Hemoglobinase activity of a cysteine protease from the ixodid tick Haemaphysalis longicornis

We report here the molecular characterization and possible function of a cysteine protease (termed HlCPL-A) identified in the midgut of the hard tick Haemaphysalis longicornis. HlCPL-A is a 333 amino acid protein belonging to the papain family of the cysteine protease. A construct encoding proHlCPL-A was expressed in Escherichia coli and purified as both procathepsin L and active processed cathepsin L forms. The HlCPL-A gene expression was up-regulated by blood-feeding process. HlCPL-A exhibited substrate specificity against synthetic peptidyl substrates (Z-Phe-Arg-MCA and Z-Arg-Arg-MCA; k_cat / K_m = 0.19 and 0.0023 M^− 1 S^− 1, respectively). The proteolytic activity of HlCPL-A was inhibited by leupeptin, antipain and E-64 but was unaffected by pepstatin. HlCPL-A was capable of degrading bovine hemoglobin at pH 3.2 to 5.6. These results suggest that HlCPL-A may play important roles in the digestion of host hemoglobin in ticks.     

Molecular cloning of a multidomain cysteine protease and protease inhibitor precursor gene from the tobacco hornworm (Manduca sexta) and functional expression of the cathepsin F-like cysteine protease domain

A Manduca sexta (tobacco hornworm) cysteine protease inhibitor, MsCPI, purified from larval hemolymph has an apparent molecular mass of 11.5 kDa, whereas the size of the mRNA is very large (9 kilobases). MsCPI cDNA consists of a 9,273 nucleotides that encode a polypeptide of 2,676 amino acids, which includes nine tandemly repeated MsCPI domains, four cystatin-like domains and one procathepsin F-like domain. The procathepsin F-like domain protein was expressed in Escherichia coli and processed to its active mature form by incubation with pepsin. The mature enzyme hydrolyzed Z-Leu–Arg–MCA, Z-Phe–Arg–MCA and Boc–Val–Leu–Lys–MCA rapidly, whereas hydrolysis of Suc–Leu–Tyr–MCA and Z-Arg–Arg–MCA was very slow. The protease was strongly inhibited by MsCPI, egg-white cystatin and sunflower cystatin with K_i values in the nanomolar range. When the MsCPI tandem protein linked to two MsCPI domains was treated with proteases, it was degraded by the cathepsin F-like protease. However, tryptic digestion converted the MsCPI tandem protein to an active inhibitory form. These data support the hypothesis that the mature MsCPI protein is produced from the MsCPI precursor protein by trypsin-like proteases. The resulting mature MsCPI protein probably plays a role in the regulation of the activity of endogenous cysteine proteases.     

Characterization of a cysteine protease from Tritrichomonas foetus that induces host-cell apoptosis

Tritrichomonas foetus is a serious veterinary pathogen, causing bovine trichomoniasis and affecting cattle herds world-wide, resulting in inflammation of the genital tract, infertility and huge economic losses. The parasite secretes a cysteine protease (CP8), which induces cytotoxicity and apoptosis in bovine vaginal and uterine epithelial cells. Mallinson et al. [D.J. Mallinson, J. Livingstone, K.M. Appleton, S.J. Lees, G.H. Coombs, M.J. North, Microbiology 1995, 141 (12) 3077–3085.] originally reported a partial DNA sequence of T. foetus CP8 based on PCR cloning of T. foetus genomic DNA. Here we report the biochemical properties of the CP8 enzyme. Kinetic properties and the substrate specificity profile of T. foetus CP8 were studied using positional scanning synthetic combinatorial libraries and Michaelis–Menten kinetic analysis of three synthetic fluorogenic substrates. The preferred substrate Z-Leu-Arg-MCA prevented host-cell death/apoptosis induced by CP8. In addition, the DNA sequence was completed by 3′ and 5′ rapid amplification of cDNA ends (RACE) and the full-length amino acid sequence was obtained.     

Purification and partial characterisation of a novel trypsin-like cysteine protease from Metarhizium anisopliae

Abstract Trypsin-like enzymes from the entomopathogenic fungus Metarhizium anisopliae have been characterised. Two proteases with tryptic activity were purified by narrow range isoelectric focussing and affinity chromatography. One of these proteases, with an isoelectric point of 5.4 and a molecular mass of 28.8 kDa is a 'classical' trypsin belonging to the serine protease class. The other protease, with an isoelectric point of 4.6 and a molecular mass of 26.7 kDa, demonstrates trypsin-like specificity but, on the basis of inhibition and activation studies, belongs to the cysteine protease family and as such is the first fungal protease to be found of this type. The amino acid composition, kinetic constants and activity against proteinaceous substrates, including locust cuticle have been determined.     

Kininogens: More than cysteine protease inhibitors and kinin precursors

Two kininogens are found in mammalian sera: HK (high molecular weight kininogen) and LK (low molecular weight kininogen) with the exception of the rat which encompasses a third kininogen, T-Kininogen (TK). Kininogens are multifunctional glycosylated molecules related to cystatins (clan IH, family I25). They harbor three cystatin domains but only two of them are tight-binding inhibitors of cysteine cathepsins. HK and LK, but not TK, are precursors of potent peptide hormones, the kinins, which are released proteolytically by tissue and plasma kallikreins. Besides these classical features novel functions of kininogens have been recently discovered; they are described in the second part of this review. HKa, which corresponds to the kinin-free two-chain HK and its isolated domain D5 (kininostatin), possesses angiostatic and pro-apoptotic properties, inhibits the proliferation of endothelial cells and participates in the regulation of angiogenesis. Moreover, some HK-derived peptides display potent and broad-spectrum microbicidal properties against both Gram-positive and Gram-negative bacteria, and thus may offer a promising alternative to conventional antibiotic therapy. Of seminal interest, a kininogen-derived peptide inhibits activation of the contact phase system of coagulation and protects mice with invasive Streptococcus pyogenes infection from pulmonary lesions. On the other hand, TK is a biomarker of aging at the end of lifespan of elderly rats. However, although TK has been initially identified as an acute phase reactant, and earlier known as alpha-l-acute phase globulin, the increase of TK in liver and plasma is not known to relate to any inflammatory event during the senescence process.     

Evaluation of dipeptide nitriles as inhibitors of rhodesain,a major cysteine protease of Trypanosoma brucei

A series of dipeptide nitriles known as inhibitors of mammalian cathepsins were evaluated for inhibition of rhodesain, the cathepsin L-like protease of Trypanosoma brucei. Compound 35 consisting of a Leu residue fitting into the S2 pocket and a triarylic moiety consisting of thiophene, a 1,2,4-oxadiazole and a phenyl ring fitting into the S3 pocket, and compound 33 with a 3-bromo-Phe residue (S2) and a biphenyl fragment (S3) were found to inhibit rhodesain in the single-digit nanomolar range. The observed steep structure-activity relationship could be explained by covalent docking simulations. With their high selectivity indices (ca. 200) and the good antitrypanosomal activity (8 μM) the compounds represent promising starting points for new rhodesain inhibitors.     

Vinyl sulfone-based inhibitors of trypanosomal cysteine protease rhodesain with improved antitrypanosomal activities

The number of reported cases of Human African Trypanosmiasis (HAT), caused by kinetoplastid protozoan parasite Trypanosoma brucei, is declining in sub-Saharan Africa. Historically, such declines are generally followed by periods of higher incidence, and one of the lingering public health challenges of HAT is that its drug development pipeline is historically sparse. As a continuation of our work on new antitrypanosomal agents, we found that partially saturated quinoline-based vinyl sulfone compounds selectively inhibit the growth of T. brucei but displayed relatively weak inhibitory activity towards T. brucei’s cysteine protease rhodesain. While two nitroaromatic analogues of the quinoline-based vinyl sulfone compounds displayed potent inhibition of T. brucei and rhodesain. The quinoline derivatives and the nitroaromatic-based compounds discovered in this work can serve as leads for ADME-based optimization and pre-clinical investigations.     

Degradation of the S. frugiperda peritrophic matrix by an inducible maize cysteine protease

A unique 33-kDa cysteine protease (Mir1-CP) rapidly accumulates at the feeding site in the whorls of maize (Zea mays L.) lines that are resistant to herbivory by Spodoptera frugiperda and other lepidopteran species. When larvae were reared on resistant plants, larval growth was reduced due to impaired nutrient utilization. Scanning electron microscopy (SEM) indicated that the peritrophic matrix (PM) was damaged when larvae fed on resistant plants or transgenic maize callus expressing Mir1-CP. To directly determine the effects of Mir1-CP on the PM in vitro, dissected PMs were treated with purified, recombinant Mir1-CP and the movement of Blue Dextran 2000 across the PM was measured. Mir1-CP completely permeabilized the PM and the time required to reach full permeability was inversely proportional to the concentration of Mir1-CP. Inclusion of E64, a specific cysteine protease inhibitor prevented the damage. The lumen side of the PM was more vulnerable to Mir1-CP attack than the epithelial side. Mir1-CP damaged the PM at pH values as high as 8.5 and more actively permeabilized the PM than equivalent concentrations of the cysteine proteases papain, bromelain and ficin. The effect of Mir1-CP on the PMs of Helicoverpa zea, Danaus plexippus, Ostrinia nubilalis, Periplaneta americana and Tenebrio molitor also was tested, but the greatest effect was on the S. frugiperda PM. These results demonstrate that the insect-inducible Mir1-CP directly damages the PM in vitro and is critical to insect defense in maize.     

New functions of lactoferrin and beta-casein in mammalian milk as cysteine protease inhibitors

We found new inhibitory function of lactoferrin and beta-casein in milk against cysteine proteases using reverse zymography. The inhibition of cathepsin L by lactoferrin was strongest and the inhibition kinetics were of a non-competitive type. Heat denatured lactoferrin lost the inhibitory activity completely, therefore the tertiary structure is essential to show the inhibition. Native lactoferrin was not degraded by papain during the assay condition. The intramolecular peptide, Y(679)-K(695), of lactoferrin is an active domain and the synthesized peptide inhibited cysteine proteases. The Y(679)-K(695) peptide showed 90% homology with the sequences of a common active site of cystatin family. beta-Casein and the active domain, synthesized L(133)-Q(151), peptide inhibited cysteine proteases. Lactoferrin and beta-casein in milk might play a role in antiseptic and antiinfectious functions due to cysteine protease inhibition of bacteria and viruses.     

Procerain,a stable cysteine protease from the latex of Calotropis procera

A protease was purified to homogeneity from the latex of medicinal plant Calotropis procera (Family-Asclepiadaceae). The molecular mass and isoelectric point of the enzyme are 28.8 kDa and 9.32, respectively. Hydrolysis of azoalbumin by the enzyme was optimal in the range of pH 7.0-9.0 and temperature 55-60 degree C. The enzyme hydrolyses denatured natural substrates like casein, azoalbumin, and azocasein with high specific activity. Proteolytic and amidolytic activities of the enzyme were activated by thiol protease activators and inhibited by thiol protease inhibitors, indicating the enzyme to be a cysteine protease. The enzyme named as procerain, cleaves N-succinyl-Ala-Ala-Ala-p-nitroanilide but not -Ala-Ala-p-nitroanilide, -Ala p-nitroanilide and N-d-Benzoyl--Arg-p-nitroanilide and appears to be peptide length dependent. The extinction coefficient (epsilon 1% 280 nm) of the enzyme was 24.9 and it had no detectable carbohydrate moiety. Procerain contains eight tryptophan, 20 tyrosine and seven cysteine residues forming three disulfide bridges, and the remaining one being free. Procerain retains full activity over a broad range of pH 3.0-12.0 and temperatures up to 70 degree C, besides being stable at very high concentrations of chemical denaturants and organic solvents. Polyclonal antibodies against procerain do not cross-react with other related proteases. Procerain unlike most of the plant cysteine proteases has blocked N-terminal residue.     

Identification of EhICP1, a chagasin-like cysteine protease inhibitor of Entamoeba histolytica

Based on the Entamoeba histolytica genome project (www.sanger.ac.uk/Project/E_histolytical/) we have identified a cysteine protease inhibitor, EhICP1 (amoebiasin 1), with significant homology to chagasin. Recombinant EhICP1 inhibited the protease activity of papain and that of a trophozoite lysate with Ki's in the picomolar range. By immunocytology, we localized the endogenous approximately 13 kDa EhICP1 in a finely dotted subcellular distribution discrete from the vesicles containing the amoebic cysteine protease, EhCP1 (amoebapain). In an overlay assay, we observed binding of recombinant EhICP1 to EhCP1. As a heptapeptide (GNPTTGF) corresponding to the second conserved chagasin motif inhibited the protease activity of both papain (K) 1.5 microM) and trophozoite extract (Ki in sub-mM range), it may be a candidate for the rational development of anti-amoebiasis drugs.     

Characterization of the cysteine protease domain of Semliki Forest virus replicase protein nsP2 by in vitro mutagenesis

The function of Semliki Forest Virus nsP2 protease was investigated by site-directed mutagenesis. Mutations were introduced in its protease domain, Pro39, and the mutated proteins were expressed in Escherichia coli, purified and their activity in vitro was compared to that of the wild type Pro39. Mutations M781T, A662T and G577R, found in temperature-sensitive virus strains, rendered the enzyme temperature-sensitive in vitro as well. Five conserved residues were required for the proteolytic activity of Pro39. Changes affecting Cys⁴⁷⁸, His⁵⁴⁸, and Trp⁵⁴⁹ resulted in complete inactivation of the enzyme, whereas the replacements N600D and N605D significantly impaired its activity. The importance of Trp⁵⁴⁹ for the proteolytic cleavage specificity is discussed and a new structural motif involved in substrate recognition by cysteine proteases is proposed.     

Ginger rhizome as a potential source of milk coagulating cysteine protease

A milk coagulating protease was purified ∼10.2-fold to apparent homogeneity from ginger rhizomes in 34.9% recovery using ammonium sulfate fractionation, together with ion exchange and size exclusion chromatographic techniques. The molecular mass of the purified protease was estimated to be ∼36 kDa by SDS-PAGE, and exhibited a pI of 4.3. It is a glycoprotein with 3% carbohydrate content. The purified enzyme showed maximum activity at pH 5.5 and at a temperature of ∼60 °C. Its protease activity was strongly inhibited by iodoacetamide, E-64, PCMB, Hg²⁺ and Cu²⁺. Inhibition studies and N-terminal sequence classified the enzyme as a member of the cysteine proteases. The cleavage capability of the isolated enzyme was higher for α_s-casein followed by β- and κ-casein. The purified enzyme differed in molecular mass, pI, carbohydrate content, and N-terminal sequence from previously reported ginger proteases. These results indicate that the purified protease may have potential application as a rennet substitute in the dairy industry.     

Babesia bovis: effects of cysteine protease inhibitors on in vitro growth

In the present study, we examined the effects of four kinds of cysteine protease inhibitors (E64, E64d, leupeptin, and ALLN) on the in vitro asexual growth of Babesia bovis. Of these, only the lipophilic inhibitors, E64d and ALLN, were found to effectively inhibit the growth of B. bovis. In further experiments, E64d, but not ALLN, significantly suppressed the parasite’s invasion of host erythrocytes, while both chemicals, especially ALLN, inhibited the parasite’s replication within the infected erythrocytes. These data suggested the presence of cysteine protease(s) derived from B. bovis, in which the protease(s) would play important roles in the erythrocyte invasion and/or replication processes of the parasite.     

Development of cell-active non-peptidyl inhibitors of cysteine cathepsins

Cysteine cathepsins are an important class of enzymes that coordinate a variety of important cellular processes, and are implicated in various types of human diseases. However, small molecule inhibitors that are cell-permeable and non-peptidyl in nature are scarcely available. Herein the synthesis and development of sulfonyloxiranes as covalent inhibitors of cysteine cathepsins are reported. From a library of compounds, compound 5 is identified as a selective inhibitor of cysteine cathepsins. Live cell imaging and immunocytochemistry of metastatic human breast carcinoma MDA-MB-231 cells document the efficacy of compound 5 in inhibiting cysteine cathepsin activity in living cells. A cell-motility assay demonstrates that compound 5 is effective in mitigating the cell-migratory potential of highly metastatic breast carcinoma MDA-MB-231 cells.     

The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis

The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.     

Potential role in development of the major cysteine protease in larvae of the brine shrimp Artemia franciscana

Encysted embryos and larvae of the brine shrimp Artemia franciscana contain a cysteine protease which represents over 90% of the protease activity in these organisms. We have used immunocytochemical methods to determine the localization and potential role of the cysteine protease in development of young larvae. In prenauplius larvae, there is intense staining for the protease on the basal side of the epidermal layer in the posterior region and diffuse staining for the protease throughout the embryo. In first instar larvae, cysteine-protease staining becomes intense in the midgut-forming area where a reticulum-like pattern emerges in cells with an abundance of yolk platelets. Cysteine-protease staining in second instar larvae becomes intense in the apical side of epidermal cells and in the basal and apical zones of midgut cells. Subcellular localization of the protease in the epidermis and midgut of young larvae using immunogold electron microscopy suggests that most is located in the cytosol and extracellular matrix adiacent to these cells. Addition of cysteine-protease inhibitors to the growth medium, especially the fluoromethyl ketone Z-Phe-Ala-CH₂F, inhibits growth and segmentation of the thorax. Collectively, these observations suggest that the major cysteine protease in embryos and larvae functions in yolk utilization, as a hatching enzyme, in apolysis during the molt cycle, and as a digestive enzyme when the swimming larvae begin to feed.     

Recombinant Der p 1 and Der f 1 exhibit cysteine protease activity but no serine protease activity

Although mite major group 1 allergens, Der p 1 and Der f 1, were first isolated as cysteine proteases, some studies reported that natural Der p 1 exhibits mixed cysteine and serine protease activity. Clarifying whether the serine protease activity originates from Der p 1 or is due to contamination is important for distinguishing between the pathogenic proteolytic activities of group 1 allergens and mite-derived serine proteases. Recombinant mite group 1 allergens would be useful tool for addressing this issue, because they are completely free from contamination by mite serine proteases. Recombinant Der p 1 and Der f 1, and highly purified natural forms exhibited only cysteine protease activity. However, commercially available natural forms exhibited both activities, but the two activities were eluted into different fractions in size-exclusion column chromatography. The substrate specificity associated with the serine protease activity was similar to that of Der f 3. These results indicate that the serine protease activity does not originate from group 1 allergens.