Fasciola hepatica Gastrodermal Cells Selectively Release Extracellular Vesicles via a Novel Atypical Secretory Mechanism
Abstract
:1. Introduction
2. Results
2.1. The Gastrodermal Cell Proteome Reflects Its Role in Secretion and Haematophagy
2.2. The Proteome of the Tegument Supports Its Barrier Function
2.3. Comparisons of the Tegument, Gut, and Secretome Indicate That the Gastrodermal Cells Are the Principal Source of Secreted Proteins
2.4. Microscopical Examination Suggests an Atypical Mechanism of Extracellular Vesicle Release by Some Gastrodermal Cells
3. Discussion
4. Materials and Methods
4.1. Laser Microdissection
4.2. Mass Spectrometry of Laser Microdissected Tissue
4.3. Database Searching
4.4. Criteria for Protein Identification
4.5. Functional Annotation of Proteins
4.6. Isolation and Mass Spectrometry Analysis of Adult F. hepatica 15K Extracellular Vesicles
4.7. Histology and Immunofluorescent Labelling of F. hepatica Tissue Sections
4.8. Processing of F. hepatica for Transmission Electron Microscopy
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Protein | Identifier | Gastrodermal Cell | LMD Tegument 1 | Previously Detected | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Unique Peptides | Surface Protein Fraction 2 | Tegument 3 | Vomitus 3 | ESP Minus Vomitus 3 | Soluble ESP 4 | 15K EVs 1,5 | 120K EVs 4,5 | ||||||
R1 | R2 | R3 | R4 | ||||||||||
Proteases and inhibitors | |||||||||||||
Proteases | |||||||||||||
Leucine aminopeptidase 2 | BN1106_s617B000566 | 15 | 11 | 18 | 17 | ● | ● | ● | ● | ● | |||
Xaa-Pro dipeptidase (M24 family) | BN1106_s468B000343 | 6 | 4 | 5 | 13 | ● | ● | ● | ● | ● | |||
Leucine aminopeptidase 2 | BN1106_s617B000567 | 5 | 4 | 5 | 6 | ● | ● | ● | ● | ● | |||
Lysosomal Pro-X carboxypeptidase | BN1106_s1620B000120 | 5 | 4 | 5 | 3 | ● | ● | ● | ● | ● | |||
Leucine aminopeptidase 1 | BN1106_s7079B000034 | 5 | 4 | 4 | 12 | ● | ● | ● | ● | ● | |||
Cathepsin B6/8 | BN1106_s793B000177 | 5 | 3 | 4 | 5 | ● | ● | ● | |||||
Cathepsin L1 | BN1106_s8490B000026 | 4 | 4 | 5 | 5 | ● | ● | ● | ● | ● | ● | ● | |
Cathepsin L2 | BN1106_s8098B000020 | 3 | 3 | 5 | 7 | ● | ● | ● | ● | ● | ● | ||
Natterin-4 (DM9 domain-containing protein) | BN1106_s586B000374 | 3 | 3 | 5 | 6 | ● | ● | ● | ● | ||||
Cathepsin L1 | BN1106_s10332B000010 | 3 | 2 | 3 | 1 | ● | ● | ● | ● | ||||
Cathepsin L5 | BN1106_s4636B000039 | 2 | 3 | 2 | 5 | ● | ● | ||||||
Cathepsin B4/5/7 | BN1106_s13444B000002 | 2 | 2 | 3 | 5 | ● | ● | ● | |||||
Cathepsin L5 | BN1106_s6354B000017 | 2 | 2 | 2 | 2 | ● | ● | ● | ● | ||||
Cysteine protease-related protein | BN1106_s1772B000188 | 1 | 2 | 2 | 5 | ● | ● | ||||||
Aspartyl aminopeptidase | BN1106_s2165B000367 | 1 | 2 | 2 | 4 | ● | |||||||
Legumain-like | BN1106_s1861B000097 | 1 | 1 | 3 | 4 | ● | ● | ● | ● | ● | |||
Mitochondrial processing peptidase | BN1106_s37B000342 | 1 | 0 | 2 | 2 | ● | |||||||
Protease inhibitors | |||||||||||||
Cystatin-1 | BN1106_s4651B000094 | 4 | 3 | 4 | 7 | ● | ● | ● | ● | ● | |||
Serpin | BN1106_s3864B000104 | 3 | 2 | 5 | 2 | ● | ● | ● | ● | ||||
FH-KTM kunitz-type proteinase inhibitor | BN1106_s318B000274 | 2 | 2 | 3 | 2 | ● | ● | ● | ● | ● | |||
FH-KTM kunitz-type proteinase inhibitor | BN1106_s8826B000029 | 1 | 2 | 2 | 2 | ● | ● | ● | ● | ||||
Structural proteins | |||||||||||||
Motor/cytoskeletal | |||||||||||||
Myosin heavy chain | BN1106_s323B000258 | 16 | 12 | 19 | 29 | ● | |||||||
Paramyosin | BN1106_s1922B000122 | 8 | 4 | 11 | 21 | ● | |||||||
Filamin | BN1106_s296B000186 | 5 | 0 | 5 | 6 | ● | ● | ● | |||||
Actin | BN1106_s658B000223 | 4 | 4 | 6 | 9 | ● | ● | ● | ● | ||||
Filamin-C | BN1106_s1515B000336 | 4 | 3 | 3 | 5 | ● | ● | ||||||
Myosin heavy chain | BN1106_s323B000257 | 3 | 1 | 4 | 5 | ● | ● | ||||||
Myosin heavy chain | BN1106_s3182B000117 | 3 | 0 | 2 | 9 | ● | ● | ● | |||||
Beta-tubulin 4 | BN1106_s1153B000359 | 2 | 2 | 3 | 3 | ● | ● | ● | |||||
Actin | BN1106_s101B000531 | 2 | 2 | 3 | 2 | ● | ● | ● | |||||
Alpha-tubulin 2 | BN1106_s925B000543 | 2 | 1 | 2 | 0 | ● | ● | ||||||
Talin | BN1106_s149B000360 | 2 | 0 | 2 | 1 | ||||||||
Alpha-actinin sarcomeric | BN1106_s4069B000247 | 1 | 2 | 1 | 4 | ● | ● | ● | ● | ● | |||
Tropomyosin | BN1106_s647B000405 | 1 | 1 | 5 | 3 | ● | |||||||
Lamin | BN1106_s1106B000091 | 1 | 1 | 2 | 5 | ||||||||
Tropomyosin-2 | BN1106_s4130B000080 | 1 | 1 | 1 | 7 | ● | ● | ● | |||||
Tubulin beta-3 | BN1106_s4860B000047 | 1 | 1 | 0 | 7 | ● | ● | ● | |||||
Titin | BN1106_s98B000745 | 1 | 0 | 2 | 2 | ● | |||||||
Cytoskeletal-binding/modifying | |||||||||||||
Spectrin beta chain, brain 4 | BN1106_s4255B000066 | 7 | 3 | 8 | 8 | ● | ● | ||||||
Merlin/moesin/ezrin/radixin | BN1106_s1300B000145 | 6 | 8 | 9 | 18 | ● | ● | ● | ● | ||||
Ezrin-radixin-moesin-binding phosphoprotein 50 | BN1106_s1037B000175 | 4 | 3 | 6 | 6 | ● | ● | ||||||
Calpain (C02 family) | BN1106_s204B000249 | 4 | 0 | 4 | 7 | ● | ● | ● | |||||
Rho GDP-dissociation inhibitor | BN1106_s4672B000098 | 2 | 2 | 3 | 1 | ● | |||||||
Myophilin | BN1106_s3747B000111 | 2 | 1 | 2 | 3 | ● | ● | ● | ● | ||||
Ras-like GTP-binding protein Rho1 | BN1106_s1908B000177 | 1 | 2 | 2 | 1 | ● | ● | ● | |||||
Calponin homolog | BN1106_s2140B000163 | 1 | 1 | 4 | 2 | ● | ● | ||||||
Translationally controlled tumour protein | BN1106_s17035B000006 | 3 | 2 | 3 | 5 | ● | ● | ||||||
Abnormal long morphology protein 1 | BN1106_s1644B000226 | 0 | 0 | 2 | 2 | ||||||||
Extracellular matrix/cell adhesion | |||||||||||||
Heparan sulphate proteoglycan | BN1106_s25B000189 | 5 | 3 | 12 | 10 | ● | ● | ● | |||||
Alpha 1 type IIA collagen | BN1106_s849B000266 | 3 | 2 | 4 | 4 | ● | |||||||
Collagen alpha-1(V) chain | BN1106_s457B000392 | 2 | 2 | 2 | 3 | ● | |||||||
Fasciclin-1 | BN1106_s100B000380 | 2 | 2 | 2 | 2 | ||||||||
Collagen alpha-1(V) chain | BN1106_s2714B000202 | 2 | 0 | 2 | 3 | ● | |||||||
Fasciclin I-like protein | BN1106_s1922B000120 | 1 | 1 | 3 | 3 | ● | ● | ||||||
Collagen alpha-2(I) chain | BN1106_s104B000457 | 1 | 1 | 2 | 2 | ● | |||||||
Collagen alpha-1(V) chain | BN1106_s849B000265 | 1 | 0 | 2 | 3 | ● | |||||||
Signal transduction | |||||||||||||
Signalling | |||||||||||||
Tyrosine 3-monooxygenase | BN1106_s3172B000053 | 4 | 4 | 6 | 6 | ● | ● | ● | ● | ||||
14-3-3 epsilon | BN1106_s686B000273 | 4 | 2 | 4 | 5 | ● | ● | ● | ● | ● | |||
14-3-3 protein beta | BN1106_s3904B000042 | 2 | 2 | 1 | 3 | ● | ● | ● | ● | ● | |||
Nucleoside diphosphate kinase | BN1106_s344B000191 | 1 | 1 | 2 | 2 | ● | ● | ● | |||||
Protein CBR-FTT-2 | BN1106_s4074B000042 | 1 | 1 | 2 | 2 | ● | |||||||
Camp-dependent protein kinase type II-alpha | BN1106_s417B000229 | 1 | 0 | 2 | 3 | ● | ● | ||||||
Calcium-binding | |||||||||||||
CaBP4 | BN1106_s214B000744 | 6 | 4 | 8 | 11 | ● | ● | ● | ● | ● | ● | ||
22.4 kDa tegument protein | BN1106_s214B000748 | 4 | 4 | 4 | 4 | ● | ● | ● | |||||
Tegumental calcium-binding EF-hand protein | BN1106_s214B000742 | 1 | 1 | 2 | 3 | ● | ● | ● | ● | ● | |||
Ubiquitination pathway | |||||||||||||
Ubiquitin-activating enzyme E1 | BN1106_s5276B000036 | 1 | 3 | 1 | 6 | ● | ● | ● | |||||
Ubiquitin-protein ligase BRE1 | BN1106_s208B000185 | 1 | 1 | 2 | 3 | ● | ● | ● | ● | ||||
Secretory pathway | |||||||||||||
ER/Golgi apparatus-related | |||||||||||||
Venom allergen-like 11 protein | BN1106_s1956B000118 | 5 | 4 | 5 | 6 | ● | |||||||
Calreticulin | BN1106_s2673B000071 | 2 | 1 | 3 | 6 | ● | |||||||
Transitional endoplasmic reticulum ATPase | BN1106_s5369B000082 | 2 | 1 | 1 | 2 | ● | ● | ● | |||||
Preprotein translocase secy subunit Sec61 | BN1106_s2995B000137 | 1 | 1 | 2 | 2 | ||||||||
Atlastin-1 | BN1106_s3190B000098 | 0 | 1 | 2 | 2 | ||||||||
Vesicle biogenesis and trafficking | |||||||||||||
Annexin | BN1106_s819B000364 | 8 | 8 | 9 | 11 | ● | ● | ● | ● | ● | ● | ● | |
Annexin | BN1106_s945B000218 | 4 | 2 | 5 | 8 | ● | ● | ● | ● | ● | ● | ||
Coatomer subunit beta | BN1106_s771B000471 | 3 | 0 | 3 | 2 | ● | |||||||
ADP-ribosylation factor 1 | BN1106_s4512B000085 | 2 | 2 | 2 | 2 | ● | ● | ||||||
SAR1 gene homolog B | BN1106_s1405B000146 | 2 | 2 | 2 | 0 | ||||||||
Coatomer protein complex subunit alpha | BN1106_s649B000500 | 2 | 1 | 2 | 3 | ● | |||||||
Annexin | BN1106_s500B000161 | 1 | 3 | 2 | 5 | ● | ● | ● | ● | ● | ● | ||
Coatomer subunit gamma | BN1106_s5131B000049 | 1 | 0 | 3 | 7 | ● | |||||||
Ras-related protein Rab-11B | BN1106_s844B000259 | 1 | 0 | 3 | 3 | ● | ● | ● | |||||
Protein transport protein Sec23A | BN1106_s2174B000116 | 1 | 0 | 2 | 3 | ● | |||||||
Pumps and transporters | |||||||||||||
Smdr2 | BN1106_s274B000296 | 8 | 7 | 12 | 18 | ● | ● | ● | |||||
ATP synthase subunit beta | BN1106_s1866B000129 | 7 | 5 | 7 | 8 | ● | ● | ● | |||||
ATP synthase subunit alpha | BN1106_s4332B000087 | 6 | 6 | 5 | 10 | ● | ● | ● | ● | ● | |||
ATP:ADP antiporter | BN1106_s3313B000078 | 2 | 2 | 5 | 8 | ● | ● | ● | |||||
V-type H+-transporting ATPase subunit A | BN1106_s2110B000156 | 2 | 2 | 3 | 4 | ● | ● | ||||||
Innexin | BN1106_s503B000225 | 2 | 2 | 2 | 2 | ||||||||
Phosphatidylcholine transfer protein | BN1106_s538B000493 | 2 | 2 | 1 | 2 | ● | ● | ||||||
Sodium potassium transporting ATPase alpha subunit | BN1106_s521B000167 | 2 | 1 | 1 | 3 | ● | ● | ● | |||||
Vacuolar H+-ATPase SFD subunit | BN1106_s2350B000136 | 1 | 2 | 2 | 2 | ● | ● | ||||||
Vacuolar ATP synthase subunit e | BN1106_s1399B000513 | 1 | 1 | 2 | 3 | ● | ● | ||||||
ATP synthase subunit beta vacuolar | BN1106_s1633B000182 | 0 | 0 | 3 | 5 | ● | ● |
Protein | Identifier | LMD Tegument | Previously Detected | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Gastrodermal Cells 1 | Surface Protein Fraction 2 | Tegument 3 | Vomitus 3 | ESP Minus Vomitus 3 | Soluble ESP 4 | 15K EVs 1,5 | 120K EVs 4,5 | ||||||
Unique Peptides | |||||||||||||
R1 | R2 | R3 | R4 | ||||||||||
Structural proteins | |||||||||||||
Motor/cytoskeletal | |||||||||||||
Myosin heavy chain * | BN1106_s323B000258 | 17 | 17 | 23 | 24 | ● | |||||||
Paramyosin * | BN1106_s1922B000122 | 9 | 10 | 9 | 10 | ● | |||||||
Filamin | BN1106_s296B000186 | 4 | 3 | 4 | 2 | ● | ● | ● | |||||
Filamin-C * | BN1106_s1515B000336 | 3 | 3 | 2 | 2 | ● | ● | ||||||
Myosin heavy chain | BN1106_s323B000257 | 5 | 3 | 2 | 3 | ● | ● | ||||||
Myosin heavy chain | BN1106_s3182B000117 | 8 | 8 | 0 | 3 | ● | ● | ● | |||||
Alpha-actinin sarcomeric | BN1106_s4069B000247 | 8 | 10 | 3 | 10 | ● | ● | ● | ● | ● | |||
Tropomyosin-2 | BN1106_s4130B000080 | 1 | 2 | 1 | 5 | ● | ● | ● | |||||
Actin | BN1106_s2907B000133 | 4 | 3 | 1 | 3 | ● | ● | ● | ● | ● | |||
Titin | BN1106_s1119B000202 | 3 | 2 | 3 | 1 | ● | |||||||
Myosin regulatory light chain | BN1106_s527B000393 | 2 | 0 | 2 | 1 | ● | |||||||
Dynein heavy chain * | BN1106_s1314B000437 | 2 | 2 | 0 | 0 | ● | |||||||
Dynein light chain | BN1106_s949B000142 | 2 | 2 | 2 | 2 | ● | ● | ● | |||||
Dynein light chain | BN1106_s1582B000145 | 1 | 2 | 2 | 1 | ● | ● | ● | |||||
Dynein light chain | BN1106_s3147B000076 | 2 | 3 | 2 | 1 | ● | ● | ● | |||||
Cytoskeletal-binding/modifying | |||||||||||||
Spectrin beta chain, brain 4 | BN1106_s4255B000066 | 3 | 5 | 2 | 1 | ● | ● | ||||||
Calpain (C02 family) | BN1106_s204B000249 | 8 | 8 | 3 | 2 | ● | ● | ● | |||||
Calponin homolog | BN1106_s2140B000163 | 4 | 4 | 1 | 1 | ● | ● | ||||||
Gelsolin | BN1106_s2349B000191 | 1 | 1 | 3 | 6 | ● | ● | ● | ● | ||||
Lymphocyte cytosolic protein 1 | BN1106_s1403B000129 | 11 | 8 | 8 | 8 | ● | ● | ● | ● | ● | ● | ||
Gelsolin | BN1106_s2349B000188 | 5 | 5 | 3 | 8 | ● | ● | ● | ● | ||||
Myophilin | BN1106_s3026B000096 | 2 | 2 | 0 | 0 | ● | ● | ● | |||||
Adenylyl cyclase-associated protein | BN1106_s4290B000110 | 2 | 2 | 0 | 0 | ● | ● | ||||||
Extracellular matrix/cell adhesion | |||||||||||||
Heparan sulphate proteoglycan | BN1106_s25B000189 | 4 | 3 | 8 | 3 | ● | ● | ● | |||||
Alpha 1 type IIA collagen * | BN1106_s849B000266 | 4 | 4 | 4 | 0 | ● | |||||||
Collagen alpha-1(V) chain * | BN1106_s457B000392 | 3 | 3 | 1 | 2 | ● | |||||||
Collagen alpha-1(V) chain * | BN1106_s2714B000202 | 4 | 3 | 3 | 1 | ● | |||||||
Fasciclin I-like protein * | BN1106_s1922B000120 | 6 | 5 | 4 | 2 | ● | ● | ||||||
Collagen alpha-2(I) chain * | BN1106_s104B000457 | 4 | 3 | 2 | 2 | ● | |||||||
Collagen alpha-1(V) chain * | BN1106_s849B000265 | 2 | 3 | 3 | 2 | ● | |||||||
Collagen alpha-2(V) chain * | BN1106_s1528B000125 | 3 | 3 | 3 | 1 | ||||||||
Collagen type XV alpha * | BN1106_s26B000447 | 2 | 1 | 2 | 2 | ||||||||
Signal transduction | |||||||||||||
Signalling | |||||||||||||
Tyrosine 3-monooxygenase | BN1106_s3172B000053 | 2 | 2 | 2 | 1 | ● | ● | ● | ● | ||||
14-3-3 epsilon | BN1106_s686B000273 | 2 | 3 | 1 | 0 | ● | ● | ● | ● | ● | |||
cAMP-dependent protein kinase * | BN1106_s2316B000078 | 2 | 2 | 0 | 3 | ● | |||||||
Bcl-2 homologous antagonist | BN1106_s5167B000050 | 2 | 2 | 0 | 1 | ● | |||||||
Calcium-binding | |||||||||||||
CaBP4 | BN1106_s214B000744 | 3 | 5 | 4 | 7 | ● | ● | ● | ● | ● | ● | ||
Tegumental calcium-binding EF-hand protein | BN1106_s214B000742 | 2 | 2 | 3 | 2 | ● | ● | ● | ● | ● | |||
Tegumental calcium-binding EF-hand protein | BN1106_s214B000741 | 2 | 2 | 0 | 1 | ● | ● | ● | |||||
Calmodulin-like protein 2 | BN1106_s2277B000048 | 2 | 3 | 2 | 5 | ● | ● | ● | ● | ||||
Ubiquitination pathway | |||||||||||||
Ubiquitin-activating enzyme E1 | BN1106_s5276B000036 | 2 | 2 | 1 | 2 | ● | ● | ● |
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Bennett, A.P.S.; de la Torre-Escudero, E.; Dermott, S.S.E.; Threadgold, L.T.; Hanna, R.E.B.; Robinson, M.W. Fasciola hepatica Gastrodermal Cells Selectively Release Extracellular Vesicles via a Novel Atypical Secretory Mechanism. Int. J. Mol. Sci. 2022, 23, 5525. https://doi.org/10.3390/ijms23105525
Bennett APS, de la Torre-Escudero E, Dermott SSE, Threadgold LT, Hanna REB, Robinson MW. Fasciola hepatica Gastrodermal Cells Selectively Release Extracellular Vesicles via a Novel Atypical Secretory Mechanism. International Journal of Molecular Sciences. 2022; 23(10):5525. https://doi.org/10.3390/ijms23105525
Chicago/Turabian StyleBennett, Adam P. S., Eduardo de la Torre-Escudero, Susan S. E. Dermott, Lawrence T. Threadgold, Robert E. B. Hanna, and Mark W. Robinson. 2022. "Fasciola hepatica Gastrodermal Cells Selectively Release Extracellular Vesicles via a Novel Atypical Secretory Mechanism" International Journal of Molecular Sciences 23, no. 10: 5525. https://doi.org/10.3390/ijms23105525