Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.
Animals ; Cystatins/pharmacology ; Cysteine Endopeptidases/metabolism ; Cysteine Proteinase Inhibitors/chemistry/*metabolism/*pharmacology ; Helminth Proteins/*metabolism/*pharmacology ; Spirometra/*metabolism

A 29 kDa cysteine protease of Taenia solium metacestodes was purified by Mono Q anion-exchanger and Superose 6 HR gel filtration chromatography. The enzyme was effectively inhibited by cysteine protease inhibitors, such as iodoacetic acid (IAA) and trans-epoxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) while inhibitors acting on serine- or metallo-proteases did not affect the enzyme activity. The purified enzyme degraded human immunoglobulin G (IgG), collagen and bovine serum albumin (BSA), but human IgG was more susceptible for proteolysis by the enzyme. To define the precise biological roles of the enzyme, more detailed biochemical and functional studies would be required.
Taenia solium/*enzymology ; Serum Albumin, Bovine/metabolism ; Leucine/analogs & derivatives/pharmacology ; Iodoacetic Acid/pharmacology ; Immunoglobulin G/metabolism ; Humans ; Cysteine Proteinase Inhibitors/pharmacology ; Cysteine Endopeptidases/chemistry/*isolation & purification/metabolism ; Collagen/metabolism ; Chromatography, Ion Exchange ; Chromatography, Gel ; Animals

Because of the complexity of the cathepsin B-like (CBL) family, an information on the biological and biochemical characteristics of individual CBL genes is lacking. In this study, we investigated the degradative effects of the recombinant HC58 protein isolated from Haemonchus contortus parasites on protein substrates over a broad pH range in vitro. This protein, which hydrolyzed the synthetic peptide substrates Z-FR-AMC and Z-RR-AMC, had characteristics of the cysteine protease class of proteins. In the acidic pH range, the isolated protein actively degraded hemoglobin (Hb), the heavy chain of goat immunoglobulin G, and azocasein. By contrast, it degraded fibrinogen in the alkaline pH range. These activities were strongly inhibited in the presence of the cysteine protease inhibitor E-64. While the protein digested Hb, it did not induce the agglutination of erythrocytes from its natural host. These results suggest that the HC58 protein may play a role in the nutrition of this parasite.
Animals ; Caseins/metabolism ; Cathepsin B/antagonists&inhibitors/*genetics/isolation & purification/*metabolism ; Cysteine Proteinase Inhibitors/pharmacology ; DNA, Complementary/genetics ; Goat Diseases/*parasitology ; Goats ; Haemonchiasis/parasitology/*veterinary ; Haemonchus/*enzymology/genetics/isolation & purification ; Hemagglutination Tests/veterinary ; Hemoglobins/metabolism ; Hydrogen-Ion Concentration ; Immunoglobulin G/metabolism ; Leucine/analogs & derivatives/pharmacology ; RNA, Helminth/chemistry/genetics ; Recombinant Proteins/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction/veterinary

OBJECTIVETo investigate the regulatory mechanisms of integrin α5 and β1 in osteoblast in the process of gingipains-induced apoptosis.

METHODSGingipains were isolated and purified from supernatants of Porphyromonas gingivalis W83 which was cultured under standard anaerobic conditions. MC3T3-E1 was challenged with or without 8.3480 U/L gingipains for 48 h and apoptosis was examined by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (TUNEL-DAPI) staining. The expression of integrin α5 and β1 was analyzed by Western blotting after MC3T3-E1 was treated under different conditions.

RESULTSArginine-specific proteinases(Rgp) activity was (41.74 ± 2.11) U/L and lysine-specific proteinase(Kgp) was (1.02 ± 0.25) U/L.Gingipains induced MC3T3-E1 cells apoptosis after 48 h. Compared with control group, expression of integrin α5 and β1 was down-regulated by gingipains in a time-dependent manner within short periods ( ≤ 72 h), integrin α5 and β1 relative expression was (0.485 ± 0.039),(0.504 ± 0.002) at 48 h,(0.398 ± 0.058),(0.179 ± 0.001) at 72 h respectively (P < 0.05). After 72 h, integrin α5 expression in MC3T3-E1 cells was stable compared with control group while integrin β1 was still lower(control group:1.000 ± 0.000, 96 h:0.604 ± 0.003, 120 h: 0.357 ± 0.002) (P < 0.05). Proteinase inhibitor tosyl- L- lysine-chloromethyl-ketone(TLCK) effectively blocked the activity of gingipain and inhibited down-regulation of integrin α5 and β1 induced by gingipains from (0.398 ± 0.058,0.179 ± 0.001 ) to (0.781 ± 0.012, 0.857 ± 0.060) (P < 0.05). TLCK alone did not have any effect on integrin α5 and β1(P > 0.05). Gingipains also decreased integrin α5 and β1 in a dose-dependent manner.When cells were treated with 20.8700 U/L gingipains, integrin α5 and β1 relative expression reached to the lowest(0.105 ± 0.004,0.020 ± 0.000) (P < 0.05).

CONCLUSIONSGingipains inhibited the expression of integrin α5 and β1 in a time- and dose- dependent manner in osteoblasts in the process of apoptosis, which may not be mediated by direct proteolytic effect.

Adhesins, Bacterial ; administration & dosage ; isolation & purification ; pharmacology ; Animals ; Apoptosis ; drug effects ; Cysteine Endopeptidases ; administration & dosage ; isolation & purification ; pharmacology ; Dose-Response Relationship, Drug ; Down-Regulation ; Integrin alpha5 ; metabolism ; Integrin beta1 ; metabolism ; Mice ; Osteoblasts ; cytology ; metabolism ; Porphyromonas gingivalis ; chemistry ; Serine Proteinase Inhibitors ; pharmacology ; Time Factors ; Tosyllysine Chloromethyl Ketone ; pharmacology