Table 1.
IntFam-241 proteins related to Haemonchus contortus, Ascaris suum and Caenorhabditis elegans intestinal peptidases.
Figure 1.
Intestinal peptidase activity.
A. Whole intestinal cell fractionation of peptidase activity. Peptidase activity was determined using a Bodipy casein substrate with 4 µg of sample, as described in methods, for whole intestinal homogenates (WL), 5,000×g pellet (P1) and supernatant (S1), and a further 50,000×g pellet (P2) and supernatant (S2) derived from S1. All samples were solubilized in 1% TX-100 prior to running assays. pH at which reactions were run is indicated on the x axis. Peptidase activity (y axis) is reported as Relative Fluorescent Units (RFU) µg-protein−1. Means that differ from one another (p<0.05) are indicated by different letter designations (A, B, C, D) below the x axis, and were analyzed according to pH conditions. B, C. Inhibition of peptidase activity in the whole lysate (B) and P2 (C) fractions by pepstatin (aspartic proteases, Pep), phenylmethylsulfonyl chloride (serine peptidases, PMSF), 1,10 phenanthroline (metallopeptidases, 1, 10 Phen) and trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane, L-trans-3-carboxyoxiran-2-carbonyl-L-leucylagmatine, N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide (C1 cysteine peptidases (including cathepsin B) cysteine peptidases, E-64). Percentage inhibition (y axis) of the mean activity uninhibited compared to mean activity of the inhibited is shown for each pH (x axis) tested. An asterisk denotes treatments that were significantly different (p<0.05) from the untreated control group for each pH indicated. All assays were conducted with three replicates.
Figure 2.
Concanavalin A (ConA) binding proteins from Adult A. suum intestine.
A, B. Histological sections were treated without (A) or with sodium periodate (B), as described in Methods, and then incubated with the ConA-horse radish peroxidase conjugate (ConA-HRPO). Binding was localized by histochemical detection. Black arrows point to intestinal microvilli, the white arrow points to material extending from microvilli into the lumen. C. Intestinal proteins separated by non-reducing SDS-PAGE were transferred to nitrocellulose filters, and then untreated (NP) or treated (P) with sodium periodate. Filters were incubated with Con A-HRPO and ConA binding bands localized by chemiluminescence. Molecular weight markers are indicated on the left of the panel. D. Peptidase activity was evaluated for intestinal proteins bound to ConA-agarose beads, as described in Methods, and conducted at the pH conditions indicated on the x axis. Relative fluorescence units (RFU) µg-protein−1 from hydrolysis of Bodipy casein is indicated on the y axis. A 95% confidence interval was constructed for group means at each pH tested, which in each case was greater than zero. E. Inhibition of peptidase activity isolated on ConA agarose beads. Assays were conducted at pH 5.0. Percentage inhibition indicated on the y axis was determined with inhibitors, as described in Figure 1, and means for each inhibitor treatment tested were significantly lower (p<0.05) than the uninhibited control group. All peptidase assays were conducted with three replicates. F. Coomassie blue stained SDS PAGE gel, as in panel C, of intestinal proteins isolated on ConA agarose beads. ConA, indicates heavy ConA proteins released from the beads. No bands were excised for mass spectrometry at or below this position in the gel.
Figure 3.
Cannulation of the Ascaris suum intestine.
A. Three cannulated female A. suum with intact posterior ends. Numbers and arrows refer to steps in the cannulation process: 1, removal of the anterior end below the esophagus with a scalpel; 2, insertion of the blunt needle cannula (25 g), with superglue gel applied to the cannula, into the intestinal lumen; 3, removal of the posterior 1/6th of the worm; 4, resection of the body wall to expose the intestine, as in panels B and C. B. Cannulated worm processed as in A, but with resected posterior body and exposed posterior region of the intestine (arrow, 5), laid onto parafilm for collection of perfusate. A syringe (6) is attached to the cannula hub to deliver perfusate. C. Enlargement of the exposed posterior region of the intestine is shown in panel B. D. Cannulated worm, with intact posterior end, injected with methylene blue dye. White arrow points to reproductive organs within the pseudocoelomic body cavity, black arrow points to the intestine filled with dye. Note that the dye is confined to the intestine.
Figure 4.
Peptidase activity in intestinal perfusates.
A. ConA-HRPO blot of proteins obtained from whole intestinal lysate (W), and PBS (P) or 4 M urea (4MU) perfusates of the intestinal lumen after separation by non-reducing SDS-PAGE and transfer to nitrocellulose. B. Samples (4 µg) of the PBS and 4MU perfusates, and pseudocoelomic fluid (Pf) were incubated with bodipy casein in peptidase assays, as described in Figs. 1 and 2. Measurements in Relative fluorescence units (RFU) µg-protein−1 are shown on the y axis. Means that differ from one another (p<0.05) are indicated by different letter designations (A, B, C) below the x axis, C, D. Inhibition of peptidase activity in PBS (B) and 4MU (C) perfusates. Assays were conducted at pH 5.0. Percentage inhibition indicated on the y axis was determined for with inhibitors, as described in Fig. 1. Means for each inhibitor treatment that were significantly lower (p<0.05) than the uninhibited control group are indicated by an asterisk. All peptidase assays were conducted with three replicates.
Table 2.
All predicted Ascaris suum peptidases in intestinal fractions and identified by mass spectrometry.
Table 3.
Summary of predicted Ascaris suum peptidases identified in Concanavalin A (ConA) binding and intestinal perfusate fractions.
Table 4.
Peptidase intestinal gene expression and presence among A. suum proteomic datasets.