In order to evaluate the possible roles of secretory proteases in the pathogenesis of amoebic keratitis, we purified and characterized a serine protease secreted by Acanthamoeba lugdunensis KA/E2, isolated from a Korean keratitis patient. The ammonium sulfate-precipitated culture supernatant of the isolate was purified by sequential chromatography on CM-Sepharose, Sephacryl S-200, and mono Q-anion exchange column. The purified 33 kDa protease had a pH optimum of 8.5 and a temperature optimum of 55 degrees C. Phenylmethylsulfonylfluoride and 4- (2- Aminoethyl) -benzenesulfonyl-fluoride, both serine protease specific inhibitors, inhibited almost completely the activity of the 33 kDa protease whereas other classes of inhibitors did not affect its activity. The 33 kDa enzyme degraded various extracellular matrix proteins and serum proteins. Our results strongly suggest that the 33 kDa serine protease secreted from this keratopathogenic Acanthamoeba play important roles in the pathogenesis of amoebic keratitis, such as in corneal tissue invasion, immune evasion and nutrient uptake.
Acanthamoeba/*enzymology/isolation & purification/pathogenicity ; Acanthamoeba Keratitis/*parasitology ; Animals ; Cornea/parasitology ; Humans ; Hydrogen-Ion Concentration ; Korea ; Serine Endopeptidases/chemistry/*isolation & purification/*metabolism ; Substrate Specificity ; Temperature ; Virulence Factors

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

Serine protease activity of high temperature requrement 2 (HtrA2) is essential for promoting cell death, as well as for protecting against cellular stresses. An X-ray crystallographic study described the formation of a pyramid shaped homotrimer that is a proteolytically competent form of HtrA2; however, little is known about effects of the trimeric structure of HtrA2 on the natural substrates. In this study, we generated the HtrA2 protein that has a single point mutation at the homotrimerization motif to assess relationship between structure and the proteolytic activity of HtrA2 on its substrates. Using gel filtration, a native gel electrophoresis system, and a co-precipitation assay, we confirm that phenylalanine 149 in HtrA2 is a crucial determinant for the formation of the HtrA2 homotrimeric structure. Moreover, we described that the HtrA2 monomeric form abolished not only autoproteolytic activity, but also the proteolytic activity against XIAP (X-linked inhibitor of apoptosis protein) known as the HtrA2 substrate. Taken together, the results indicate that the homotrimeric structure of HtrA2 is required for executing its serine protease activity.
Alanine/metabolism ; Amino Acid Motifs ; Amino Acid Sequence ; Amino Acid Substitution ; Cell Line ; Chromatography, Gel ; Crystallography, X-Ray ; Escherichia coli/genetics ; Glutathione Transferase/metabolism ; Hydrolysis ; Molecular Sequence Data ; Phenylalanine/metabolism ; Point Mutation ; Precipitin Tests ; Protein Structure, Tertiary ; Recombinant Fusion Proteins/metabolism ; Research Support, Non-U.S. Gov't ; Sequence Homology, Amino Acid ; Serine Endopeptidases/*chemistry/genetics/isolation & purification/*metabolism ; Structure-Activity Relationship ; Transfection