Identification and characterization of a cathepsin L-like cysteine protease from Taenia solium metacestode

Taenia solium metacestode, a larval pork tapeworm, is a causative agent of neurocysticercosis, one of the most common parasitic diseases in the human central nervous system. In this study, we identified a cDNA encoding for a cathepsin L-like cysteine protease from the T solium metacestode (TsCL-1) and characterized the biochemical properties of the recombinant enzyme. The cloned cDNA of 1216 bp encoded 339 amino acids with an approximate molecular weight of 37.6 kDa which containing a typical signal peptide sequence (17 amino acids), a pro-domain (106 amino acids), and a mature domain (216 amino acids). Sequence alignments of TsCL-1 showed low sequence similarity of 27.3-44.6 to cathepsin L-like cysteine proteases from other helminth parasites, but the similarity was increased to 35.9-55.0 when compared to mature domains. The bacterially expressed recombinant protein (rTsCL-1) did not show enzyme activity; however, the rTsCL-1 expressed in Pichia pastoris showed typical biochemical characteristics of cysteine proteases. It degraded human immunoglobulin G (IgG) and bovine serum albumin (BSA), but not collagen. Western blot analysis of the rTsCL-1 showed antigenicity against the sera from patients with cysticercosis, sparganosis or fascioliasis, but weak or no antigenicity against the sera from patients with paragonimiasis or clonorchiasis. (c) 2006 Published by Elsevier B.V.

Expressional induction of Paralichthys olivaceus cathepsin B gene in response to virus, poly I:C and lipopolysaccharide

Cathepsin B is a lysosomal cysteine protease of the papain-like enzyme family with multiple biological functions. In this study, Paralichthys olivaceus cathepsin B (PoCatB) cDNA was isolated from flounder embryonic cells (FEC) treated with UV-inactivated grass carp hemorrhage virus (GCHV) and subsequently identified as a vitally induced gene. The full length cDNA of PoCatB is 1801 bp encoding 330-amino acids. The deduced protein has high homology to all known cathepsin B proteins, containing an N-terminal signal peptide, cysteine protease active sites, the occluding loop segment and a glycosylation site, all of which are conserved in the cathepsin B family. PoCatB transcription of FEC cells could be induced by turbot (Scophthalmus maximus) rhabdovirus (SMRV), UV-inactivated SMRV, UV-inactivated GCHV, poly I:C or lipopolysaccharide (LPS), and SMRV or poly I:C was revealed to be most effective among the five inducers. In normal flounder, PoCatB mRNA was detectable in all examined tissues. Moreover, SMRV infection could result in significant upregulation of PoCatB mRNA, predominantly in spleen, head kidney, posterior kidney, intestine, gill and muscle with 18.2,10.9, 24.7,12, 31.5 and 18 fold increases at 72 h post-infection respectively. These results provided the first evidence for the transcriptional induction of cathepsin B in fish by virus and LPS, indicating existence of a novel function in viral defense. (C) 2008 Elsevier Ltd. All rights reserved.

Scophthalmus maximus cystatin B enhances head kidney macrophage-mediated bacterial killing

Cystatins form a large family of cysteine protease inhibitors found in a wide arrange of organisms. Studies have indicated that mammalian cystatins play important roles under both physiological and pathological conditions. However, much less is known about fish cystatins. In this report, we described the identification and analysis of a cystatin B homologue, SmCytB, from turbot Scophthalmus maximus. The open reading frame of SmCytB is 300 bp, which encodes a 99-residue protein that shares high levels of sequence identities with the cystatin B of a number of fish species and contains the conserved cysteine protease inhibitor motif of cystatin B. Constitutive expression of SmCytB is high in muscle, brain, heart and liver, and low in spleen. blood, gill and kidney. Bacterial infection upregulates SmCytB expression in kidney, spleen, liver and brain but not in muscle or heart. Functional analysis showed that recombinant SmCytB purified from Escherichia colt exhibits apparent cysteine protease inhibitor activity. Transient overexpression of SmCytB in head kidney macrophages enhances macrophage bactericidal activity probably through a nitric oxide-independent mechanism. These results indicate that SmCytB is involved in the immune defense of turbot against bacterial infection. (C) 2010 Elsevier Ltd. All rights reserved.

A clip domain serine protease (cSP) from the Chinese mitten crab Eriocheir sinensis: cDNA characterization and mRNA expression

Clip domain serine protease (cSP), characterized by conserved clip domains, is a new serine protease family identified mainly in arthropod, and plays important roles in development and immunity. In the present study, the full-length cDNA of a cSP (designated EscSP) was cloned from Chinese mitten crab Eriocheir sinensis by expressed sequence tags (ESTs) and PCR techniques. The 1380 bp EscSP cDNA contained a 1152 bp open reading frame (ORF) encoding a putative cSP of 383 amino acids, a 5'-untranslated region (UTR) of 54 bp, and a 3'-UTR of 174 bp. Multiple sequence alignment presented twelve conserved cysteine residues and a canonical catalytic triad (His(185), Asp(235) and Ser(332)) critical for the fundamental structure and function of EscSP. Two types of cSP domains, the clip domain and tryp_spc domain, were identified in the deduced amino acids sequence of EscSP. The conservation characteristics and similarities with previously known cSPs indicated that EscSP was a member of the large cSP family. The mRNA expression of EscSP in different tissues and the temporal expression in haemocytes challenged by Listonella anguillarum were measured by real-time RT-PCR. EscSP mRNA transcripts could be detected in all examined tissues, and were higher expressed in muscle than that in hepatopancreas. gill, gonad, haemocytes and heart. The EscSP mRNA expression in haemocytes was up-regulated after L anguillarum challenge and peaked at 2 h (4.96 fold, P < 0.05) and 12 h (9.90 fold, P < 0.05). Its expression pattern was similar to prophenoloxidase (EsproPO), one of the components of crab proPO system found in our previous report. These results implied that EscSP was involved in the processes of host-pathogen interaction probably as one of the proPO system members. (C) 2009 Elsevier Ltd. All rights reserved.

Identification of a silicatein(-related) protease in the giant spicules of the deep-sea hexactinellid Monorhaphis chuni

Silicateins, members of the cathepsin L family, are enzymes that have been shown to be involved in the biosynthesis/condensation of biosilica in spicules from Demospongiae (phylum Porifera), e. g. Tethya aurantium and Suberites domuncula. The class Hexactinellida also forms spicules from this inorganic material. This class of sponges includes species that form the largest biogenic silica structures on earth. The giant basal spicules from the hexactinellids Monorhaphis chuni and Monorhaphis intermedia can reach lengths of up to 3 m and diameters of 10 mm. The giant spicules as well as the tauactines consist of a biosilica shell that surrounds the axial canal, which harbours the axial filament, in regular concentric, lamellar layers, suggesting an appositional growth of the spicules. The lamellae contain 27 kDa proteins, which undergo post-translational modification (phosphorylation), while total spicule extracts contain additional 70 kDa proteins. The 27 kDa proteins cross-reacted with anti-silicatein antibodies. The extracts of spicules from the hexactinellid Monorhaphis displayed proteolytic activity like the silicateins from the demosponge S. domuncula. Since the proteolytic activity in spicule extracts from both classes of sponge could be sensitively inhibited by E-64 (a specific cysteine proteinase inhibitor), we used a labelled E-64 sample as a probe to identify the protein that bound to this inhibitor on a blot. The experiments revealed that the labelled E-64 selectively recognized the 27 kDa protein. Our data strongly suggest that silicatein(-related) molecules are also present in Hexactinellida. These new results are considered to also be of impact for applied biotechnological studies.

ISOLATION AND PROPERTIES OF A BLOOD-COAGULATION FACTOR-X ACTIVATOR FROM THE VENOM OF KING COBRA (OPHIOPHAGUS HANNAH)

A specific blood coagulation factor X activator was purified from the venom of Ophiophagus hannah by gel filtration and two steps of FPLC Mono-Q column ion-exchange chromatography. It showed a single protein band both in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline polyacrylamide gel electrophoresis. The mol. wt was estimated to be 62,000 in non-reducing conditions and 64,500 in reducing conditions by SDS-PAGE. The isoelectric point was found to be pH 5.6. The enzyme had weak amidolytic activities toward CBS 65-25, but it showed no activities on S-2266, S-2302, thrombin substrate S-2238, plasmin substrate S-2251 or factor Xa substrate S-2222. It had no arginine esterase activity toward substrate benzoylarginine ethylester (BAEE). The enzyme activated factor X in vitro and the effect was absolutely Ca2+ dependent, with a Hill coefficient of 6.83. It could not activate prothrombin nor had any effect on fibrinogen and thus appeared to act specifically on factor X. The procoagulant activity of the enzyme was almost completely inhibited by serine protease inhibitors like PMSF, TPCK and soybean trypsin inhibitor; partially inhibited by L-cysteine. Metal chelator EDTA did not inhibit its procoagulant activity. These results suggest that the factor X activator from O. hannah venom is a serine protease.

Trypsin inhibitory loop is an excellent lead structure to design serine protease inhibitors and antimicrobial peptides

The disulfide-bridged hendecapeptide ( CWTKSIPPKPC) loop, derived from an amphibian skin peptide, is found to have strong trypsin inhibitory capability. This loop, called the trypsin inhibitory loop ( TIL), appears to be the smallest serine protease inhib

Purification and characterization of an irreversible serine protease inhibitor from skin secretions of Bufo andrewsi

Amphibian skin secretions contain many bioactive compounds. In the present work, an irreversible serine protease inhibitor, termed baserpin, was purified for the first time from the skin secretions of toad Bufo andrewsi by Successive ion-exchange and gelfiltration chromatography. Baserpin is a single chain glycoprotein, with an apparent molecular weight of about 60 kDa in SDS-PAGE. Baserpin is an irreversible inhibitor and effectively inhibits the catalytic activity of trypsin, chymotrypsin and elastase. SDS-stable baserpin-trypsin complex could be seen in SDS-PAGE indicates that it possibly belongs to the serpin superfamily. According to the association rates determined, baserpin is a potent inhibitor of bovine trypsin (4.6 X 10(6) M-1 S-1), bovine chymotrypsin (8.9 X 10(6) M-1 s(-1)) and porcine elastase (6.8 X 10(6) M-1 s(-1)), whereas it shows no inhibitory effect on thrombin. The N-terminal sequence of baserpin is HTQYPDILIAKPXDK, which shows no similarity with other known serine protease inhibitors. (c) 2005 Elsevier Ltd. All rights reserved.

An anticoagulant serine protease from the wasp venom of Vespa magnifica

Wasp is an important venomous animal that can induce human fatalities. Coagulopathy is a clinical symptom after massive wasp stings, but the reason leading to the envenomation manifestation is still not known. In this paper, a toxin protein is purified and characterized by Sephadex G-75 gel filtration, CM-Sephadex C-25 cationic exchange and fast protein liquid chromatography (FPLC) from the venom of the wasp, Vespa magnifica (Smith). This protein, named magnvesin. contains serine protease-like activity and inhibits blood coagulation. The cDNA encoding magnvesin is cloned from the venom sac cDNA library of the wasp. The deduced protein from the cDNA is composed of 305 amino acid residues. Magnvesin shares 52% identity with allergen serine protease from the wasp Polistes dominulus. Magnvesin exerted its anti-coagulant function by hydrolyzing coagulant factors TF, VII, VIII, IX and X. (c) 2008 Elsevier Ltd. All rights reserved.

Two serine protease inhibitors from the skin secretions of the toad, Bombina microdeladigitora

Two serine protease inhibitors (named BMSI 1 and BMSI 2, respectively) were identified from the skin secretions of the toad, Bombina microdeladigitora. The cDNAs encoding BMSIs were cloned from a cDNA library prepared from the toad skin. The deduced complete amino acid sequences of BMSIs indicate that mature BMSI1 and BMSI2 are composed of 60 amino acids including 10 half-cystines to form 5 disulfide bridges. A FASTA search in the databanks revealed that BMSIs exhibit sequence similarity with other serine protease inhibitors from amphibians of the genus Bombina. BMSI1 potently inhibited trypsin and thrombin with a K(i) value of 0.02 mu M and 0.15 mu M, respectively. Sequence analysis revealed that all serine protease inhibitors from five amphibians of the genus Bombina share highly conserved primary structures. (c) 2007 Elsevier Inc. All rights reserved.

Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom

Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibito

A unique group of inactive serine protease homologues from snake venom

A number of inactive serine protease homologues (SPHs), which have poorly understood functions, have been identified in invertebrates and vertebrates. Recently, several SPH transcripts have been reported from snake venom glands, which provide potential ne

Purification and characterization of jerdofibrase, a serine protease from the venom of Trimeresurus jerdonii snake

A fibrin(ogen)olytic serine protease from Trimeresurus jerdonii venom was identified and purified to SDS-polyacrylamide gel electrophoresis homogeneity. It is a single chain polypeptide with a molecular weight of 32 kDa under reduced condition and 28 kDa

Purification and cloning of cysteine-rich proteins from Trimeresurus jerdonii and Naja atra venoms

Three 26 kDa proteins, named as TJ-CRVP, NA-CRVP1 and NA-CRVP2, were isolated from the venoms of Trimeresurus jerdonii and Naja atra, respectively. The N-terminal sequences of TJ-CRVP and NA-CRVPs were determined. These components were devoid of the enzymatic activities tested, such as phospholipase A(2), arginine esterase, proteolysis, L-amino acid oxidase, 5' nucleotidase, acetylcholinesterase. Furthermore, these three components did not have the following biological activities: coagulant and anticoagulant activities, lethal activity, myotoxicity, hemorrhagic activity, platelet aggregation and platelet aggregation-inhibiting activities. These proteins are named as cysteine-rich venom protein (CRVP) because their sequences showed high level of similarity with mammalian cysteine-rich secretory protein (CRISP) family. Recently, some CRISP-like proteins were also isolated from several different snake venoms, including Agkistrodon blomhoffi, Trimeresurus flavoviridis, Lanticauda semifascita and king cobra. We presumed that CRVP might be a common component in snake venoms. Of particular interest, phylogenetic analysis and sequence alignment showed that NA-CRVP1 and ophanin, both from elapid snakes, share higher similarity with CRVPs from Viperidae snakes. (C) 2003 Elsevier Ltd. All rights reserved.