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.

Characterization of a fibrinogen-clotting enzyme from Trimeresurus stejnegeri venom, and comparative study with other venom proteases

Trimeresurus stejnegeri venom, which contains TSV-PA (a specific plasminogen activator sharing 60-70% sequence homology with venom fibrinogen-clotting enzymes), also possesses fibrinogen-clotting activity in vitro. A fibrinogen-clotting enzyme (stejnobin) has been purified to homogeneity by gel filtration and ion-exchange chromatography on a Mono-Q column. It is a single-chain glycoprotein with a mol. wt of 44,000. The NH2-terminal amino acid sequence of stejnobin shows great homology with venom fibrinogen-clotting enzymes and TSV-PA. Like TSV-PA, stejnobin was able to hydrolyse several chromogenic substrates. Comparative study of substrate specificities of stejnobin and other venom proteases purified in our laboratory was carried out on five chromogenic substrates. Stejnobin clotted human fibrinogen with a specific activity of 122 NIH thrombin-equivalent units/mg protein. However, stejnobin did not act on other blood coagulation factors, such as factor X, prothrombin and plasminogen. Diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride inhibited its activity, whereas ethylenediamine tetracetic acid had no effect on it, indicating that it is a serine protease. Although stejnobin showed strong immunological cross-reaction with polyclonal antibodies raised against TSV-PA, it was interesting to observe that, unlike the case of TSV-PA, these antibodies did not inhibit the amidolytic and fibrinogen-clotting activities of stejnobin. (C) 1998 Elsevier Science Ltd. All rights reserved.

Molecular cloning of serine proteases from elapid snake venoms

Serine proteases are widely distributed in viperid snake venoms, but rare in elapid snake venoms. Previously, we have identified a fibrinogenolytic enzyme termed OhS1 from the venom of Ophiophagus hannah. The results indicated that OhS1 might be a serine

Actions of two serine proteases from Trimeresurus jerdonii venom on chromogenic substrates and fibrinogen

Jerdonobin and jerdofibrase are two serine proteases purified from the venom of Trimeresurus jerdonii. The Michaelis constant K-m and the catalytic rate constant K-cat of jerdonobin or jerdofibrase on three chromogenic substrates, H-D-Pro-Phe-Arg-pNA (S2302), H-D-Phe-pipecolyl-Arg-pNA (S2238), and H-D-Val-Leu-Lys-pNA (S2251) were obtained from lineweaver-Burk plots. Jerdofibrase could hydrolyze all three substrates, but jerdonobin had no detectable activity on S2251, suggesting a relatively broader substrate specificity for jerdofibrase than jerdonobin. By SDS-PAGE, jerdofibrase preferentially degraded Bbeta-chain of fibrinogen. It also degraded Aalpha-chain of fibrinogen with relatively slow activity, but did not act on the gamma-chain. In contrast, jerdonobin did not degrade fibrinogen within 12 h. Fibrinopeptides liberation test, identified by HPLC, showed jerdonobin released fibrinopeptide A and a small amount of fibrinopeptide B. Unlike jerdonobin, jerdofibrase mainly released fibrinopeptide B. These results indicate that the two enzymes differ in their ability to hydrolyze chromogenic substrates and in their actions on fibrinogen. (C) 2002 Elsevier Science Inc. All rights reserved.

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.

Identification of an additional two-cysteine containing type I interferon in rainbow trout Oncorhynchus mykiss provides evidence of a major gene duplication event within this gene family in teleosts

Multiple type I interferons (IFNs) have recently been identified in salmonids, containing two or four conserved cysteines. In this work, a novel two-cysteine containing (2C) IFN gene was identified in rainbow trout. This novel trout IFN gene (termed IFN5) formed a phylogenetic group that is distinct from the other three salmonid IFN groups sequenced to date and had a close evolutionary relationship with IFNs from advanced fish species. Our data demonstrate that two subgroups are apparent within each of the 2C and 4C type I IFNs, an evolutionary outcome possibly due to two rounds of genome duplication events that have occurred within teleosts. We have examined gene expression of the trout 2C type I IFN in cultured cells following stimulation with lipopolysaccharide, phytohaemagglutinin, polyI:C or recombinant IFN, or after transfection with polyI:C. The kinetics of gene expression was also studied after viral infection. Analysis of the regulatory elements in the IFN promoter region predicted several binding sites for key transcription factors that potentially play an important role in mediating IFN5 gene expression.

A study of low-energy ion induced radiolysis of thiol-containing amino acid cysteine in the solid and aqueous solution states

The radiolysis of cysteine under plasma discharge and irradiation of low-energy Ion beam was investigated. The damage of cysteine in aqueous solution under discharge was assessed via the acid ninhydrin reagent and the yield of cystine produced from the reaction was analyzed by FTIR In addition, the generation of hydrogen sulfide was also identified The destruction of solid cysteine under low-energy ion beam irradiation was estimated via monitoring IR bands of different functional groups (-SH, -NH3, -COO-) of cysteine. and the production of cystine from ion-irradiated solid cysteine after dissolution in water was also verified These results may help us to understand the inactivation of sulphydryl enzymes under direct and indirect interaction with the low-energy ion irradiation (C) 2010 Elsevier B V All rights reserved.

Formation of reversible shell cross-linked micelles from the biodegradable amphiphilic diblock copolymer poly(L-cysteine)-block-poly(L-lactide)

A novel biodegradable diblock copolymer, poly(L-cysteine)-b-Poly(L-lactide) (PLC-b-PLLA), was synthesized by ring-opening polymerization (ROP) of N-carboxyanhydride of beta-benzyloxycarbonyl-L-Cysteine (ZLC-NCA) with amino-terminated Poly(L-lactide) (NH2-PLLA) as a macroinitiator in a convenient way. The diblock copolymer and its precursor were characterized by H-1 NMR, Fourier transform infrared (FT-IR), gel permeation chromatography (GPC), and X-ray photoelectron spectroscopy (XPS) measurements. The length of each block polymer could be tailored by molecular design and the ratios of feeding monomers.

Sensitive detection of cysteine based on fluorescent silver clusters

In this work,we report the application of novel, water-soluble fluorescent Ag clusters in fluorescent sensors for detecting cysteine, an important biological analyte. The fluorescence of poly(methacrylic acid) (PMAA)templated Ag clusters was found to be quenched effectively by cysteine, but not when the other alpha-amino acids were present. By virtue of the specific response, a new, simple, and sensitive fluorescent method for detecting cysteine has been developed based on Ag clusters. The present assay allows for the selective determination of cysteine in the range of 2.5 x 10(-8) to 6.0 x 10(-6) M with a detection limit of 20 nM at a signal-to-noise ratio of 3. Based on the absorption and fluorescence studies, we suggested that cysteine quenched the emission by the thiol-adsorption-accelerated oxidation of the emissive Ag clusters. The present study shows a promising step toward the application of silver clusters, a new class of attractive fluorescence probes.