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GPIomics: global analysis of glycosylphosphatidylinositol-anchored molecules of Trypanosoma cruzi.

Nakayasu ES, Yashunsky DV, Nohara LL, Torrecilhas AC, Nikolaev AV, Almeida IC - Mol. Syst. Biol. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, The Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX 79968, USA.

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.

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Fractionation of the synthetic trypomastigote mucin GPI and by-products using POROS R1 ziptip. The synthetic mucin GPI (Man-[EtNP]Man-Man2-[AEP]GlcN-InsP-1-O-alkyl-C16:0-2-O-acyl -C18:2-Gro) was purified through POROS R1 50 resin as described in Materials and methods. The top panel represents the MS spectrum of the starting material, whereas the bottom four panels represent the MS spectra of the ziptip fractions eluted with 40, 50, 60, and 70% 2-propanol, respectively. All observed GPI species are doubly charged, plus or minus sodium adduct. lyso-GPI, GPI species missing the C18:2-fatty acid at C-2 of the glycerol backbone; m/z, mass-to-charge ratio.
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f1: Fractionation of the synthetic trypomastigote mucin GPI and by-products using POROS R1 ziptip. The synthetic mucin GPI (Man-[EtNP]Man-Man2-[AEP]GlcN-InsP-1-O-alkyl-C16:0-2-O-acyl -C18:2-Gro) was purified through POROS R1 50 resin as described in Materials and methods. The top panel represents the MS spectrum of the starting material, whereas the bottom four panels represent the MS spectra of the ziptip fractions eluted with 40, 50, 60, and 70% 2-propanol, respectively. All observed GPI species are doubly charged, plus or minus sodium adduct. lyso-GPI, GPI species missing the C18:2-fatty acid at C-2 of the glycerol backbone; m/z, mass-to-charge ratio.

Mentions: Next, we developed a polystyrene-based RPC and tested it using a synthetic GPI (Yashunsky et al, 2006) containing several by-products resulting from unsaturated fatty acid oxidation or removal during its prolonged storage as nuclear magnetic resonance (NMR) sample in [2H]6-DMSO solution. The GPI sample was loaded onto a ziptip manufactured with POROS R1 resin (C4 groups linked to polystyrene-divinylbenzene beads) and eluted step-wisely with increasing (40, 50, 60, and 70%) 2-propanol concentrations. Each fraction was then analyzed by negative-ion mode electrospray ionization (ESI)-MS (Materials and methods). The results clearly showed an efficient separation between the bona fide GPI species and lyso-GPIs, oxidized GPIs, and other by-products (Figure 1).


GPIomics: global analysis of glycosylphosphatidylinositol-anchored molecules of Trypanosoma cruzi.

Nakayasu ES, Yashunsky DV, Nohara LL, Torrecilhas AC, Nikolaev AV, Almeida IC - Mol. Syst. Biol. (2009)

Fractionation of the synthetic trypomastigote mucin GPI and by-products using POROS R1 ziptip. The synthetic mucin GPI (Man-[EtNP]Man-Man2-[AEP]GlcN-InsP-1-O-alkyl-C16:0-2-O-acyl -C18:2-Gro) was purified through POROS R1 50 resin as described in Materials and methods. The top panel represents the MS spectrum of the starting material, whereas the bottom four panels represent the MS spectra of the ziptip fractions eluted with 40, 50, 60, and 70% 2-propanol, respectively. All observed GPI species are doubly charged, plus or minus sodium adduct. lyso-GPI, GPI species missing the C18:2-fatty acid at C-2 of the glycerol backbone; m/z, mass-to-charge ratio.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683718&req=5

f1: Fractionation of the synthetic trypomastigote mucin GPI and by-products using POROS R1 ziptip. The synthetic mucin GPI (Man-[EtNP]Man-Man2-[AEP]GlcN-InsP-1-O-alkyl-C16:0-2-O-acyl -C18:2-Gro) was purified through POROS R1 50 resin as described in Materials and methods. The top panel represents the MS spectrum of the starting material, whereas the bottom four panels represent the MS spectra of the ziptip fractions eluted with 40, 50, 60, and 70% 2-propanol, respectively. All observed GPI species are doubly charged, plus or minus sodium adduct. lyso-GPI, GPI species missing the C18:2-fatty acid at C-2 of the glycerol backbone; m/z, mass-to-charge ratio.
Mentions: Next, we developed a polystyrene-based RPC and tested it using a synthetic GPI (Yashunsky et al, 2006) containing several by-products resulting from unsaturated fatty acid oxidation or removal during its prolonged storage as nuclear magnetic resonance (NMR) sample in [2H]6-DMSO solution. The GPI sample was loaded onto a ziptip manufactured with POROS R1 resin (C4 groups linked to polystyrene-divinylbenzene beads) and eluted step-wisely with increasing (40, 50, 60, and 70%) 2-propanol concentrations. Each fraction was then analyzed by negative-ion mode electrospray ionization (ESI)-MS (Materials and methods). The results clearly showed an efficient separation between the bona fide GPI species and lyso-GPIs, oxidized GPIs, and other by-products (Figure 1).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, The Border Biomedical Research Center, University of Texas at El Paso, El Paso, TX 79968, USA.

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.

Show MeSH
Related in: MedlinePlus