Dynamic secretome of Trichomonas vaginalis: Case study of β-amylases
2017; Elsevier BV; Volume: 17; Issue: 2 Linguagem: Inglês
10.1074/mcp.ra117.000434
ISSN1535-9484
AutoresJitka Štáfková, Petr Rada, Dionigia Meloni, Vojtěch Žárský, Tamara Smutná, Nadine Zimmann, Karel Harant, Petr Pompach, Ivan Hrdý, Jan Tachezy,
Tópico(s)Syphilis Diagnosis and Treatment
ResumoThe secretion of virulence factors by parasitic protists into the host environment plays a fundamental role in multifactorial host–parasite interactions. Several effector proteins are known to be secreted by Trichomonas vaginalis, a human parasite of the urogenital tract. However, a comprehensive profiling of the T. vaginalis secretome remains elusive, as do the mechanisms of protein secretion. In this study, we used high-resolution label-free quantitative MS to analyze the T. vaginalis secretome, considering that secretion is a time- and temperature-dependent process, to define the cutoff for secreted proteins. In total, we identified 2 072 extracellular proteins, 89 of which displayed significant quantitative increases over time at 37 °C. These 89 bona fide secreted proteins were sorted into 13 functional categories. Approximately half of the secreted proteins were predicted to possess transmembrane helixes. These proteins mainly include putative adhesins and leishmaniolysin-like metallopeptidases. The other half of the soluble proteins include several novel potential virulence factors, such as DNaseII, pore-forming proteins, and β-amylases. Interestingly, current bioinformatic tools predicted the secretory signal in only 18% of the identified T. vaginalis-secreted proteins. Therefore, we used β-amylases as a model to investigate the T. vaginalis secretory pathway. We demonstrated that two β-amylases (BA1 and BA2) are transported via the classical endoplasmic reticulum-to-Golgi pathways, and in the case of BA1, we showed that the protein is glycosylated with multiple N-linked glycans of Hex5HexNAc2 structure. The secretion was inhibited by brefeldin A but not by FLI-06. Another two β-amylases (BA3 and BA4), which are encoded in the T. vaginalis genome but absent from the secretome, were targeted to the lysosomal compartment. Collectively, under defined in vitro conditions, our analysis provides a comprehensive set of constitutively secreted proteins that can serve as a reference for future comparative studies, and it provides the first information about the classical secretory pathway in this parasite. The secretion of virulence factors by parasitic protists into the host environment plays a fundamental role in multifactorial host–parasite interactions. Several effector proteins are known to be secreted by Trichomonas vaginalis, a human parasite of the urogenital tract. However, a comprehensive profiling of the T. vaginalis secretome remains elusive, as do the mechanisms of protein secretion. In this study, we used high-resolution label-free quantitative MS to analyze the T. vaginalis secretome, considering that secretion is a time- and temperature-dependent process, to define the cutoff for secreted proteins. In total, we identified 2 072 extracellular proteins, 89 of which displayed significant quantitative increases over time at 37 °C. These 89 bona fide secreted proteins were sorted into 13 functional categories. Approximately half of the secreted proteins were predicted to possess transmembrane helixes. These proteins mainly include putative adhesins and leishmaniolysin-like metallopeptidases. The other half of the soluble proteins include several novel potential virulence factors, such as DNaseII, pore-forming proteins, and β-amylases. Interestingly, current bioinformatic tools predicted the secretory signal in only 18% of the identified T. vaginalis-secreted proteins. Therefore, we used β-amylases as a model to investigate the T. vaginalis secretory pathway. We demonstrated that two β-amylases (BA1 and BA2) are transported via the classical endoplasmic reticulum-to-Golgi pathways, and in the case of BA1, we showed that the protein is glycosylated with multiple N-linked glycans of Hex5HexNAc2 structure. The secretion was inhibited by brefeldin A but not by FLI-06. Another two β-amylases (BA3 and BA4), which are encoded in the T. vaginalis genome but absent from the secretome, were targeted to the lysosomal compartment. Collectively, under defined in vitro conditions, our analysis provides a comprehensive set of constitutively secreted proteins that can serve as a reference for future comparative studies, and it provides the first information about the classical secretory pathway in this parasite. Trichomonas vaginalis is an anaerobic, aerotolerant pathogen that causes trichomoniasis, the most widespread nonviral sexually transmitted disease in humans. Although the majority of infections are asymptomatic, approximately one-third of infected women develop symptoms such as vaginitis and urethritis (1.Petrin D. Delgaty K. Bhatt R. Garber G. Clinical and microbiological aspects of Trichomonas vaginalis.Clin. Microbiol. Rev. 1998; 11: 300-317Crossref PubMed Google Scholar). In addition, trichomonad infection has been associated with poor birth outcomes and increased risk of Human Immunodeficiency Virus (HIV) acquisition (2.Kissinger P. Adamski A. Trichomoniasis and HIV interactions: A review.Sex Transm. Infect. 2013; 89: 426-433Crossref PubMed Scopus (144) Google Scholar). In men, the infection is rarely symptomatic; however, the parasite can damage sperm cells (3.Tuttle Jr, J.P. Holbrook T.W. Derrick F.C. Interference of human spermatozoal motility by Trichomonas vaginalis.J. Urol. 1977; 118: 1024-1025Crossref PubMed Scopus (37) Google Scholar, 4.Benchimol M. d Andrade R.I. da Silva F.R. Burla Dias A.J. 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Thus, the establishment of trichomonad infection within such hostile environments is dependent on multifactorial host–parasite interactions that involve both contact-dependent and contact-independent mechanisms (10.Ryan C.M. de Miguel N. Johnson P.J. Trichomonas vaginalis: Current understanding of host-parasite interactions.Essays Biochem. 2011; 51: 161-175Crossref PubMed Google Scholar). The former include the adherence of the parasite to vaginal epithelial cells, the contact-dependent extracellular killing of host cells (11.Krieger J.N. Ravdin J.I. Rein M.F. Contact-dependent cytopathogenic mechanisms of Trichomonas vaginalis.Infect. Immun. 1985; 50: 778-786Crossref PubMed Google Scholar, 12.Lin W.C. Chang W.T. Chang T.Y. Shin J.W. The pathogenesis of human cervical epithelium cells induced by interacting with Trichomonas vaginalis.PLoS One. 2015; 10: e0124087PubMed Google Scholar, 13.Fiori P.L. Rappelli P. Addis M.F. Mannu F. Cappuccinelli P. 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Trichomonas vaginalis exosomes deliver cargo to host cells and mediate hostratioparasite interactions.PLoS Pathog. 2013; 9: e1003482Crossref PubMed Scopus (168) Google Scholar). With regard to nutrients, the energy metabolism of T. vaginalis is dependent on glucose to generate ATP via anaerobic fermentation in the cytosol and via the extended glycolytic pathway in hydrogenosomes, an anaerobic form of mitochondria (20.Müller M. Mentel M. van Hellemond J.J. Henze K. Woehle C. Gould S.B. Yu R.Y. van der Giezen M. Tielens A.G. Martin W.F. Biochemistry and evolution of anaerobic energy metabolism in eukaryotes.Microbiol. Mol. Biol. Rev. 2012; 76: 444-495Crossref PubMed Scopus (501) Google Scholar, 21.Hrdý I. Tachezy J. Müller M. Metabolism of trichomonad hydrogenosomes.in: Tachezy J. Hydrogenosomes and Mitosomes:Mitochondria of Anaerobic Euakryotes. Springer-Verlag, Berlin, Heidelberg2008: 114-145Crossref Google Scholar). The main source of glucose in the vaginal fluid is likely free glycogen derived from vaginal epithelial cells (VECs) (22.Mirmonsef P. Hotton A.L. Gilbert D. Gioia C.J. Maric D. Hope T.J. Landay A.L. Spear G.T. Glycogen levels in undiluted genital fluid and their relationship to vaginal pH, estrogen, and progesterone.PLoS One. 2016; 11: e0153553Crossref PubMed Scopus (54) Google Scholar, 23.Mirmonsef P. Hotton A.L. Gilbert D. Burgad D. Landay A. Weber K.M. Cohen M. Ravel J. Spear G.T. Free glycogen in vaginal fluids is associated with Lactobacillus colonization and low vaginal pH.PLoS One. 2014; 9: e102467Crossref PubMed Scopus (128) Google Scholar, 24.Rajan N. Cao Q. Anderson B.E. Pruden D.L. Sensibar J. Duncan J.L. Schaeffer A.J. Roles of glycoproteins and oligosaccharides found in human vaginal fluid in bacterial adherence.Infect. Immun. 1999; 67: 5027-5032Crossref PubMed Google Scholar, 25.Gregoire A.T. Carbohydrates of human vaginal tissue.Nature. 1963; 198: 996Crossref PubMed Scopus (7) Google Scholar, 26.Sumawong V. Gregoire A.T. Johnson W.D. Rakoff A.E. Identification of carbohydrates in the vaginal fluid of normal females.Fertil. Steril. 1962; 13: 270-280Abstract Full Text PDF PubMed Google Scholar). To be utilized by T. vaginalis, glycogen and glucose-containing polymers must be extracellularly digested to monomeric glucose, which is then transported into the cells. Glycogen-hydrolyzing enzymes include endo-acting α-amylases (EC 3.2.1.1) that randomly hydrolyze α-1,4-linkages of glycogen, exo-acting β-amylases (EC 3.2.1.2) that hydrolyze α-1,4-linkages of glycogen at the nonreducing end to liberate β-maltose, and α-glucosidases (EC 3.2.1.20) that act on α-1,4-linkages of oligosaccharides to liberate d-glucose. Early studies suggested that T. vaginalis secretes α-glucosidase to hydrolyze maltose to glucose (27.ter Kuile B.H. Müller M. Maltose utilization by extracellular hydrolysis followed by glucose transport in Trichomonas vaginalis.Parasitol. 1995; 110: 37-44Crossref PubMed Scopus (12) Google Scholar). More recently, enzymes with α-amylase and β-amylase activities that utilize glycogen as a substrate were found to be released by T. vaginalis (28.Huffman R.D. Nawrocki L.D. Wilson W.A. Brittingham A. Digestion of glycogen by a glucosidase released by Trichomonas vaginalis.Exp. Parasitol. 2015; 159: 151-159Crossref PubMed Scopus (4) Google Scholar, 29.Smith R.W. Brittingham A. Wilson W.A. Purification and identification of amylases released by the human pathogen Trichomonas vaginalis that are active towards glycogen.Mol. Biochem. Parasitol. 2016; 210: 22-31Crossref PubMed Scopus (4) Google Scholar). High-resolution mass-spectrometry-based proteomic studies have been used to analyze the T. vaginalis surface proteome (30.de Miguel N. Lustig G. Twu O. Chattopadhyay A. Wohlschlegel J.A. Johnson P.J. Proteome analysis of the surface of Trichomonas vaginalis reveals novel proteins and strain-dependent differential expression.Mol. Cell. Proteomics. 2010; 9: 1554-1566Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) and the exosome proteome (19.Twu O. de Miguel N. Lustig G. Stevens G.C. Vashisht A.A. Wohlschlegel J.A. Johnson P.J. Trichomonas vaginalis exosomes deliver cargo to host cells and mediate hostratioparasite interactions.PLoS Pathog. 2013; 9: e1003482Crossref PubMed Scopus (168) Google Scholar), which have revealed a number of new candidate proteins with potential roles in T. vaginalis–host interactions and in the parasite's pathogenicity. More recent quantitative proteomic analyses have identified surface membrane proteins that are released to the T. vaginalis environment upon cleavage by rhomboid protease (31.Riestra A.M. Gandhi S. Sweredoski M.J. Moradian A. Hess S. Urban S. Johnson P.J. A Trichomonas vaginalis rhomboid protease and its substrate modulate parasite attachment and cytolysis of host cells.PLoS Pathog. 2015; 11: e1005294Crossref PubMed Scopus (29) Google Scholar). The best-studied group of secreted proteins is the proteases, including cysteine proteases and metalloproteases (17.Sommer U. Costello C.E. Hayes G.R. Beach D.H. Gilbert R.O. Lucas J.J. Singh B.N. Identification of Trichomonas vaginalis cysteine proteases that induce apoptosis in human vaginal epithelial cells.J. Biol. Chem. 2005; 280: 23853-23860Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 32.Hernández H.M. Sariego I. Alvarez A.B. Marcet R. Vancol E. Alvarez A. Figueredo M. Sarracent J. Trichomonas vaginalis 62 kDa proteinase as a possible virulence factor.Parasitol. Res. 2011; 108: 241-245Crossref PubMed Scopus (11) Google Scholar, 33.Arroyo R. Cárdenas-Guerra R.E. Figueroa-Angulo E.E. Puente-Rivera J. Zamudio-Prieto O. Ortega-López J. Trichomonas vaginalis cysteine proteinases: Iron response in gene expression and proteolytic activity.Biomed. Res. Int. 2015; 2015: 946787Crossref PubMed Scopus (27) Google Scholar). Kucknoor et al. (34.Kucknoor A.S. Mundodi V. Alderete J.F. The proteins secreted by Trichomonas vaginalis and vaginal epithelial cell response to secreted and episomally expressed AP65.Cell Microbiol. 2007; 9: 2586-2597Crossref PubMed Scopus (47) Google Scholar) identified 32 various secreted proteins, including a putative adhesin, AP65, via 2-D SDS-PAGE followed by MALDI-TOF (34.Kucknoor A.S. Mundodi V. Alderete J.F. The proteins secreted by Trichomonas vaginalis and vaginal epithelial cell response to secreted and episomally expressed AP65.Cell Microbiol. 2007; 9: 2586-2597Crossref PubMed Scopus (47) Google Scholar). In addition, Twu et al. showed that the parasite secretes a macrophage migration inhibitory factor (5.Twu O. Dessí D. Vu A. Mercer F. Stevens G.C. de M.N. Rappelli P. Cocco A.R. Clubb R.T. Fiori P.L. Johnson P.J. Trichomonas vaginalis homolog of macrophage migration inhibitory factor induces prostate cell growth, invasiveness, and inflammatory responses.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 8179-8184Crossref PubMed Scopus (118) Google Scholar). However, information about the T. vaginalis secretome remains rather incomplete. The major challenge for studies of the secretome using high-resolution MS is to identify bona fide secreted proteins and avoid artifacts caused by protein contamination. Here, we used quantitative MS and considered the fact that secretion is a time- and temperature-dependent process in defining the cutoff for T. vaginalis-secreted proteins. After bioinformatic sorting of the secreted proteins, we focused on β-amylases as model secreted proteins to investigate the T. vaginalis secretory pathway. Moreover, β-amylases are absent from humans and animals and may provide a suitable target for the development of novel antiparasitic strategies. T. vaginalis strain Tv17–48 was isolated from a symptomatic patient, and the axenic culture was immediately stored in liquid nitrogen (35.Kulda J. Vojtěchovská M. Tachezy J. Demes P. Kunzová E. Metronidazole resistance of Trichomonas vaginalis as a cause of treatment failure in trichomoniasis—A case report.Br. J. Vener. Dis. 1982; 58: 394-399PubMed Google Scholar). The strain was cultivated in tryptone-yeast extract-maltose medium (TYM) supplemented with 10% inactivated horse serum (36.Diamond L.S. The establishment of various trichomonads of animals and man in axenic cultures.J. Parasitol. 1957; 43: 488-490Crossref PubMed Google Scholar). T. vaginalis cells in the logarithmic phase of growth were harvested by centrifugation and washed twice in isotonic Doran's medium (37.Doran D.J. Studies on trichomonads: III. Inhibitors, acid production, and substrate utilization by 4 strains of Tritrichomonas foetus.J. Protozool. 1959; 6: 177-182Crossref Scopus (16) Google Scholar) with 15 mm maltose (Doran's medium with maltose). The cells were then resuspended at a concentration of 1 × 106 cells/ml, and 15 ml of suspension was incubated in 15 ml tubes for 10, 30, 60, and 120 min at 37 °C. Control cells were incubated for 60 and 120 min on ice. After incubation, the cells were removed by centrifugation at 1,000 × g for 5 min at 4 °C, and then the supernatant was centrifuged at 10,000 × g for 10 min to remove cell debris, filtered through a 0.22 μm filter, and centrifuged at 100,000 × g for 75 min to remove microvesicles (5.Twu O. Dessí D. Vu A. Mercer F. Stevens G.C. de M.N. Rappelli P. Cocco A.R. Clubb R.T. Fiori P.L. Johnson P.J. Trichomonas vaginalis homolog of macrophage migration inhibitory factor induces prostate cell growth, invasiveness, and inflammatory responses.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 8179-8184Crossref PubMed Scopus (118) Google Scholar). The proteins in the final supernatant were precipitated with TCA for 10 min at 4 °C (one volume of TCA to four volumes of supernatant). The precipitated proteins were pelleted at 12,000 × g for 20 min at 4 °C, washed with cold acetone, dried, and stored at −80 °C. During the incubation described above, the cell integrity was monitored under a light microscope using the trypan blue exclusion test (38.Strober W. Trypan blue exclusion test of cell viability.Curr. Protoc. Immunol. 2001; (Appendix 3)Crossref Scopus (978) Google Scholar). In parallel, at each time point, we determined the free activity of the cytosolic enzyme NADH oxidase in the cell suspension (39.Linstead D.J. Bradley S. The purification and properties of two soluble reduced nicotinamide: Acceptor oxidoreductases from Trichomonas vaginalis.Mol. Biochem. Parasitol. 1988; 27: 125-133Crossref PubMed Scopus (45) Google Scholar). In addition, aliquots of trichomonad suspensions taken at each time point were processed for transmission electron microscopy. The cell samples were centrifuged at 3 000 × g for 10 min and fixed in 2.5% glutaraldehyde and 5 mm CaCl2 in 0.1 m cacodylate buffer, pH 7.2, overnight at 4 °C. The cells were then postfixed in 0.1 m cacodylate buffer containing 1.6% ferricyanide, 10 mm CaCl2, and 2% OsO4 at 4 °C for 15 min, dehydrated in acetone, and embedded in the epoxy resin EMBed 812 (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin sections were stained with uranyl acetate and observed using a JEOL JEM-1011. The cell-free samples of TCA-precipitated proteins were dissolved in 100 mm triethylammonium bicarbonate buffer with 2% sodium deoxycholate, reduced with 5 mm tris(2-carboxyethyl)phosphine for 30 min at 60 °C, and alkylated with 10 mm S-methyl methanethiosulfonate for 10 min at room temperature. Total protein concentrations were measured via the bicinchoninic acid assay (Sigma-Aldrich, St. Louis, MO, USA). Next, 100 μg of proteins were digested with trypsin (trypsin:protein ratio 1:50) overnight at 37 °C. After digestion, 1% trifluoroacetic acid (TFA) was added. Sodium deoxycholate was removed by extraction to ethyl acetate as previously described (40.Masuda T. Tomita M. Ishihama Y. Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis.J. Proteome. Res. 2008; 7: 731-740Crossref PubMed Scopus (417) Google Scholar). The remaining ethyl acetate was removed using vacuum centrifugation at 45 °C for 10 min, and then 1% TFA was added. The samples were desalted using C18 sorbent (Supelco 66883-U, supplied by Sigma-Aldrich). The eluents were dried and resuspended in 20 μl of 1% TFA. A nano reversed-phase column (EASY-Spray column, 50 cm × 75 μm inner diameter, PepMap C18, 2 μm particles, 100 Å pore size) was used for nanoLC-MS analysis. Mobile phase buffer A consisted of water, 2% acetonitrile, and 0.1% formic acid. Mobile phase B consisted of 80% acetonitrile and 0.1% formic acid. Two micrograms of each sample were loaded onto the trap column (Acclaim PepMap300, C18, 5 μm, 300 Å Wide Pore, 300 μm × 5 mm) at a flow rate of 15 μl/min. The loading buffer consisted of water, 2% acetonitrile, and 0.1% TFA. Peptides were eluted with a gradient from 2% to 40% B over 60 min at a flow rate of 300 nl/min. The peptide cations eluted were converted to gas-phase ions via electrospray ionization and analyzed on a Thermo Orbitrap Fusion (Q-OT- qIT, Thermo Fisher Scientific, Waltham, MA, USA). Spectra were acquired with a 2 s duty cycle. Full MS spectra were acquired in the Orbitrap within a mass range of 350–1,400 m/z, at a resolution of 120,000 at 200 m/z and with a maximum injection time of 50 ms. The most intense precursors were isolated by quadrupole ion trapping with a 1.6 m/z isolation window and fragmented via higher-energy collisional dissociation with the collision energy set to 30%. Fragment ions were detected in the ion trap with the scan range mode set to normal and the scan rate set to rapid with a maximum injection time of 35 ms. The fragmented precursors were excluded from fragmentation for 60 s. For label-free quantification (LFQ), the data were processed in MaxQuant LFQ version 1.5.8.3 (41.Cox J. Hein M.Y. Luber C.A. Paron I. Nagaraj N. Mann M. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ.Mol. Cell. Proteomics. 2014; 13: 2513-2526Abstract Full Text Full Text PDF PubMed Scopus (2710) Google Scholar). Searches were performed using the latest version of the T. vaginalis database from UniProt (release 2017_4, 60,330 entries) and a common contaminant database. Trypsin was used to generate the peptides, and two missed cleavages were allowed. The protein modifications were set as follows: cysteine (unimod nr: 39) as static and methionine oxidation (unimod: 1384) and protein N terminus acetylation (unimod: 1) as variable. The precursor ion mass tolerance in the initial search was 20 ppm, the tolerance in the main search was 4.5 ppm, and the fragment ion mass tolerance was 0.5 Da. The false discovery rates for peptides and for proteins were set to 1%. For each identified protein, the cell localization and secretory pathway signal were predicted using the SignalP 4.1 server (http://www.cbs.dtu.dk/services/TMHMM/), TargetP 1.1 server (http://www.cbs.dtu.dk/services/TargetP/), and SecretomeP 2.0 server (http://www.cbs.dtu.dk/services/SecretomeP/). Transmembrane helixes and topology were predicted using the TMHMM server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Conserved domains were predicted using Pfam 31.0 (http://pfam.xfam.org/), and distant homologies were detected using the HHpred search against the CD database https://toolkit.tuebingen.mpg.de and Evolutionary Classification of Protein Domains (42.Vizcaíno J.A. Csordas A. Del-Toro N. Dianes J.A. Griss J. Lavidas I. Mayer G. Perez-Riverol Y. Reisinger F. Ternent T. Xu Q.W. Wang R. Hermjakob H. 2016 update of the PRIDE database and its related tools.Nucleic Acids Res. 2016; 44: 11033Crossref PubMed Scopus (18) Google Scholar) Molecular function gene ontology (http://geneontology.org/page/molecular-function-ontology-guidelines) and manual curation were used to sort the identified proteins. Three independent biological experiments were performed, and each biological sample was analyzed in three technical replicates using LFQ mass spectrometry. Proteins with LFQ values determined in at least two biological replicates with two valid values within the technical replicates were used for further processing. Changes in the LFQ values between two consecutive time points were calculated for each biological replicate as the difference in the LFQ binary logarithm between the means of technical replicates. The significance of each change was estimated using Student's t test. To distinguish actively released proteins from contaminants, we used two criteria: (i) the secreted protein displayed more significant increases than decreases in LFQ values over time, and (ii) the difference between the LFQ values for a given protein at 37 °C and 4 °C was greater than 1 LFQ unit. The secretion score (SecS) was then calculated as the sum of all decreases and significant increases (p value < 0.05). Hierarchical clustering was performed using the standard UPGMA hierarchical clustering method with the Scipy package (https://www.scipy.org/). Secreted proteins were ordered according to the means of LFQ values of technical replicates at 37 °C. Boxplot analysis of dominant clusters was performed based on the ratio [sum LFQ values (10 min, 30 min)+1]/[sum LFQ values (60 min, 120 min)+1]. The median, 25th, and 75th percentiles were computed for each cluster using the Scipy package (https://www.scipy.org/). The BA1 coding gene was subcloned into the modified TagVag vector (43.Hrdy I. Hirt R.P. Dolezal P. Bardonová L. Foster P.G. Tachezy J. Embley T.M. Trichomonas hydrogenosomes contain the NADH dehydrogenase module of mitochondrial complex I.Nature. 2004; 432: 618-622Crossref PubMed Scopus (208) Google Scholar) to allow for the expression of BA1 in T. vaginalis with a streptavidin tag at the C terminus. The logarithmic cell culture (2.5 liter) was harvested, the cells were broken by sonication, and the cell lysate was spun down by ultracentrifugation (100,000 × g for 25 min at 4 °C). Tagged BA1 was isolated from the supernatant using the Strep-Tactin system (IBA GmbH, Göttingen, Germany). The purity of the isolated protein was checked by SDS-PAGE. Recombinant BA1 protein was transferred to 50 mm ammonium bicarbonate buffer, pH 7.8, with Amicon Ultra 0.5 ml centrifugal filters MWCO 3 kDa (Merck, Darmstadt, Germany). Twenty micrograms of BA1 protein were reduced by dithiothreitol (Sigma-Aldrich) and alkylated by iodoacetamide (Sigma-Aldrich). The protein was digested by trypsin (Promega, Madison, WI, USA) overnight at 37 °C. To reduce the size of certain tryptic peptides, the endoproteinase Glu-C (Roche, Basel, Switzerland) was added to the sample and incubated overnight at 25 °C. The peptide mixture was separated by a reversed-phase HPLC connected to a 15 T solariX XR mass spectrometer (Bruker Daltonics, Billerica, MA USA) operating in data-dependent mode. Data were processed by the DataAnalysis 4.2 software (Bruker Daltonics), and glycopeptides were identified by manual data curation based upon measured mass values. Annotated, mass-labeled spectra for all glycopeptides identified are presented in Fig. S1A–S1G. The T. vaginalis BA1 protein sequence was used as a query for a BLAST search in the NCBI RefSeq protein database (GenBank release 220.0), and 1,312 homologues were used to build a preliminary phylogeny using Fast Tree (44.Price M.N. Dehal P.S. Arkin A.P. FastTree 2—Approximately maximum-likelihood trees for large alignments.PLoS One. 2010; 5: e9490Crossref PubMed Scopus (7090) Google Scholar). Then, 166 representative sequences were manually selected and aligned using MAFFT (45.Katoh K. Standley D.M. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability.Mol. Biol Evol. 2013; 30: 772-780Crossref PubMed Scopus (21
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