Proteomic Analysis of Trichomonas vaginalis Phagolysosome, Lysosomal Targeting, and Unconventional Secretion of Cysteine Peptidases
| Autor: | Nadine, Zimmann, Petr, Rada, Vojtěch, Žárský, Tamara, Smutná, Kristína, Záhonová, Joel, Dacks, Karel, Harant, Ivan, Hrdý, Jan, Tachezy |
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| Rok vydání: | 2021 |
| Předmět: |
Proteomics
CP cysteine protease ST sucrose-tris buffer LAMP lysosome-associated membrane glycoprotein BFA Brefeldin A MPR mannose 6-phopshate receptor SNAP soluble N-ethylmaleimide-sensitive-factor attachment protein DMAP DNA methyltransferase-associated protein TMD transmembrane domain BA β-amylase TEAB triethylammonium bicarbonate Cysteine Proteases Phagosomes TSP tetraspanin MPR300 mannose 6-phopshate receptor 300 mCLCP mutated cathepsin L-like cysteine peptidase CD-MPR cation-dependent mannose 6-phosphate receptor LTS lysosomal targeting sequence LFQ MS label-free quantitative mass spectrometry MRH mannose 6-phosphate receptor homology SNARE SNAP receptor CPBF1 cysteine protease-binding protein family 1 TYM tryptone-yeast extract-maltose medium nanoLC-MS nano reverse-phase liquid chromatography mass spectrometry NHS N-hydroxysuccinimide bHX β-hexosaminidase FITC fluorescein isothiocyanate SDC sodium deoxycholate MES 2-[n-morpholino]-ethanesulfonic acid buffer TLCK tosyl-L-lysyl-chloromethane hydrochloride glycosylation proteome PBS phosphate-buffered saline vATPase vacuolar ATPase CLCP cathepsin L-like cysteine peptidase LGF large granule fraction ER endoplasmic reticulum mannose 6-phosphate receptor TCA trichloracetic acid Trichomonas vaginalis Humans GGA Golgi-localized γ-ear-containing ADP ribosylation factor-binding protein Cysteine GlcNAc-PT N-acetylglucosamine-1-phosphotransferase s-LTS lysosomal targeting sequence of soluble protein TGN trans Golgi network UCE uncovering enzyme N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase AP acid phosphatase Research M6P mannose 6-phosphate CI-MPR cation-independent mannose 6-phosphate receptor LERP lysosomal enzyme receptor protein EDC 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride mBA mutated β-amylase CLQ chloroquine TBSR Trichomonas beta-sandwich repeat protein MS mass spectrometry BBS borate buffered saline phagolysosome t-LTS lysosomal targeting sequence of transmembrane protein Lysosomes cysteine peptidase LF lactoferrin Peptide Hydrolases HA hemagglutinin |
| Zdroj: | Molecular & Cellular Proteomics : MCP |
| ISSN: | 1535-9484 |
| Popis: | The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases. Graphical Abstract Highlights • Trichomonas vaginalis phagolysosome consist of over 460 proteins. • Lysosomes are involved in secretion of virulence factors such as TvCP2. • N-glycosylation is required for lysosomal protein targeting. • T. vaginalis possesses homologs of mannose 6-phosphate receptor. In Brief Lysosomes represent a central degradative compartment of eukaryotes, yet little is known about biogenesis and function of this organelle in the parasitic protist Trichomonas vaginalis. We analyzed the phagolysosomal proteome that consists of over 460 proteins including important virulence factors. We demonstrated that glycosylation is involved in lysosomal protein targeting in T. vaginalis, which is unprecedented in parasitic protists. In addition to the classical secretory pathway, lysosomes are involved in unconventional protein secretion. |
| Databáze: | OpenAIRE |
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