GPIomics: global analysis of glycosylphosphatidylinositol‐anchored molecules ofTrypanosoma cruzi

Abstract

Glycosylphosphatidylinositol (GPI) anchoring is a common, relevant posttranslational modification of eukaryotic surface proteins. Here, we developed a fast, simple, and highly sensitive (high attomole-low femtomole range) method that uses liquid chromatography-tandem mass spectrometry (LC-MS n ) for the first large-scale analysis of GPI-anchored molecules (i.e., the GPIome) of a eukaryote, Trypanosoma cruzi, the etiologic agent of Chagas disease. Our genome-wise prediction analysis revealed that approximately 12% of T. cruzi genes possibly encode GPI-anchored proteins. By analyzing the GPIome of T. cruzi insect-dwelling epimastigote stage using LC-MS n , we identified 90 GPI species, of which 79 were novel. Moreover, we determined that mucins coded by the T. cruzi small mucin-like gene (TcSMUG S) family are the major GPI-anchored proteins expressed on the epimastigote cell surface. TcSMUG S mucin mature sequences are short (56-85 amino acids) and highly O-glycosylated, and contain few proteolytic sites, therefore, less likely susceptible to proteases of the midgut of the insect vector. We propose that our approach could be used for the high throughput GPIomic analysis of other lower and higher eukaryotes.

The Protein Lipidation and Its Analysis

“…Alternatively, a LC-MS-based method was recently developed by Almeida and co-workers and applied for the structural characterization of the GPI units present in proteins. Using this approach, 90 GPI species could be identified of which 79 were novel [22]. Despite these advances, much work is still needed to elucidate the role of the GPI anchor and its involvement in cell function as well as in disease.…”

Structures of Glycolipids Found in Trypanosomatids: Contribution to Parasite Functions

“…cruzi GPI-anchored proteins are found in all evolutive forms and are encoded by thousands of members of multigene families, such as trans-sialidase (TS)/gp85 glycoprotein, mucin, mucin-associated surface proteins (MASP) and metalloproteinase gp63 [13]. Some of them, such as the TS/gp85 and mucins, have been shown to be very important for the infectivity of the parasite and for escaping the host immune response [8,[66][67][68][69][70].…”

“…TcSMUG comprises 70-80 genes that were originally divided in two groups (S for Small and L for Large) according to the size of their encoded mRNAs. Recently, the group S gene products have been identified as the major 35-50 kDa mucins expressed during the epimastigote stage [13].…”

“…A combination of isolation and separation technologies, as well as the use of mass spectrometry, for glycolipid structural characterization is required to unravel functional aspects of these cell surface molecules and to gain a better understanding of their role in infectious diseases. Considerable advances have recently been made in fields such as liquidchromatography-Mass spectrometry (LC-MS) of glycolipids from mammalian cells [139], GPI-anchored proteomics of Plamodium falciparum [140], GPIomics of T.cruzi [13] and glycolipid arrays to study antitoxic malaria response [141]. The combination of these sensitive and powerful techniques has allowed us to increase our structural and functional knowkedge of a wide variety of glycoconjugates an other macromolecules expressed by different protozoa.…”

Back to the future in Chagas disease: from animal models to patient cohort studies, progress in immunopathogenesis research

“…It is presently unknown if parasite clones from the major filogenetic lineages express variable levels of tGPI-mucin (Nakayasu et al 2009), other TLR ligands (Ouaissi et al 2002, Mathieu-Daudé et al 2007) and/or cruzipain isoforms . If so, parasite clones may display differential power to activate microvascular beds via the TLR2/CXCR2/cruzipain > kinin/B 2 R/B 1 R pathway.…”