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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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Related in: MedlinePlus

Analysis by LC-MS2 and LC-MS3 of GPI-peptides released by trypsin treatment. (A) MS2 spectrum of the GPI-peptide species at m/z 1188.5, which corresponds to APTPGD-EtNP-Hex4-[AEP]HexN-InsP-1-O-alkyl-C16:0-2-O -acyl-C16:0-Gro. (B) MS3 spectrum of the fragment corresponding to the peptide attached to dehydrated EtN at m/z 582.4. The peptide fragments are indicated. (C) Proposed fragmentation and structure of the GPI-peptide APTPGD-EtNP-Hex4-[AEP]HexN-InsP-1-O-alkyl-C16:0-2-O -acyl-C16:0-Gro. APTPGD, Ala-Pro-Thr-Pro-Gly-Asp.
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f4: Analysis by LC-MS2 and LC-MS3 of GPI-peptides released by trypsin treatment. (A) MS2 spectrum of the GPI-peptide species at m/z 1188.5, which corresponds to APTPGD-EtNP-Hex4-[AEP]HexN-InsP-1-O-alkyl-C16:0-2-O -acyl-C16:0-Gro. (B) MS3 spectrum of the fragment corresponding to the peptide attached to dehydrated EtN at m/z 582.4. The peptide fragments are indicated. (C) Proposed fragmentation and structure of the GPI-peptide APTPGD-EtNP-Hex4-[AEP]HexN-InsP-1-O-alkyl-C16:0-2-O -acyl-C16:0-Gro. APTPGD, Ala-Pro-Thr-Pro-Gly-Asp.

Mentions: In T. cruzi, mucins are one of the major GPI-anchored antigens expressed on the parasite cell surface, and they are involved in the escape from host immune response as well as adhesion and invasion of host cells (Acosta-Serrano et al, 2001; Buscaglia et al, 2006; Acosta-Serrano et al, 2007). T. cruzi mucins are encoded by at least 863 genes, grouped in two major families (i.e., TcMUC and TcSMUG) (Buscaglia et al, 2006; Acosta-Serrano et al, 2007). We had shown earlier that we could identify the mucins expressed by the infective trypomastigote stage through the analysis of a short (3–4-mer) peptide containing the ω site still attached to the GPI anchor (GPI-peptide) (Buscaglia et al, 2004). Here, we exploited this approach to sequence the GPI-peptide derived from mucins of noninfective epimastigotes and, therefore, to determine which gene family(ies) is(are) expressed on this parasite stage. Although epimastigote mucins are somewhat resistant to different proteases (e.g., trypsin, proteinase K) as observed by SDS–PAGE (not shown), the C-terminal region seems to be less glycosylated, thus, susceptible to proteolytic digestion. Thus, the mucin-rich (9% n-butanol) extract from T. cruzi epimastigotes was digested with trypsin and analyzed by LC-MS2 and LC-MS3 (Figure 3A). On the basis of the structures identified from the proteinase K-treated samples, we could rapidly match several fragments, such as AAG (m/z 537.6), AEP-HexN-PI (m/z 1065.5), Hex(AEP)HexN-PI (m/z 1227.6), and Hex2(AEP)HexN-PI (m/z 1389.7), thus partially determining the GPI-peptide structure (Figure 4A). By combining the partially determined GPI-structure and the possible peptides that could be generated by tryptic digestion and being attached to a GPI anchor (using the GPI-anchoring prediction), one peptide candidate could be the APTPGD sequence from mucin TcSMUG S. Indeed, the fragmentation of GPI-peptide species showed abundant fragments corresponding to the mass of this peptide attached to dehydrated EtN, EtNP, or EtNP-Hex1–4 (Figure 4A), similar to the fragmentation pattern described elsewhere (Redman et al, 1994). However, the fragmentation of the peptide moiety in the MS2 was very poor impairing the sequence confirmation. To ultimately sequence the peptide and determine the ω site, fragments corresponding to the peptide attached to dehydrated EtN or AEP were subjected to data-independent MS3 analysis (Figure 4B; Supplementary Figure 4). The quality of MS3 spectra were good enough to enable the de novo sequencing of the peptide. With this approach, we could sequence peptides attached to five different GPI structures, four of them also detected after proteinase K digestion (Figure 4A–C; Table II; Supplementary Table I; Supplementary Figure 4). Although five GPI species were found, all these species were attached to the same peptide sequence (APTPGD), which corresponds to the carboxyl terminus of the TcSMUG S subfamily of mucins from T. cruzi (El-Sayed et al, 2005; Buscaglia et al, 2006; Acosta-Serrano et al, 2007). This result was corroborated by amino-acid compositional analysis (not shown). The TcSMUG S subfamily comprises eight unique sequences (GenBank accession numbers XP_804663.1, XP_807370.1, XP_807371.1, XP_821038.1, XP_821039.1, XP_821040.1, XP_821041.1, and XP_821042.1), with predicted mature protein sequences varying from 56 to 85 amino acids. TcSMUG S sequences contain approximately 40% threonine in their composition (Buscaglia et al, 2004), suggesting that they could be heavily O-glycosylated and, therefore, quite resistant to protease digestion as we have already observed (data not shown).


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)

Analysis by LC-MS2 and LC-MS3 of GPI-peptides released by trypsin treatment. (A) MS2 spectrum of the GPI-peptide species at m/z 1188.5, which corresponds to APTPGD-EtNP-Hex4-[AEP]HexN-InsP-1-O-alkyl-C16:0-2-O -acyl-C16:0-Gro. (B) MS3 spectrum of the fragment corresponding to the peptide attached to dehydrated EtN at m/z 582.4. The peptide fragments are indicated. (C) Proposed fragmentation and structure of the GPI-peptide APTPGD-EtNP-Hex4-[AEP]HexN-InsP-1-O-alkyl-C16:0-2-O -acyl-C16:0-Gro. APTPGD, Ala-Pro-Thr-Pro-Gly-Asp.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Analysis by LC-MS2 and LC-MS3 of GPI-peptides released by trypsin treatment. (A) MS2 spectrum of the GPI-peptide species at m/z 1188.5, which corresponds to APTPGD-EtNP-Hex4-[AEP]HexN-InsP-1-O-alkyl-C16:0-2-O -acyl-C16:0-Gro. (B) MS3 spectrum of the fragment corresponding to the peptide attached to dehydrated EtN at m/z 582.4. The peptide fragments are indicated. (C) Proposed fragmentation and structure of the GPI-peptide APTPGD-EtNP-Hex4-[AEP]HexN-InsP-1-O-alkyl-C16:0-2-O -acyl-C16:0-Gro. APTPGD, Ala-Pro-Thr-Pro-Gly-Asp.
Mentions: In T. cruzi, mucins are one of the major GPI-anchored antigens expressed on the parasite cell surface, and they are involved in the escape from host immune response as well as adhesion and invasion of host cells (Acosta-Serrano et al, 2001; Buscaglia et al, 2006; Acosta-Serrano et al, 2007). T. cruzi mucins are encoded by at least 863 genes, grouped in two major families (i.e., TcMUC and TcSMUG) (Buscaglia et al, 2006; Acosta-Serrano et al, 2007). We had shown earlier that we could identify the mucins expressed by the infective trypomastigote stage through the analysis of a short (3–4-mer) peptide containing the ω site still attached to the GPI anchor (GPI-peptide) (Buscaglia et al, 2004). Here, we exploited this approach to sequence the GPI-peptide derived from mucins of noninfective epimastigotes and, therefore, to determine which gene family(ies) is(are) expressed on this parasite stage. Although epimastigote mucins are somewhat resistant to different proteases (e.g., trypsin, proteinase K) as observed by SDS–PAGE (not shown), the C-terminal region seems to be less glycosylated, thus, susceptible to proteolytic digestion. Thus, the mucin-rich (9% n-butanol) extract from T. cruzi epimastigotes was digested with trypsin and analyzed by LC-MS2 and LC-MS3 (Figure 3A). On the basis of the structures identified from the proteinase K-treated samples, we could rapidly match several fragments, such as AAG (m/z 537.6), AEP-HexN-PI (m/z 1065.5), Hex(AEP)HexN-PI (m/z 1227.6), and Hex2(AEP)HexN-PI (m/z 1389.7), thus partially determining the GPI-peptide structure (Figure 4A). By combining the partially determined GPI-structure and the possible peptides that could be generated by tryptic digestion and being attached to a GPI anchor (using the GPI-anchoring prediction), one peptide candidate could be the APTPGD sequence from mucin TcSMUG S. Indeed, the fragmentation of GPI-peptide species showed abundant fragments corresponding to the mass of this peptide attached to dehydrated EtN, EtNP, or EtNP-Hex1–4 (Figure 4A), similar to the fragmentation pattern described elsewhere (Redman et al, 1994). However, the fragmentation of the peptide moiety in the MS2 was very poor impairing the sequence confirmation. To ultimately sequence the peptide and determine the ω site, fragments corresponding to the peptide attached to dehydrated EtN or AEP were subjected to data-independent MS3 analysis (Figure 4B; Supplementary Figure 4). The quality of MS3 spectra were good enough to enable the de novo sequencing of the peptide. With this approach, we could sequence peptides attached to five different GPI structures, four of them also detected after proteinase K digestion (Figure 4A–C; Table II; Supplementary Table I; Supplementary Figure 4). Although five GPI species were found, all these species were attached to the same peptide sequence (APTPGD), which corresponds to the carboxyl terminus of the TcSMUG S subfamily of mucins from T. cruzi (El-Sayed et al, 2005; Buscaglia et al, 2006; Acosta-Serrano et al, 2007). This result was corroborated by amino-acid compositional analysis (not shown). The TcSMUG S subfamily comprises eight unique sequences (GenBank accession numbers XP_804663.1, XP_807370.1, XP_807371.1, XP_821038.1, XP_821039.1, XP_821040.1, XP_821041.1, and XP_821042.1), with predicted mature protein sequences varying from 56 to 85 amino acids. TcSMUG S sequences contain approximately 40% threonine in their composition (Buscaglia et al, 2004), suggesting that they could be heavily O-glycosylated and, therefore, quite resistant to protease digestion as we have already observed (data not shown).

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