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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220409	1/2	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers ME	2023	67%
Study summary Full title Life stage-specific glycosylation of extracellular vesicles from schistosomula and adult worms drives differential interaction with C-type lectin receptors DC-SIGN and MGL. All authors Kuipers ME, Nguyen DL, van Diepen A, Mes L, Bos E, Koning RI, Nolte-'t Hoen ENM, Smits HH, Hokke CH Journal Front Mol Biosci Abstract Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released para (show more...)Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released parasite products that modulate the host's immune system. Many of these products are glycosylated and interact with host cells C-type lectin receptors (CLRs). We previously reported on specific fucose-containing glycans present on extracellular vesicles (EVs) released by schistosomula, the early juvenile life stage of the schistosome, and the interaction of these EVs with the C-type lectin receptor Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN or CD209). EVs are membrane vesicles with a size range between 30-1,000 nm that play a role in intercellular and interspecies communication. Here, we studied the glycosylation of EVs released by the adult schistosome worms. Mass spectrometric analysis showed that GalNAcβ1-4GlcNAc (LacDiNAc or LDN) containing N-glycans were the dominant glycan type present on adult worm EVs. Using glycan-specific antibodies, we confirmed that EVs from adult worms were predominantly associated with LDN, while schistosomula EVs displayed a highly fucosylated glycan profile. In contrast to schistosomula EV that bind to DC-SIGN, adult worm EVs are recognized by macrophage galactose-type lectin (MGL or CD301), and not by DC-SIGN, on CLR expressing cell lines. The different glycosylation profiles of adult worm- and schistosomula-derived EVs match with the characteristic glycan profiles of the corresponding life stages and support their distinct roles in schistosome life-stage specific interactions with the host. (hide) EV-METRIC 67% (96th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type whole parasite culture Sample origin adult worm Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: S. mansoni TSP2 non-EV: None Proteomics no EV density (g/ml) 1.09-1.18 Show all info Study aim Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 41.60% Total gradient volume, incl. sample (mL) 2.113 Sample volume (mL) 0.293 Orientation Bottom-up Speed (g) 166,18 Duration (min) 120 Fraction volume (mL) 0.1 Fraction processing Centrifugation Pelleting: volume per fraction 2.75 Pelleting: speed (g) 187,813 Characterization: Protein analysis Protein Concentration Method microBCA Protein Yield (µg) yes, per 100 adult worms Western Blot Antibody details provided? No Detected EV-associated proteins S. mansoni TSP2 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 30-250 EV concentration Yes Particle yield number per 100 adult worms EM EM-type Cryo-EM Image type Close-up, Wide-field Report size (nm) 30-180 Extra information All data on protein concentration and NTA of the schistosomula EVs can be found in EV track ID EV190032. More information on the protocol for the cryo EM of this publication can be found in EV track ID EV220119. Furthermore, we performed glycomics, western blots targeting glycan structures, and lectin blots of the adult worm EVs. The western blots targeting glycan structures were also performed on the schistosomula EV (glycomics on these EV are linked to EV track ID EV190032).

	EV220409	2/2	Schistosoma mansoni	whole parasite culture	(d)(U)C DG	Kuipers ME	2023	50%
Study summary Full title Life stage-specific glycosylation of extracellular vesicles from schistosomula and adult worms drives differential interaction with C-type lectin receptors DC-SIGN and MGL. All authors Kuipers ME, Nguyen DL, van Diepen A, Mes L, Bos E, Koning RI, Nolte-'t Hoen ENM, Smits HH, Hokke CH Journal Front Mol Biosci Abstract Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released para (show more...)Schistosomes can survive in mammalian hosts for many years, and this is facilitated by released parasite products that modulate the host's immune system. Many of these products are glycosylated and interact with host cells C-type lectin receptors (CLRs). We previously reported on specific fucose-containing glycans present on extracellular vesicles (EVs) released by schistosomula, the early juvenile life stage of the schistosome, and the interaction of these EVs with the C-type lectin receptor Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN or CD209). EVs are membrane vesicles with a size range between 30-1,000 nm that play a role in intercellular and interspecies communication. Here, we studied the glycosylation of EVs released by the adult schistosome worms. Mass spectrometric analysis showed that GalNAcβ1-4GlcNAc (LacDiNAc or LDN) containing N-glycans were the dominant glycan type present on adult worm EVs. Using glycan-specific antibodies, we confirmed that EVs from adult worms were predominantly associated with LDN, while schistosomula EVs displayed a highly fucosylated glycan profile. In contrast to schistosomula EV that bind to DC-SIGN, adult worm EVs are recognized by macrophage galactose-type lectin (MGL or CD301), and not by DC-SIGN, on CLR expressing cell lines. The different glycosylation profiles of adult worm- and schistosomula-derived EVs match with the characteristic glycan profiles of the corresponding life stages and support their distinct roles in schistosome life-stage specific interactions with the host. (hide) EV-METRIC 50% (21st percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type whole parasite culture Sample origin schistosomula Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Density gradient Protein markers EV: S. mansoni TSP2 non-EV: None Proteomics no EV density (g/ml) 1.09-1.18 Show all info Study aim Mechanism of uptake/transfer/Identification of content (omics approaches) Sample Species Schistosoma mansoni Sample Type whole parasite culture Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 100,000 g and 150,000 g Pelleting performed No Density gradient Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 10% Highest density fraction 41.60% Total gradient volume, incl. sample (mL) 2.113 Sample volume (mL) 0.293 Orientation Bottom-up Speed (g) 166,18 Duration (min) 120 Fraction volume (mL) 0.1 Fraction processing Centrifugation Pelleting: volume per fraction 2.75 Pelleting: speed (g) 187,813 Characterization: Protein analysis Protein Concentration Method Not determined Western Blot Antibody details provided? No Detected EV-associated proteins S. mansoni TSP2 Characterization: Lipid analysis No Characterization: Particle analysis None Extra information All data on protein concentration and NTA of the schistosomula EVs can be found in EV track ID EV190032. More information on the protocol for the cryo EM of this publication can be found in EV track ID EV220119. Furthermore, we performed glycomics, western blots targeting glycan structures, and lectin blots of the adult worm EVs. The western blots targeting glycan structures were also performed on the schistosomula EV (glycomics on these EV are linked to EV track ID EV190032).

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EV-TRACK ID	EV220409
species	Schistosoma mansoni
sample type	whole parasite culture
condition	adult worm	schistosomula
separation protocol	dUC/ Density gradient	dUC/ Density gradient
Exp. nr.	1	2
EV-METRIC %	67	50