Characterisation of a cysteine protease from poultry red mites and its potential use as a vaccine for chickens.

Poultry red mites (PRMs, Dermanyssus gallinae) are ectoparasites that negatively affect farmed chickens, leading to serious economic losses worldwide. Acaricides have been used to control PRMs in poultry houses. However, some PRMs have developed resistance to acaricides, and therefore different approaches are required to manage the problems caused by PRMs. Vaccination of chickens is one of the methods being considered to reduce the number of PRMs in poultry houses. In a previous study, a cysteine protease, Deg-CPR-1, was identified as a candidate vaccine against PRMs distributed in Europe. In this study, we investigated the characteristics of Deg-CPR-1. A phylogenetic analysis revealed that Deg-CPR-1 is closely related to the digestive cysteine proteases of other mite species, and it was classified into a cluster different from that of chicken cathepsins. Deg-CPR-1 of PRMs in Japan has an amino acid substitution compared with that of PRMs in Europe, but it showed efficacy as a vaccine, consistent with previous findings. Deg-CPR-1 exhibited cathepsin L-like enzyme activity. In addition, the Deg-CPR-1 mRNA was expressed in the midgut and in all stages of PRMs that feed on blood. These results imply that Deg-CPR-1 in the midgut may have important functions in physiological processes, and the inhibition of its expression may contribute to the efficacy of a Deg-CPR-1-based vaccine. Further research is required to fully understand the mechanisms of vaccine efficacy.

TITLE: Caractérisation d'une cystéine protéase des poux rouges de la volaille et son utilisation potentielle comme vaccin pour les poulets. ABSTRACT: Les acariens communément appelés poux rouges de la volaille (PRV, Dermanyssus gallinae) sont des ectoparasites qui affectent négativement les poulets d'élevage, entraînant de graves pertes économiques au niveau mondial. Des acaricides ont été utilisés pour contrôler les PRV dans les poulaillers. Cependant, certains PRV ont développé une résistance aux acaricides, et par conséquent, différentes approches sont nécessaires pour gérer les problèmes qu'ils causent. La vaccination des poulets est l'une des méthodes envisagées pour réduire le nombre de PRV dans les poulaillers. Dans une étude précédente, une cystéine protéase, Deg-CPR-1, a été identifiée comme un vaccin candidat contre les PRV distribués en Europe. Dans cette étude, nous avons étudié les caractéristiques de Deg-CPR-1. L'analyse phylogénétique a révélé que Deg-CPR-1 est étroitement liée aux cystéine protéases digestives d'autres espèces d'acariens, et elle a été classée dans un groupe différent de celui des cathepsines de poulet. La Deg-CPR-1 des PRV au Japon a une substitution d'acide aminé par rapport à celle des PRV en Europe, mais elle a montré une efficacité en tant que vaccin, conformément aux résultats précédents. Deg-CPR-1 a présenté une activité enzymatique de type cathepsine L. De plus, l'ARNm de Deg-CPR-1 était exprimé dans l'intestin moyen et à tous les stades où les PRV se nourrissent de sang. Ces résultats impliquent que Deg-CPR-1 dans l'intestin moyen peut avoir des fonctions importantes dans les processus physiologiques, et que l'inhibition de son expression peut contribuer à l'efficacité d'un vaccin basé sur Deg-CPR-1. Des recherches supplémentaires sont nécessaires pour comprendre pleinement les mécanismes de l'efficacité du vaccin.

Effects of UV-B radiation on the survival, egg hatchability and transcript expression of antioxidant enzymes in a high-temperature adapted strain of Neoseiulus barkeri.

Biological control of spider mites in hot and dry weather is a serious technical issue. A high-temperature adapted strain (HTAS) of the predatory mite Neoseiulus barkeri Hughes was selected from its conventional strain (CS), via long-term heat acclimation and frequent heat hardenings in our previous studies. However, the environment of high temperature is usually associated with enhanced ultraviolet (UV) radiation. In the present study, the physiological effects of UV-B radiation on survival rate and egg damage of N. barkeri were investigated, as well as the activities and expression profiles of antioxidant enzymes to UV-B radiation stress. UV-B radiation had deleterious effects on egg hatchability and survival of N. barkeri. Adults of the HTAS strain were less UV-B resistant than those of the CS strain; they also had lower levels of enzymatic activity of superoxide dismutase (SOD) and catalase against oxidative damage and weaker upregulation of SOD genes. The mRNA expression of three SOD genes of CS adult females immediately increased whereas that of HTAS showed almost no difference under UV-B stress for 1 h. The results showed the HTAS of N. barkeri had lower fitness under UV-B stress compared with the CS of N. barkeri. These results suggested that long-term heat acclimation may exert a profound impact on the developmental physiology of N. barkeri.

Effects of heat stress on development, reproduction and activities of protective enzymes in Mononychellus mcgregori.

Mononychellus mcgregori is a pest mite of cassava. Since its invasion into China in 2008 it has spread rapidly. In order to determine the potential distribution and to analyze its invasion, diffusion and ecological adaptation mechanisms, we investigated the effect of high-temperature stress (30, 33, 36, 39 and 42 °C) on its development and reproduction, and the activity of protective enzymes in the mite. The results indicated significant influences: (1) adults could not lay eggs after they had been exposed to 42 °C for 4 h or longer; (2) egg development was slower and egg hatchability decreased after exposure of adults to 33-42 °C for 1 h; (3) offspring development (all stages) was slower after exposure of adults to 33-42 °C for 2 h or more; and (4) polyphenol oxidase (PPO), peroxidase (POD), ascorbate peroxidase (APX) and catalase (CAT) activities in the adults increased to high levels after exposure to 33-42 °C for 1 h, and superoxide dismutase activity increased only after exposure to 42 °C for 1 h. In conclusion, exposure to high temperatures for only 1 h probably has an important impact on the mite's population growth. The significant increase of PPO, POD, APX, and CAT activities in adults may partially explain how M. mcgregori survive exposure to a relatively high temperature.

Assessment of cathepsin D and L-like proteinases of poultry red mite, Dermanyssus gallinae (De Geer), as potential vaccine antigens.

Vaccination is a feasible strategy for controlling the haematophagous poultry red mite Dermanyssus gallinae. A cDNA library enriched for genes upregulated after feeding was created to identify potential vaccine antigens. From this library, a gene (Dg-CatD-1) encoding a 383 amino acid protein (Dg-CatD-1) with homology to cathepsin D lysosomal aspartyl proteinases was identified as a potential vaccine candidate. A second gene (Dg-CatL-1) encoding a 341 amino acid protein (Dg-CatL-1) with homology to cathepsin L cysteine proteinases was also selected for further study. IgY obtained from naturally infested hens failed to detect Dg-CatD-1 suggesting that it is a concealed antigen. Conversely, Dg-CatL-1 was detected by IgY derived from natural-infestation, indicating that infested hens are exposed to Dg-CatL-1. Mortality rates 120 h after mites had been fed anti-Dg-CatD-1 were significantly higher than those fed control IgY (PF<0·01). In a survival analysis, fitting a proportional hazards model to the time of death of mites, anti-Dg-CatD-1 and anti-Dg-CatL-1 IgY had 4·42 and 2·13 times higher risks of dying compared with controls (PF<0·05). Dg-CatD-1 and L-1 both have potential as vaccine antigens as part of a multi-component vaccine and have the potential to be improved as vaccine antigens using alternative expression systems.

Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases.

Phytocystatins are inhibitors of cysteine-proteases from plants putatively involved in plant defence based on their capability of inhibit heterologous enzymes. We have previously characterised the whole cystatin gene family members from barley (HvCPI-1 to HvCPI-13). The aim of this study was to assess the effects of barley cystatins on two phytophagous spider mites, Tetranychus urticae and Brevipalpus chilensis. The determination of proteolytic activity profile in both mite species showed the presence of the cysteine-proteases, putative targets of cystatins, among other enzymatic activities. All barley cystatins, except HvCPI-1 and HvCPI-7, inhibited in vitro mite cathepsin L- and/or cathepsin B-like activities, HvCPI-6 being the strongest inhibitor for both mite species. Transgenic maize plants expressing HvCPI-6 protein were generated and the functional integrity of the cystatin transgene was confirmed by in vitro inhibitory effect observed against T. urticae and B. chilensis protein extracts. Feeding experiments impaired on transgenic lines performed with T. urticae impaired mite development and reproductive performance. Besides, a significant reduction of cathepsin L-like and/or cathepsin B-like activities was observed when the spider mite fed on maize plants expressing HvCPI-6 cystatin. These findings reveal the potential of barley cystatins as acaricide proteins to protect plants against two important mite pests.

Enzymatic activity in body and fecal extracts of the storage mite Chortoglyphus arcuatus.

BACKGROUND: Chortoglyphus arcuatus has been described in many countries. Many allergens are potent enzymes, which may promote a Th2 immune response. The aim of this study was to evaluate the enzymatic activity of body and fecal extracts of C. arcuatus. MATERIAL AND METHODS: Feces and bodies of full-grown C. arcuatus cultures were separated by sieving, extracted in PBS, dialyzed and lyophilized. The antigenic profile of both extracts was determined by SDS-PAGE. Immunoblot experiments were conducted using a pool of sera from allergic individuals residing in Galicia, a region of Spain, where this species is abundant. The enzymatic activity of the extracts was evaluated by the zymogram technique. Serine and cysteine protease activity was measured using in vitro methods. The API Zym system was used to determine the enzymatic properties of the extracts. RESULTS: The antigenic profile showed that the body extract contained more and better defined bands than the fecal extract. Allergens were detected in both extracts in a molecular weight range between 14 and 100 kDa. Gelatinolytic gels confirmed that fecal extracts contain more hydrolytic enzymatic activity than body extracts. Serine protease activity in fecal extracts was higher than in body extracts (5.98 vs. 2.701 IU of trypsin/mg of freeze-dried material). No cysteine protease activity was detected. CONCLUSION: C. arcuatus extracts contain several allergens and proteins with high enzymatic activity, especially in the feces. Some of these allergens may be enzymes. Fecal extracts have more enzymatic activity than body extracts.

The proteinases of Psoroptes ovis, the sheep scab mite--their diversity and substrate specificity.

The sheep scab mite, Psoroptes ovis, causes severe dermatitis in infected sheep with severe welfare and production implications. The dermatitis has the characteristics of an immediate hypersensitivity type reaction which, by analogy to other mite species, including the house dust mites (Dermatophagoides spp.), is likely to be invoked by a variety of allergens including mite-derived proteinases. Here, the proteinases in P. ovis extracts were characterised using substrate gel analysis, inhibitor sensitivity and their ability to degrade a variety of potential natural protein substrates. These analyses showed that mites contain several proteinases which could be differentiated on the basis of molecular size and inhibitor sensitivity with cysteine, metalloproteinases and aspartyl proteinases predominating. These proteinases degraded collagen and fibronectin, possibly indicative of a role in lesion initiation, they degraded several blood proteins, a property which may aid mite feeding and they degraded immunoglobulin G, possibly aiding immuno-evasion. Because proteinases, particularly the cysteine class, are demonstrably allergenic in other mite infestations, these proteinases clearly merit further immunological and biochemical definition.

Host plant-induced changes in detoxification enzymes and susceptibility to pesticides in the twospotted spider mite (Acari: Tetranychidae).

Adult female twospotted spider mites, Tetranychus urticae Koch, reared on lima bean plants were moved to cucumber, maize, or new lima bean plants (the latter being a control) and evaluated after 24 h or 7 d for changes in susceptibility to three pesticides and in levels of related detoxification enzymes. The largest and most consistent changes were observed in mites feeding on cucumber. Susceptibility of mites on cucumber to the synthetic pyrethroids bifenthrin and lambda-cyhalothrin was greater than that of mites reared on lima bean and maize after only 24 h on the plants, and remained higher after 7 d. Mites on cucumber also were more susceptible to the organophosphate dimethoate than were mites on lima bean, but only after 7 d on the host. Susceptibility was inversely related to activities of both general esterase and glutathione S-transferase (GST) in mites on cucumber; general esterase and GST activities were 60 and 25% lower, respectively, than activities of twospotted spider mite on lima bean after 7 d of feeding. Mites on maize were slightly but significantly more susceptible than those on lima bean to bifenthrin, but not to lambda-cyhalothrin, after 7 d and to dimethoate after 24 h but not after 7 d. General esterase and GST activities in twospotted spider mite fed on maize for 24 h were 20 and 16% higher, respectively, than activities in twospotted spider mite on lima bean, but general esterase activity was 30% lower than lima bean-fed mites and GST was not different after 7 d. Thus, plant-induced changes in general esterase activity, perhaps in combination with GST activity, in twospotted spider mite appear to be inversely related to, and possibly responsible for, changes in susceptibility of twospotted spider mite to several pesticides, particularly the synthetic pyrethroids. General esterases appear to play less of a role in the detoxification of the organophosphate insecticide dimethoate.

The transmembrane protein occludin of epithelial tight junctions is a functional target for serine peptidases from faecal pellets of Dermatophagoides pteronyssinus.

There have been only a few studies of how allergens cross the airway epithelium to cause allergic sensitization. House dust mite fecal pellets (HDMFP) contain several proteolytic enzymes. Group 1 allergens are cysteine peptidases, whilst those of groups 3, 6 and 9 have catalytic sites indicative of enzymes that mechanistically behave as serine peptidases. We have previously shown that the group 1 allergen Der p 1 leads to cleavage of tight junctions (TJs), allowing allergen delivery to antigen presenting cells. In this study we determined whether HDMFP serine peptidases similarly compromise the airway epithelium by attacking TJs, desmosomes and adherens junctions. Experiments were performed in monolayers of MDCK, Calu-3 or 16HBE14o-epithelial cells. Cell junction morphology was examined by 2-photon molecular excitation microscopy and digital image analysis. Barrier function was measured as mannitol permeability. Cleavage of cell adhesion proteins was studied by immunoblotting and mass spectrometry. HDMFP serine peptidases led to a progressive cleavage of TJs and increased epithelial permeability. Desmosomal puncta became more concentrated. Cleavage of TJs involved proteolysis of the TJ proteins, occludin and ZO-1. This was associated with activation of intracellular proteolysis of ZO-1. In contrast to occludin, E-cadherin of adherens junctions was cleaved less extensively. Although Calu-3 and 16HBE14o-cells expressed tethered ligand receptors for serine peptidases, these were not responsible for transducing the changes in TJs. HDMFP serine peptidases cause cleavage of TJs. This study identifies a second general class of HDM peptidase capable of increasing epithelial permeability and thereby creating conditions that would favour transepithelial delivery of allergens.

Oncostatin M synergises with house dust mite proteases to induce the production of PGE(2) from cultured lung epithelial cells.

The release of PGE(2) and nitric oxide (NO) from the respiratory epithelium may act to dampen inflammation. In other tissues, oncostatin M (OSM), a potent inducer of epithelial antiproteases, has also been shown to interact with IL-1beta to stimulate PGE(2) release. However, whether OSM interacts with pro-inflammatory cytokines and proteases in the production of anti-inflammatory eicosanoids and NO from airway epithelium is unknown. The effect of OSM and the related cytokine leukaemia inhibitory factor (LIF) on PGE(2) and NO production by the respiratory epithelial cell line, A549 in response to pro-inflammatory cytokines as well as protease-rich house dust mite (HDM) fractions and a protease-deficient rye grass pollen extract was examined by immunohistochemistry, cell culture, ELISA and enzyme-immunoassay. Cells treated with a mixture of IL-1beta, IFNgamma and LPS for 48 h produced a 9 fold increase in PGE(2) and a 3 fold increase in NO levels (both P<0.05). Both OSM and LIF were without effect. However, OSM added together with the cytokine mixture synergistically enhanced PGE(2) production (22 fold, P<0.05). OSM also synergistically enhanced PGE(2) production in response to a cysteine protease-enriched, but not serine protease-enriched HDM fraction (P<0.05). Rye grass extract, neither alone nor in combination with OSM, induced PGE(2) or NO production, although it did induce the release of GM-CSF. These observations suggest that OSM is an important co-factor in the release of PGE(2) and NO from respiratory epithelial cells and may play a role in defense against exogenous proteases such as those derived from HDM.

A comparative survey of the hydrolytic enzymes of ectoparasitic and free-living mites.

Extracts of ectoparasitic mites of birds (Dermanyssus gallinae), sheep (Psoroptes ovis) and plants (Tetranychus urticae) and of free-living mites (Acarus siro) contained acid and alkaline phosphatase, C4 and C8 esterases, lipase, leucine and valine aminopeptidases and a range of glycosidase activities. Dermanyssus gallinae and P. ovis, species highly adapted to an animal parasitic lifestyle, had very similar profiles and contained low activities of glycosidases. In contrast, the polyphagous species A. siro contained moderate to high activities of every glycosidase examined, whereas the phytophagous species, T. urticae, displayed high activities of only beta-galactosidase and beta-glucuronidase. All extracts hydrolysed haemoglobin with optima below pH6, and this hydrolysis was associated with an aspartic proteinase and variable cysteine proteinase activity dependent on species. Inhibitor-labelling with biotinyl-Phe-Ala-FMK revealed the presence of cysteine proteinases with molecular masses of 25-33.5kDa. Each mite species contains the enzymes necessary to complete digestion of the diet in the intracellular lysosomal compartment. The absolute and relative activities of each enzyme varied, and are discussed according to phylogeny and dietary habit.

Characterization of proteases from a stored product mite, Tyrophagus putrescentiae.

Extracts of Tyrophagus putrescentiae feces exhibited higher (>50-fold) specific protease activity rates than those measured using mite body extracts for the substrates azocasein, BApNa, SA(2)PPpNa, HA, and HPA. This suggests that trypsin, chymotrypsin, and carboxypeptidases A and B are involved in mite digestion. Hydrolysis of the substrates ZAA(2)MNA and LpNa was only 3 times higher in fecal extracts, suggesting that levels of cathepsin B and aminopeptidases in the lumen of the digestive tract are low compared to the other enzymes. The hydrolysis of hemoglobin was only detected in body extracts indicating that cathepsin D is not a digestive protease in this species. Protease inhibitors of different specificity were tested invivo to establish their potential as control agents. We found that development from larvae to adult was significantly retarded in larvae fed on brewers' yeast containing inhibitors of serine proteases, whereas no such effect was found with inhibitors of cysteine and aspartyl proteases. Interestingly, when dietary mixtures of serine protease, aminopeptidase and carboxypeptidase inhibitors were fed to T.putrescentiae, a synergistic effect was observed that retarded development. Several plant lectins were also tested, but none affected development.

Inhibition of Df-protease associated with allergic diseases by polyphenol.

It was reported that Df-protease from house dust mite (Dermatophagoides farinae) catalyzes the activation of the kallikrein-kinin system in human plasma and is closely associated with mite-induced allergy. Therefore, to prevent the release of kinin by Df-protease, the inhibitory activity of polyphenols including catechins and flavonols was tested in vitro and in vivo. Among them, myricetin and epigallocatechin gallate (EGCg) effectively inhibited the amidase activity of Df-protease with K(i) values of 1 x 10(-)(8) and 6 x 10(-)(4) M, respectively. The kinin release in human plasma was extensively inhibited by the addition of EGCg in comparison with myricetin. Enhancement of vascular permeability in guinea pigs caused by Df-protease was markedly suppressed by EGCg.

Proteinase and gelatinolytic activities of house dust mite and cockroach extracts.

The presence of gelatinolytic activity in dust mite and Periplaneta americana allergenic crude extracts were studied. The former presented major activity in a broad band between 45 and 66 kDa and minor activity at 32 kDa, while the latter showed a more complex pattern with gelatinolytic activity at 90, 78, 65, 34, 32 and 24 kDa. When the proteolytic activity patterns of dust mites and cockroach crude extracts were analyzed at three different pH levels, the proteases in both cases were optimally active at pH 6, showed no activity at pH 3.5 and little activity at pH 8.5. The susceptibility of both extracts to a set of well-known protease inhibitors suggested that they are composed of cysteine and serine proteinases, the latter probably being a trypsin-like type. When immunochemical properties were studied, dust mite bands of about 200, 110, 65, 60 and 43 kDa showed immunoreactivity against a polyclonal human anti-dust mite serum, with the band of approximately 200 kDa presenting the highest antigenicity. A similar analysis was applied to the cockroach extract, which exhibited immunoreactive bands at 90, 78, 65 and 34 kDa when incubated with a polyclonal rabbit anti-Blatta serum. Only those of 90, 78 and 65 kDa reacted against a polyclonal human anti-Blatta serum. These results suggested a correlation between some proteases with gelatinolytic activity and the allergenicity of both extracts.

Immunological control of scab mites: digestive enzymes as candidate compounds.

Phosphatases, C4 and C8 esterases, leucine and valine aminopeptidases, n-acetyl-beta-glucosaminidase, beta-glucosidase, beta-galactosidase and beta-glucuronidase were active in extracts of scab mites (Psoroptes spp.) raised on sheep or rabbits. Trypsin and chymotrypsin activities were not detected. Haemoglobin was hydrolysed by a detergent-soluble fraction of the mite extracts in a pH-dependent fashion with an optimum of pH 3-5. Acid proteinase activity was greater in mites raised on rabbits than in those raised on sheep. Inhibitors of cysteine, serine and metallo-proteinases failed to inhibit the hydrolysis of H-Pro-Thr-Glu-Phe-Phe(NO2)Arg-Leu-OH while pepstatin A, a specific inhibitor of aspartic proteinases, totally inhibited its hydrolysis at a concentration of 1 nM.

Enzymatic analyses of house dust mite extracts from Dermatophagoides pteronyssinus and Dermatophagoides farinae (Acari: Pyroglyphidae) during different phases of culture growth.

The majority of clinically important allergens of Dermatophagoides pteronyssinus (Trouessart, 1897) and Dermatophagoides farinae Hughes, 1961 present enzymatic activity. The allergenic enzymes described include cysteine proteases in group 1 allergens, trypsins in group 3, amylases in group 4, and chymotrypsins in group 6. Apart from these, other possibly allergenic enzymes also have been identified. Therefore, enzymatic profiles were studied during the 3 growth periods of the mite population--latency phase, exponential growth phase, and death phase. The activity of 19 different enzymes was analyzed by means of the Api Zym system, a method that has been used to study both mite extracts and other allergenic materials. Our study has demonstrated that the extracts contain a large variety of enzymes. It has been observed that enzymatic activity is caused exclusively by mites because the control carried out on the culture medium was negative for all the enzymes studied. Generally, the levels of diverse enzymatic activity increased with the growth of the culture, and decreased later, in both species. However, proteases are the exception; they maintain a high level of activity during the death phase of the cultured mites. The ratio between trypsin and chymotrypsin activity can be used as an excellent tool for quality control parameters during obtention of allergenic mite extracts.

Hydrolytic enzymes of Psoroptes cuniculi (Delafond).

Phosphatases, C4 and C8 esterases, leucine and valine aminopeptidases, N-acetyl-beta-glucosaminidase, beta-glucosidase, beta-galactosidase and beta-glucuronidase were detected in extracts of the parasitic mite Psoroptes cuniculi. Lipase, trypsin-like and chymotrypsin-like activities were not present. Haemoglobin was hydrolysed by a detergent-soluble fraction of the mite extracts with a maximum hydrolysis between pH 3 and 5. Acid proteinase activity was greater against haemoglobin than bovine serum albumin. Inhibitors of cysteine, serine and metallo-proteinases failed to inhibit the hydrolysis of H-Pro-Thr-Glu-Phe-Phe(NO2)-Arg-Leu-OH while pepstatin A inhibited its hydrolysis in a dose-dependent manner (IC50 8.02 x 10(-11) M (+/- 0.30 x 10(-11). Thermal inactivation of the proteolytic activity followed an exponential decay pattern. Typical K(m) and Vmax values were 7.2 x 10(-5) (+/- 0.7 x 10(-5) M-1 and 1.13 x 10(-3) (+/- 0.05 x 10(-3) OD unit-1 min-1 respectively. Acid proteinase activity eluted from a size exclusion column in a single, major peak representing a molecular weight range of 21-24.5 kDa. The major endoproteinase of P. cuniculi therefore appears to be a cathepsin D-like aspartic proteinase.

A chestnut seed cystatin differentially effective against cysteine proteinases from closely related pests.

Cystatin CsC, a cysteine proteinase inhibitor from chestnut (Castanea sativa) seeds, has been purified and characterized. Its full-length cDNA clone was isolated from an immature chestnut cotyledon library. The inhibitor was expressed in Escherichia coli and purified from bacterial extracts. Identity of both seed and recombinant cystatin was confirmed by matrix-assisted laser desorption/ionization mass spectrometry analysis, two-dimensional electrophoresis and N-terminal sequencing. CsC has a molecular mass of 11,275 Da and pI of 6.9. Its amino acid sequence includes all three motifs that are thought to be essential for inhibitory activity, and shows significant identity to other phytocystatins, especially that of cowpea (70%). Recombinant CsC inhibited papain (Ki 29 nM), ficin (Ki 65 nM), chymopapain (Ki 366 nM), and cathepsin B (Ki 473 nM). By contrast with most cystatins, it was also effective towards trypsin (Ki 3489 nM). CsC is active against digestive proteinases from the insect Tribolium castaneum and the mite Dermatophagoides farinae, two important agricultural pests. Its effects on the cysteine proteinase activity of two closely related mite species revealed the high specificity of the chestnut cystatin.

Cell lines of pulmonary and non-pulmonary origin as tools to study the effects of house dust mite proteinases on the regulation of epithelial permeability.

BACKGROUND: Allergenic and non-allergenic proteinases from house dust mites (HDM) cause loss of adhesion between airway epithelial cells that may result in a loss of functional cohesion between the cells and thus assist in allergen presentation. Improved cellular assay systems are needed to ascertain the mechanisms involved. OBJECTIVES: To survey a series of epithelial cell lines (Calu-3, 16HBE14o-, NCI-H292 and A549 from human airways, and MDCK from dog kidney) and establish their utility for studies of the effects of HDM proteinases from D. pteronyssinus on epithelial permeability. To develop an improved method for measuring changes in epithelial permeability induced by HDM proteinases and other provocants. METHODS: The permeability of epithelial monolayer cultures to mannitol was calculated from measurements of clearance using a technique that permits mathematical estimation and reduction of non-cellular diffusional constraints. Permeability was studied under control conditions and after perturbation of monolayers with HDM proteinases (separated into serine- and cysteine-proteinase classes) or chelation of extracellular Ca2+. Fluorescent antibody staining was used to investigate whether the cells expressed tight junctions (staining of ZO-1), desmosomes (staining of desmoplakin) and zonulae adherentes (staining of E-cadherin). RESULTS: The Calu-3 line was identified as an airway cell line that expressed functional tight junctions, desmosomes and zonulae adherentes. Calu-3 monolayers exhibited a low clearance and permeability to mannitol, similar to that seen in the extensively characterized MDCK cell line. Clearance and permeability were significantly increased by treatment with either HDM proteinase fraction or by calcium chelation. 16HBE14o- cells also had a low permeability to mannitol under control conditions and expressed a similar repertoire of functional proteins from major intercellular junctions. In contrast, NCI-H292 and A549 cell lines were functionally deficient in tight junctions, although they did express desmosomes and zonulae adherentes to a greater extent. Epithelial permeability was found to be a more appropriate and sensitive index of epithelial perturbation than was tracer clearance. CONCLUSION: These results suggest that the Calu-3 and 16HBE14o- cell lines are useful tools in studying the mechanism of HDM proteinases on airway epithelial cell function. HDM proteinases of both cysteine and serine mechanistic classes were found to perturb epithelial adhesion and function.