A cysteine protease from the latex of Ficus benjamina has in vitro anthelmintic activity against Haemonchus contortus / Atividade anti-helmíntica in vitro de uma protease cisteínica do látex de Ficus benjamina contra Haemonchus contortus

Abstract Haemonchus contortus is a gastrointestinal nematode that is responsible for high mortality rates in ruminant herds. The resistance of nematodes to synthetic anthelmintics is widespread and requires a continuous search for new bioactive molecules, such as proteins. The objective of this study was to evaluate the anthelmintic potential of a protease purified from the latex of Ficus benjamina against H. contortus . Fresh latex was collected from plants via small incisions in the green stems, the rubber was removed by centrifugation, and the latex protein extract (LPE) was obtained. After LPE fractionation with ammonium sulfate and chromatography of the fraction containing the highest proteolytic activity on CM-cellulose, a cysteine protease (FbP) was purified. FbP has a molecular mass of approximately 23.97 kDa, and its proteolytic activity was stable between pH 6.0 and pH 10 and over a broad temperature range, with optimum activity at 60 °C. FbP inhibited both the development and exsheathment of H. contortus larvae, with 50% effective concentrations of 0.26 and 0.79 mg/mL, respectively. We conclude that this cysteine protease from F. benjamina latex with anthelmintic activity against H. contortus could be a promising alternative for the development of products for use in parasite control programmes.

Resumo Haemonchus contortus é um nematoide gastrintestinal, responsável por altas taxas de mortalidade em rebanhos de pequenos ruminantes. A resistência dos nematoides aos anti-helmínticos sintéticos está generalizada e requer uma busca contínua por novos compostos bioativos, como as proteínas. O objetivo deste trabalho foi avaliar o potencial anti-helmíntico da protease purificada do látex de Ficus benjamina contra H. contortus . O látex fresco foi coletado das plantas por pequenas incisões nas hastes verdes e o extrato proteico de látex (EPL) foi obtido. Após o fracionamento do EPL com sulfato de amônio e cromatografia da fração contendo a maior atividade proteolítica da CM-Celulose, uma protease cisteínica (FbP) foi purificada. A FbP tem massa molecular de cerca de 23,97 kDa, a atividade proteolítica foi estável entre pH 6,0 e pH 10 e ao longo de uma ampla faixa de temperatura, com atividade ótima a 60 °C. A FbP inibiu tanto o desenvolvimento quanto o desembainhamento das larvas de H. contortus, com 50% de inibição nas concentrações de 0,26 e 0,79 mg/mL, respectivamente. Concluímos que esta protease cisteínica do látex de F. benjamina, com ação anti-helmíntica contra H. contortus, pode ser uma alternativa promissora para o desenvolvimento de produtos a serem utilizados em programas de controle de parasitos.

Potential of laticifer fluids for inhibiting Aedes aegypti larval development: evidence for the involvement of proteolytic activity

It has been shown previously that the laticifer fluid of Calotropis procera (Ait.) R.Br. is highly toxic to the egg hatching and larval development of Aedes aegypti L. In the present study, the larvicidal potential of other laticifer fluids obtained from Cryptostegia grandiflora R.Br., Plumeria rubra L. and Euphorbia tirucalli L. was evaluated. We attempted to correlate larvicidal activity with the presence of endogenous proteolytic activity in the protein fraction of the fluids. After collection, the fluids were processed by centrifugation and dialysis to obtain the soluble laticifer protein (LP) fractions and eliminate water insoluble and low molecular mass molecules. LP did not visibly affect egg hatching at the doses assayed. LP from Cr. grandiflora exhibited the highest larval toxicity, while P. rubra was almost inactive. E. tirucalli was slightly active, but its activity could not be correlated to proteins since no protein was detected in the fluid. The larvicidal effects of LP from C. procera and Cr. grandiflora showed a significant relationship with the proteolytic activity of cysteine proteinases, which are present in both materials. A purified cysteine proteinase (papain) from the latex of Carica papaya (obtained from Sigma) was similarly effective, whereas trypsin and chymotrypsin (both serine proteinases) were ineffective. The results provide evidence for the involvement of cysteine proteinase activity in the larvicidal action of some laticifer fluids. C. procera is an invasive species found in areas infested with Ae. aegypti and thus could prove useful for combating mosquito proliferation. This is the first report to present evidence for the use of proteolytic enzymes as chemical agents to destroy Ae. aegypti larvae.

Cysteine proteinases of Leishmania

Cysteine proteinases have been found in a variety of organisms, and have been implicated in many processes related to pathogenesis in parasites. In this review we discuss cysteine proteinases of Leishmania, that may be involved in mechanisms of survival and growth of these parasites in the microbicidal environment of the macrophage.

Heterogeneity of cysteine proteinases in Leishmania braziliensis and Leishmania major

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with acrylamide copolymerized with gelatin (substrate-SDS-PAGE) was used to compare promastigote proteinases from two Leishmania species (L. braziliensis and L. major-like). Substrate-SDS-PAGE resolved at least 6 distinct proteinase activities with relative molecular masses between 20 and 65 kDa. The profile of proteinase activity was the same for both species, although qualitative differences were observed. L. major-like expressed a low molecular weight (20 kDa) proteinase with maximum activity at pH 7.0, which was demonstrable from pH 5.0 to pH 8.0. The 20-kDa protease was recovered in the detergent phase of TX-114 and was inhibited by the sulfhydryl group-blocking reagent E-64 (2.5 mM) and the non-specific inhibitor iodoacetic acid (1 mM). Pepstatin (1 microM) and PMSF (2.5 mM) did not inhibit the 20-kDa enzyme. The present study suggests that both Leishmania species studied express hydrophobic cysteine proteases of different molecular weights, since about 2 x 10(7) parasites were analyzed in each lane and the proteolytic activity developed at 37 degrees C for 16 h

Purification of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi by affinity chromatography

The major cysteine proteinase from Trypanosoma cruzi, cruzipain, can be obtained essentially homogeneous, starting from crude epimastigote extracts, in one step, by affinity chromatography on Cystatin-Sepharose (specific for cysteine proteinases) or ConA-Sepharose (specific for high mannose N-linked glycoproteins). The methods offer considerable potential for enzyme purification from scarce sources, such as other parasite stages or radioactively labelled material with high specific radioactivity