Ribbon diagram superposition of the catalytic domains of the <i>E. tenella</i> cathepsin B and human cathepsin B.

<p>The X-ray crystallographic structure of the human cathepsin B (pdb 1 gmy) is colored in green and the proposed structure of homology-based model of the <i>E. tenella</i> cathepsin B is in orange. The catalytic triad residues (Cys266, His445 and Asn465) are depicted in the <i>ball-and-stick</i> representation. On the left panel, the occluding loop is represented in purple with the two adjacent histidine residues (His352, His353) in the <i>ball-and-stick</i> representation. On the right panel, the surface exposed loops specific to <i>E. tenella</i> cathepsin B are in red. The residues delimitating the loops: Asn334-Ser341, Glu377-Lys383 and Asp389-Thr395 are shown.</p

Biochemical activities of CPs throughout sporulation.

<p>(A) Activities detected on Z-FR-AMC. Lysates of oocysts (1 mg/ml) taken at 0, 6, 12, 24 and 48 h after the beginning of sporulation were incubated with the substrate Z-FR-AMC (10 µM). The morphology of oocysts (under light microscopy) throughout the course of sporulation is shown. The scale bar represents 2 µm. (B) Activities detected on Z-FR-AMC and Z-LR-AMC in presence of the global cysteine protease inhibitor E-64 or the human cathepsin B specific inhibitor CA-074. Lysates (1 mg/ml) of oocysts taken at 0 h (black bars) and 48 h (white bars) after commencement of sporulation were pre-incubated with the inhibitors before adding the substrates. The data represents two independent experiments.</p

Effect of CA-074 Me on MDBK cell invasion by <i>E. tenella</i> sporozoites.

<p>Purified sporozoites were incubated with 10 to 100 µM of CA-074ME, a specific cathepsin B inhibitor permeable to membranes or with 1% DMSO. Sporozoites were then washed and incubated with MDBK cells. Infected cells were fixed and intracellular parasites were detected by IF using specific sera against sporozoites, and counted. The data represent three independent experiments. * denotes significant differences at p<0.05 in the capacity of CA-074 Me- treated parasites to invade cells compared to the control.</p

Profile of active CPs throughout sporulation.

<p>(A) Lysates of unsporulated oocysts (0) and oocysts in sporulation for 6, 12, 24, 36 and 48 h were incubated with the probe Biot-LC-LVG-CHN₂. The complexes were revealed by Western blot using streptavidin peroxidase. (B) Lysates of oocysts obtained at 0 h or 48 h of sporulation time (St) were pre-incubated or not (−) with E-64 (28 µM) or CA-074 (100 µM) before addition of Biot-LC-LVG-CHN₂. The interaction between the probe and CP catalytic sites was detected by Western blot using streptavidin peroxidase. The arrowhead indicates the activity corresponding to the <i>E. tenella</i> cathepsin B (33 kDa).</p

Expression profile of CPs in <i>E. tenella</i>.

<p>(A) Total RNAs from unsporulated oocysts (0), oocysts in sporulation for 6 h (6) and fully sporulated oocysts (48) were extracted and RT-PCR were performed using specific primers to <i>etcpb, etcpl, etcpc1, etcpc2</i> and <i>etcpc3</i>. (B) The single-copy <i>actin</i> gene was amplified in parallel as a control (<i>etactin</i>). The down regulation of <i>etactin</i> during sporulation has been previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031914#pone.0031914-Kinnaird1" target="_blank">[17]</a>. The RT-PCR products were resolved on a 0.7% agarose gel stained with ethidium bromide. The band observed at 0.8 kb with primers specific to <i>etcpc2</i> and total RNAs from unsporulated oocyst (0) is a nonspecific amplification product.</p

Degradation of human HMWK by CF sputum.

<p>Exogenous HMWK was incubated in the activity buffer with supernatants of CF sputum at 30°C for 0–5 hours. Hydrolysis products were separated by 12.5% SDS-PAGE, transferred to nitrocellulose membranes and immunoblotted with rabbit polyclonal anti-kininogen antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025577#pone.0025577-Lalmanach3" target="_blank">[24]</a>. Lane 1, 0-h incubation; lane 2, 2-h incubation; lane 3, 5-h incubation; lane 4, 5-h incubation in the presence of CA-074; lane 5, 5-h incubation in the presence of E-64. For clarity, one representative sample is shown.</p

Maturation of human procathepsins in CF sputum.

<p>(A) Autocatalytic maturation of procathepsins B and S. Supernatants were incubated for up to 6 h in 100 mM sodium acetate buffer pH 4.3, 10 µg/ml dextran sulfate, 4 mM DTT, at 37°C. The maturation products were separated by 12% SDS/PAGE under reducing conditions, transferred to a nitrocellulose membrane and analyzed by western blotting using polyclonal antibodies specific for (a) human cathepsin B and (b) human cathepsin S. One representative sample is shown. ★, proforms; ◂, mature double-chain cathepsin B; ←, mature cathepsin S. (B) Elastase-dependent maturation of procathepsin B. Supernatants were incubated for up to 4 h in 50 mM HEPES pH 7.4, 150 mM NaCl, 0.05% NP40 at 37°C and the maturation products analyzed by immunoblotting using a polyclonal anti-cathepsin B antibody. Pefabloc was used as control (4 h). Pef, Pefabloc (AEBSF). (+): <i>P. aeruginosa</i>-positive CF sputum (one representative sample); (−): <i>P. aeruginosa</i>-negative CF sputum (one representative sample). ◃, single-chain cathepsin B; ◂, double-chain cathepsin B; ★, procathepsin B.</p

CP and HNE activities, and CP/CPI balance in CF sputum.

<p>(+): <i>P. aeruginosa</i>-colonized CF sputum; (−): <i>P. aeruginosa</i>-negative CF sputum. Cathepsins B, H, K, L and S and HNE activities were quantified as reported in details in the experimental section. Data are shown as individual points and statistically significant P values are shown. The horizontal bars indicate medians. (A) Cathepsins B, L, S, K and H. (B) CP/CPI balance. CPI: expressed as inhibitory site (cystatin-like) equivalent. (C) Elastase activities: the horizontal bars indicate medians. The western blot analysis was performed using a rabbit anti-HNE antibody (representative samples are shown). ◂, unbound HNE; ★, bound HNE.</p

Cleavage of Nidogen-1 by Cathepsin S Impairs Its Binding to Basement Membrane Partners

<div><p>Cathepsin S (catS), which is expressed in normal human keratinocytes and localized close to the dermal-epidermal junction (DEJ) degrades some of major basement membrane (BM) constituents. Among them, catS readily hydrolyzed in a time and dose dependent manner human nidogen-1 (nid-1) and nidogen-2, which are key proteins in the BM structure. CatS preferentially cleaved nid-1 at both acid and neutral pH. Hydrolysis of nid-1 was hampered in murine <em>ctss</em>^−/− spleen lysates pretreated with inhibitors of other classes of proteases. Nid-1 was cleaved within its G2 and G3 globular domains that are both involved in interactions with other BM components. Binding assays with soluble and immobilized ligands indicated that catS altered the formation of complexes between nid-1 and other BM components. Assuming that the cleavage of nid-1 impairs its ability to crosslink with BM partners and perturbs the viscoelastic properties of BM matrix, these data indicate that catS may participate in BM proteolysis, in addition to already identified proteases.</p> </div

Major BM components and cysteine cathepsins B, K, L, S in the epidermis of human skin.

<p>(A) Immunofluorescence labeling of major BM components (type IV collagen, laminin, nid-1 and -2) and cathepsins B, K, L, S. The nuclei of keratinocytes were stained with DAPI. Ep: epidermis; De: dermis, Sc: stratum corneum, Sg: stratum granulosum, Ss: stratum spinosum, Sb: stratum basale. The continuous line indicates the outermost layer of the skin, the dashed line indicates the basal lamina. Bars correspond to 25 µm. (B) Expression of pro- and mature cathepsins B, K, L and S in human skin epidermis. The dark arrow indicates the proform of cathepsin and the light arrow indicated the mature form. Intermediate bands correspond to uncompleted maturation. (C) Samples of conditioned culture medium of human primary keratinocytes were concentrated (x 200), separated (100 µg protein) by SDS-PAGE (15%) under reducing conditions. (D) Transwell BM matrix migration assays (n = 5 separate experiments) by keratinocytes (C: control) treated with E-64 (**, <i>P</i><0.01 when compared to control). (E) Double immunofluorescent labeling of catS and nid-1 in human skin and confocal laser microscopy analysis. Secondary antibodies were Alexa Fluor 546 anti-goat (catS: red) and Alexa Fluor 488 anti-mouse (nidogen-1: green). Magnification (insert) of the lower panel of the image (x 3). Arrowheads indicate cells containing catS and nid-1 close together (yellow). Scale bar = 25 µm. (F) BM matrix (5 µg) was incubated alone or with concentrated keratinocyte supernatant (10 µg protein) at pH 5.5 or 7.4 for 24 h. For controls, supernatants were pre-incubated for 30 min with E-64 or LHVS. Data represent immunoblot analysis with nid-1 antibodies from at least 3 independent experiments. (G) Pretreated lysates (10 µg) of wild-type C57/Bl6 mouse spleen (wt, lysates: 1–3) and catS-deficient spleen (<i>ctss^−/−</i>, lysates: 4–6) were incubated (pH 7.4, 37°C) for 1 h in order to inactivate all cysteine cathepsins, except catS. BM matrix (10 µg) was then incubated alone or with cell lysates (pH 7.4, 37°C) for 18 h. Immunoreactive nid-1 was revealed by western blot. Assays were performed on different samples (n = 15), which yielded similar results and a representative of two independent experiments is presented.</p