Localization and Developmental Regulation of a Dispersed Gene Family 1 Protein in Trypanosoma cruzi

The dispersed gene family 1 (DGF-1) is the fifth largest gene family in the Trypanosoma cruzi genome, with over 500 members (11). Many of the predicted DGF-1 protein products have several transmembrane domains and N-glycosylation and phosphorylation sites and were thought to localize in the plasma membrane. Here, we report that affinity-purified antibodies against a region of one of these proteins (DGF-1.2) localized it intracellularly in different stages of the parasite. DGF-1.2 is more abundant in the amastigote stage than in trypomastigotes and epimastigotes, as detected by immunofluorescence and Western blot analyses. The protein changed localization during intracellular or extracellular differentiation from the trypomastigote to the amastigote stage, where it finally localized to small bodies in close contact with the inner side of the amastigote plasma membrane. DGF-1.2 did not colocalize with markers of other subcellular organelles, such as acidocalcisomes, glycosomes, reservosomes, lipid droplets, or endocytic vesicles. During extracellular differentiation, the protein was detected in the culture medium from 0 to 22 h, peaking at 14 h. The presence of DGF-1.2 in the differentiation culture medium was confirmed by mass spectrometry analysis. Finally, when epimastigotes were subjected to starvation, there was a decrease in the labeling of the cells and, in Western blots, the appearance of bands of lower molecular mass, suggesting its cleavage. These results represent the first report of direct immunodetection and developmental expression and secretion of a DGF-1 protein.Trypanosoma cruzi is the causative agent of Chagas disease, an endemic illness affecting between 16 and 18 million people in North, Central, and South America for which no vaccine or satisfactory treatment is available (22). During its life cycle, the parasite goes through different stages in the vector (epimastigotes and metacyclic trypomastigotes) and in the mammalian host (amastigotes and bloodstream trypomastigotes). As part of its survival strategy in these varying environments, the parasite has developed a large repertoire of multigene families (9, 11, 12, 16). Among these families, the dispersed gene family 1 (DGF-1) has approximately 565 copies, ranging from 6 to 10 kbp, dispersed throughout the parasite genome (11). The members of the DGF-1 family encode proteins that share 85 to 95% sequence identity (11). Wincker and colleagues first identified clones bearing a common repeated sequence from a T. cruzi genome library (24) and later described the nucleotide sequence of a representative gene (DGF-1.1) (23). They concluded, from in silico studies, that DGF-1 genes encoded putative cell surface proteins (23). In 2005, Kim and colleagues (16) described a new member of this family (DGF-1.2) located in the subtelomeric region of a T. cruzi chromosome surrounded by mainly two kinds of sequences: genes encoding the trans-sialidase (TS) and retrotransposon hot spot (RHS) protein families. The sizes of the open reading frames (ORFs) of DGF-1 genes and their abundance in the T. cruzi genome suggested that they are essential sequences for parasite survival. Furthermore, the existence of some telomeric DGF-1 copies that were always flanked by pseudogenes suggested that these genes have been subjected to strong selective pressure and, as a consequence, that they should be expressed at some life cycle stage of the parasite (16).A glycopeptide shared by several members of the DGF-1 family was recently detected in a glycoproteomic study of T. cruzi trypomastigotes, demonstrating that at least a DGF-1 family member protein is actually expressed and N-glycosylated (3). We also detected a number of peptides corresponding to several DGF-1 family member proteins in a proteomic study of acidocalcisome fractions of epimastigotes of T. cruzi (R. Docampo, J. A. Atwood, R. Tarleton, and R. Orlando, unpublished data). However, this family of proteins has no known orthologs in other species, even in trypanosomatids, and little is known about their localization, expression patterns, and functions in T. cruzi.In the present work, we prepared affinity-purified antibodies against a peptide of the DGF-1.2 protein and investigated its subcellular localization by immunofluorescence microscopy. We also used mass spectrometry (MS) to identify specific peptides recognized by anti-DGF-1.2 antibodies by using fingerprinting analysis.We found that the antibodies preferentially labeled amastigotes. The localization of the protein was in intracellular bodies and not on the cell surface and changed during amastigote development. During the in vitro trypomastigote-to-amastigote transition, we detected continuous secretion of DGF-1.2 into the medium, peaking at 14 h. Anti DGF-1 antibodies that recognized the intracellular protein in both differentiation forms also recognized the secreted protein from trypomastigotes and amastigotes. Finally, when epimastigotes were subjected to starvation, there was a decrease in labeling of the cells and the appearance of defined bands of smaller molecular mass in Western blots, suggesting its cleavage.