Se sequences and also the recognized structure of the GPI anchor in
Se sequences as well as the identified structure on the GPI anchor in this parasite (Figure 1A) [3], we proposed that the T. cruzi GPI biosynthetic pathway occurs in the ER based on the diagram shown in Figure 1B. Dolichol-phosphate mannose synthase (DPM1), also named dolichol-phosphate-b-D-mannosyltransferase, catalyses the transfer of a mannose residue from GDP-mannose to dolicholphosphate (Dol-P) generating Dol-P-mannose, utilized as a donor for all mannosylation reactions that are a part of the GPI biosynthetic pathway [40], [41]. Comparisons among DPM1 of a variety of organisms [42], [43], [44] showed that, collectively with S. cerevisiae, T. brucei, and Leishmania mexicana [45] and in contrast to P. falciparum DPM1, T. cruzi DPM1 belongs to a group that incorporates monomeric enzymes that have a C-terminal hydrophobic tail. The glycosyltransferase complex that’s responsible for ALDH2 custom synthesis transferring Nacetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol (PI) to produce N-acetylglucosaminyl-PI (GlcNAc-PI) has six and seven proteins, respectively, in yeast and mammalian cells [16]. TcGPI3 was identified because the gene encoding the catalytic subunit with the T. cruzi glycosyltransferase complex since it shares 41 and 49 of sequence identity with the yeast GPI3 and mammalian PIG-A, respectively. Amongst other components from the glycosyltransferase complex present in yeast, we identified the T. cruzi orthologs of GPI1, GPI2, GPI15, and GPI19. In mammalian cells, DPM2, a non-catalytic subunit of dolichol-P-mannose synthase, is physically connected with PIG-A, PIG-C and PIG-Q and enhances GlcNAc-PI transferase activity [46]. A T. cruzi gene encoding a protein with 17 identity to human DPM2 and containing a DPM2 domain, which probably acts as a regulatory component of the N-acetyl-glucosamine transferase complicated, was also identified. Only 1 element of this complicated, named ERI1 in yeast [47], and PIG-Y in mammals [48], was not identified either in T. cruzi, P. falciparum or T. brucei. The T. cruzi ortholog of yeast GPI12 (named PIG-L in mammals) [49], encoding theDisruption of T. cruzi genesDNA constructs developed to delete each TcGPI8 alleles in the T. cruzi CL Brener genome by homologous recombination have been prepared soon after PCR amplification of the 59 and 39 regions from the TcGPI8 gene (for primer sequences, see Table S1). The generated PCR products (with 487 bp and 647 bp, respectively) had been cloned sequentially into the SacISpeI and XhoIXbaI web pages of pCR2.1 TOPO vector (Invitrogen), flanking the neomycin phosphotransferase (NeoR) or hygromycin phosphotransferase (HygR) resistance markers that were cloned into this vector. To enhance mRNA expression within the parasite, the 39 UTR plus downstream intergenic sequences on the T. cruzi gliceraldehyde-3-phosphate dehydrogenase (gapdh) gene was inserted downstream in the HygR marker. Similar constructs making use of 59 and 39 flanking sequences derived from TcGPI3 and TcGPI10 genes have been generated. Epimastigote transfections had been performed by electroporation with 50 mg DNA as described previously [37]. ALDH1 Storage & Stability Twenty-four hours immediately after transfection, 200 mgml of hygromycin B or G418 was added to the cultures and selected populations have been obtained around 30 days following transfection. Cloned cell lines have been obtained by plating on semisolid blood agar plates, immediately after a further 30 days of incubation at 28uC.Electron microscopy analyses of T. cruziEpimastigotes have been fixed in 5 glutaraldehyde in 0.1 M cacodylate buffer pH 7.two and processed fol.