Fig. 2.

Gastrodermal cell-derived EVs contain inactive cathepsin L zymogens. A, Soluble extracts from the 15K vesicle pellet were incubated in 100 mm citrate phosphate buffer (pH 4.5) for 3 h at 37 °C. Aliquots of the reaction mixtures were removed after 0, 30, 60, 120, and 180 min and halted with 10 μm E-64 on ice. Samples were then transferred to nitrocellulose membranes and probed with an anti-Fasciola cathepsin L1 antibody. The inactive zymogen (37 kDa) showed a progressive decrease in intensity concurrent with the appearance of a band representing the 24 kDa mature enzyme (arrowhead). B, The auto-activation of FhCL1 shown in (A) was analyzed by following the hydrolysis of the fluorogenic dipeptide substrate Z-Leu-Arg-NHMec over 60 min at 37 °C. The autoactivation was inhibited in the presence of 10 μm E-64. C, Transmission electron micrograph (TEM) showing secretory vesicles (arrowed) within the specialized gastrodermal cells that line the gut of adult F. hepatica. D, TEM showing immunogold labeling for FhCL1 localized to the secretory vesicles within a gastrodermal cell within the gut of adult F. hepatica.