Tegument Glycoproteins and Cathepsins of Newly Excysted Juvenile Fasciola hepatica Carry Mannosidic and Paucimannosidic N-glycans
| Autor: | Garcia-Campos, A., Ravida, A., Nguyen, D.L., Cwiklinski, K., Dalton, J.P., Hokke, C.H., O'Neill, S., Mulcahy, G. |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Glycosylation Glycobiology Mannose Biochemistry Cathepsin B chemistry.chemical_compound Tandem Mass Spectrometry Lectins Medicine and Health Sciences Public and Occupational Health Post-Translational Modification Gel Electrophoresis Staining chemistry.chemical_classification Vaccines biology lcsh:Public aspects of medicine Glycopeptides Helminth Proteins Vaccination and Immunization 3. Good health Infectious Diseases Sequence Analysis Research Article Silver Staining Multiple Alignment Calculation Glycan lcsh:Arctic medicine. Tropical medicine lcsh:RC955-962 Immunology Electrophoretic Staining Research and Analysis Methods Electrophoretic Techniques 03 medical and health sciences SDG 3 - Good Health and Well-being Polysaccharides Hepatica Tropical Medicine Computational Techniques Animals Fasciola hepatica Amino Acid Sequence Molecular Biology Techniques Sequencing Techniques Molecular Biology Glycoproteins Cathepsin Life Cycle Stages Public Health Environmental and Occupational Health Biology and Life Sciences Proteins lcsh:RA1-1270 biology.organism_classification Split-Decomposition Method carbohydrates (lipids) 030104 developmental biology chemistry Specimen Preparation and Treatment Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization biology.protein Preventive Medicine Peptides Glycoprotein Sequence Alignment |
| Zdroj: | PLOS Neglected Tropical Diseases PLoS Neglected Tropical Diseases, Vol 10, Iss 5, p e0004688 (2016) PLoS Neglected Tropical Diseases PLoS Neglected Tropical Diseases, 10(5) Garcia-Campos, A, Ravidà, A, Nguyen, D L, Cwiklinski, K, Dalton, J P, Hokke, C H, O'Neill, S & Mulcahy, G 2016, ' Tegument Glycoproteins and Cathepsins of Newly Excysted Juvenile Fasciola hepatica Carry Mannosidic and Paucimannosidic N-glycans ', PLoS Neglected Tropical Diseases, vol. 10, no. 5, e0004688 . https://doi.org/10.1371/journal.pntd.0004688 |
| ISSN: | 1935-2735 |
| DOI: | 10.1371/journal.pntd.0004688 |
| Popis: | Recently, the prevalence of Fasciola hepatica in some areas has increased considerably and the availability of a vaccine to protect livestock from infection would represent a major advance in tools available for controlling this disease. To date, most vaccine-target discovery research on this parasite has concentrated on proteomic and transcriptomic approaches whereas little work has been carried out on glycosylation. As the F. hepatica tegument (Teg) may contain glycans potentially relevant to vaccine development and the Newly Excysted Juvenile (NEJ) is the first lifecycle stage in contact with the definitive host, our work has focused on assessing the glycosylation of the NEJTeg and identifying the NEJTeg glycoprotein repertoire. After in vitro excystation, NEJ were fixed and NEJTeg was extracted. Matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of released N-glycans revealed that oligomannose and core-fucosylated truncated N-glycans were the most dominant glycan types. By lectin binding studies these glycans were identified mainly on the NEJ surface, together with the oral and ventral suckers. NEJTeg glycoproteins were affinity purified after targeted biotinylation of the glycans and identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). From the total set of proteins previously identified in NEJTeg, eighteen were also detected in the glycosylated fraction, including the F. hepatica Cathepsin B3 (FhCB3) and two of the Cathepsin L3 (FhCL3) proteins, among others. To confirm glycosylation of cathepsins, analysis at the glycopeptide level by LC-ESI-ion-trap-MS/MS with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) was carried out. We established that cathepsin B1 (FhCB1) on position N80, and FhCL3 (BN1106_s10139B000014, scaffold10139) on position N153, carry unusual paucimannosidic Man2GlcNAc2 glycans. To our knowledge, this is the first description of F. hepatica NEJ glycosylation and the first report of N-glycosylation of F. hepatica cathepsins. The significance of these findings for immunological studies and vaccine development is discussed. Author Summary Fasciola hepatica is a parasite responsible for the zoonotic disease fasciolosis, prevalence of which has increased in recent years because of the emergence of triclabendazole-resistant strains as well as changing climatic conditions. A number of F. hepatica protein antigens are used for assessing the immune response of the definitive host for the development of recombinant vaccines but no such vaccine has been commercialised yet. Glycans that may contribute to the antigenic, immunological and protective properties in F. hepatica have not been characterised. Using a panel of plant lectins with defined sugar binding specificities alongside mass spectrometric analysis, we found that high mannose and oligomannose N-glycans are the most abundant in the surface of the juvenile fluke and identified eighteen proteins likely to be glycosylated. Additionally, we found that the proteases cathepsin B1 and L3 contain a glycosylation site occupied by an unusually short Man2GlcNAc2 N-glycan. This work is the starting point for understanding how F. hepatica glycans interact with the definitive host at the initiation of infection. Additionally, it provides useful information for including glycans in the design of new vaccine candidates. |
| Databáze: | OpenAIRE |
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