GPIomics: global analysis of glycosylphosphatidylinositol‐anchored molecules ofTrypanosoma cruzi

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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. ### Synopsis Glycosylphosphatidylinositol (GPI) anchoring is a common modification of proteins found on the surface of eukaryotic cells. In higher eukaryotes such as mammals, GPI biosynthesis is vital for embryonic development, and GPI‐anchored proteins participate in important biological processes such as cell–cell interactions, signal transduction, endocytosis, complement regulation, and antigenic presentation ([Paulick and Bertozzi, 2008][1]). In lower eukaryotes such as protozoan parasites (e.g., Trypanosoma cruzi , Trypanosoma brucei , Leishmania spp., and Plasmodium spp.), which cause major endemic human infectious diseases worldwide (e.g., Chagas disease, sleeping sickness, leishmaniasis, malaria), GPI‐anchored molecules extensively coat the parasite cell surface and actively participate in relevant parasite‐mammalian host interactions ([Ferguson, 1999][2]). T. cruzi is the etiologic agent of Chagas disease, or American trypanosomiasis, a neglected tropical disease that affects over 11 million people and causes an estimated 50 000 annual deaths in Latin America ([Dias et al , 2002][3]; [Barrett et al , 2003][4]; [Moncayo and Ortiz Yanine, 2006][5]). More recently, Chagas disease has become a public health menace for the U.S. and some European countries, where an increasing number of chronically T. cruzi‐ infected migrants from endemic countries are residing in ([Bern et al , 2007][6]; [Piron et al , 2008][7]). There are only two commercial drugs (Benznidazole and Nifurtimox) available for the treatment of Chagas disease, and both are partially effective and highly toxic. In addition, no human vaccine is currently available for treating or preventing Chagas disease ([Garg and Bhatia, 2005][8]; [Dumonteil, 2007][9]; [Hotez et al , 2008][10]). Therefore, there is an urgent need for new therapeutic targets against T. cruzi. In this regard, GPI‐anchored proteins and free GPI anchors seem to be very attractive targets for development of new therapies for preventing or treating Chagas disease. These glycoconjugates play a central role in the parasite infectivity and host immune response against this deadly pathogen ([Almeida and Gazzinelli, 2001][11]; [Buscaglia et al , 2006][12]; [Gazzinelli and Denkers, 2006][13]; [Acosta‐Serrano et al , 2007][14]). T. cruzi has four developmental stages or forms, two (i.e., epimastigote and metacyclic trypomastigote) dwelling in the hematophagous triatomine insect vector (a Reduviidae, popularly known as the kissing bug ), and two (i.e., amastigote and trypomastigote) in the mammalian host. The parasite can be transmitted by contaminated excrement of the insect vector, blood transfusion, organ transplantation, or congenitally. Each developmental stage of T. cruzi has been proposed to express a different subset of GPI‐anchored proteins on the cell surface. These proteins are encoded by thousands of members of multigene families, such as trans ‐sialidase (TS)/gp85 glycoprotein, mucin, mucin‐associated surface protein (MASP), and metalloproteinase gp63 ([Buscaglia et al , 2006][12]; [Acosta‐Serrano et al , 2007][14]). Although some of the expressed members (proteins) of these multigene families have been shown to be modified by GPI‐anchor addition, it has not been known how many of these gene products could possibly be GPI anchored. To answer this question, we performed a genome‐wise GPI‐anchoring prediction analysis. Here we show that approximately 12% of the annotated protein sequences of T. cruzi possibly code for GPI‐anchored proteins. This number is much higher compared with other lower and higher eukaryotes that have in average from 0.5 to 2% of proteins predicted to be GPI anchored. Despite the overall importance of GPI anchors, there is no universal methodology for the systematic analysis of these molecules. One of the major hurdles to develop a method for the large scale analysis of GPIs is their complex structure. The general structure of a GPI anchor comprises a hydrophobic lipid tail and a hydrophilic carbohydrate (glycan) core, which together provide a highly amphiphilic character for these molecules ([McConville and Ferguson, 1993][15]; [Ferguson, 1999][2]). The complex structure and amphiphilic nature of GPIs make their extraction and purification more difficult, and multiple techniques are required to determine their fine structure. To overcome this problem, here we have developed a fast, simple, and highly sensitive approach that uses liquid chromatography‐tandem mass spectrometry (LC‐MS n ) for the first large‐scale analysis of GPI‐anchored...