells were then left to sediment and adhere to the surfaces of these polylysine-coated slides for 15 min at RT. Monoclonal mouse TEPC-15 (Sigma Aldrich, Taufkirchen, Germany) diluted 1:1000 in TB buffer (0.1% (promastigotes. Approximately 10 mg of pelleted promastigotes were homogenized in 30 L 0.2% (were separated on a 2D-gel as described above and stained with FlamingoTM according to.