The cathepsin L-like proteinases from the midgut of Tenebrio molitor larvae: Sequence, properties, immunocytochemical localization and function

Cristofoletti, Plı́nio T.; Ribeiro, Alberto F.; Terra, Walter R.

doi:10.1016/j.ibmb.2005.03.006

Cited by 89 publications

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References 49 publications

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“…At the present stage, a possible explanation for the broad-spectrum inhibition of coleopteran cysteine cathepsins by plant cystatins might be the predominance of cathepsin L-like enzymes in midgut extracts, including some forms able to cleave both the Z-RR-MCA and Z-FR-MCA substrates (see Bown et al 2004 andCristofoletti et al, 2005). An alternative explanation would be the existence of two cathepsin B populations in the midgut exhibiting differential sensitivity to cystatin inhibition.…”

Section: Discussionmentioning

confidence: 93%

Section: Cystatin-sensitive Cathepsins In the Banana Weevil Midgutmentioning

confidence: 99%

“…3B). Overall, the well-documented preference of coleopteran cathepsin L-like enzymes for Z-FR-MCA despite the Z-RR-MCA-hydrolyzing activity of some isoforms (Bown et al, 2004;Cristofoletti et al, 2005), the specificity of cathepsin B-like enzymes for Z-RR-MCA (Mort, 1998), and the differential inhibitory effects of Z-FY-DMK and human stefin A against banana weevil Z-FR-and Z-RR-hydrolyzing proteinases (Student t-test; P<0.01) (Fig. 3B) suggest the occurrence of cathepsin L-like and cathepsin B-like proteinase populations in the banana weevil midgut, both sensitive to E-64 and cystatins but exhibiting distinct preferences for the Z-FR-MCA and Z-RR-MCA substrates.…”

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confidence: 94%

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Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

Kiggundu

Muchwezi

Vyver

et al. 2009

Arch Insect Biochem Physiol

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The general potential of plant cystatins for the development of insect-resistant transgenic plants still remains to be established given the natural ability of several insects to compensate for the loss of active cysteine proteases following inhibitor ingestion. Here we assessed the potential of cystatins for the development of banana lines resistant to the banana weevil Cosmopolites sordidus, a major pest of banana and plantain in Africa. Protease inhibitory assays were first conducted with protein and methylcoumarin (MCA) peptide substrates to measure the inhibitory efficiency of different cystatins in vitro, followed by a diet bioassay with cystatin-infiltrated banana stem disks to monitor the impact of two plant cystatins, oryzacystatin I (OC-I, or OsCYS1)and papaya cystatin (CpCYS1), on the overall growth rate of young weevil larvae. As observed earlier for other Coleoptera, banana weevils produce a variety of proteases for dietary protein digestion, including in particular Z-Phe-Arg-MCA-hydrolyzing (cathepsin L-like) and Z-Arg-Arg- MCA-hydrolyzing (cathepsin B-like) proteases active in mildly acidic conditions. Both enzyme populations were sensitive to the diagnostic cysteine protease inhibitor E-64 and to different plant cystatins including OsCYS1. In line with these broad inhibitory effects of cystatins, OsCYS1 andCpCYS1 caused an important growth delay in young larvae developing for 10 days in cystatininfiltrated banana stem disks. These promising results, which illustrate the natural susceptibility of C. sordidus to plant cystatins, are discussed in the light of current genomic data on coleopteran cysteine cathepsins and recent hypotheses suggesting a key role for digestive cathepsin B-like enzymes as a determinant for resistance or susceptibility to plant cystatins in Coleoptera.Keywords: Banana weevil (Cosmopolites sordidus); banana (Musa spp.); cathepsin B-like proteases; cysteine cathepsins; plant cystatins; oryzacystatin I Plant cystatins against the banana weevil Page 3 INTRODUCTIONThe banana weevil Cosmopolites sordidus (Germar) (Coleoptera: Curculionidae) is an insect pest of considerable importance in Africa, associated with the rapid decline of banana and plantain plantations (Gold, 1999a(Gold, , 2000Swennen and Vuylsteke, 2001;Gold et al., 2005). At the adult stage, banana weevil females deposit their eggs inside host plant tissues, at the base of the pseudostem or on an exposed corm. On hatching, the larvae tunnel through the corm to feed and develop, where they damage tissues, compromise water and mineral uptake, and weaken the colonized organs. Banana weevil infestations cause important losses in the field by a direct negative impact on harvestable bunch weight and a weakening effect on infested organs causing plant toppling during windstorms (Sengooba, 1986;Rukazambuga et al., 1998).Banana weevil control in Africa relies essentially on cultural and sanitation practices such as the use of clean planting material, the systematic trapping of adult weevils to prevent population build-up, an...

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Section: Discussionmentioning

confidence: 93%

Section: Cystatin-sensitive Cathepsins In the Banana Weevil Midgutmentioning

confidence: 99%

mentioning

confidence: 94%

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Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

Kiggundu

Muchwezi

Vyver

et al. 2009

Arch Insect Biochem Physiol

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“…These types of peptidases develop their maximum activity under alkaline to neutral pH conditions [2]. In some insect groups the intestinal lumen is acidic and the digestion switched to cysteine-like proteinases such as cathepsin D or L [3,4]. Some Coleoptera groups (e.g.…”

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Pathways of Insect Protein Digestion: Triatominae (Kissing Bugs)

Waniek¹

2014

Entomol Ornithol Herpetol

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EditorialThe majority of insects use serine proteinases like trypsin and chymotrypsin as main enzymes to digest their food [1]. These types of peptidases develop their maximum activity under alkaline to neutral pH conditions [2]. In some insect groups the intestinal lumen is acidic and the digestion switched to cysteine-like proteinases such as cathepsin D or L [3,4]. Some Coleoptera groups (e.g. Bruchidae, Tenebrionidae) developed a digestive system based on such proteinases [2,5]. But the major group using acid proteinases are the Hemiptera. This taxon uses cysteine proteinases because ancestral groups were using plant seeds containing serine proteinases inhibitors as a food source and therefore had to evolve their digestive system [6,7]. The conquest of this food source was a challenge for the digestive system [8] but it certainly contributed to the success of this insect group opening up new niches.One of the best analyzed hemipteran groups considering their digestion is the Triatominae (Heteroptera, Reduviidae) subfamily. Many species of this group -especially from the genera Triatoma, Rhodnius and Panstrongylus [9] -are medically important because they are able to transmit Trypanosoma cruzi (Kinetoplastida, Trypanosomatidae), the causative agent of Chagas disease [10]. In contrast to mosquitoes which develop from egg to adult in few weeks and frequently show low rates of parasitic infection, some long-lived triatomine species needs several months to complete their life cycle [1]. Besides this, kissing bugs takes huge amounts of blood from their vertebrate host at each nymph stage and, consequently usually show higher infection rates (up to about 80%) [1,[11][12][13][14][15]. In this insect group several digestive enzymes and their respective genes were identified and characterized. In the beginning of the 1980s activities of cathepsin B and D, aminopeptidase and carboxypeptidase have been shown [16,17]. Later, when the appropriate molecular biology methods were available, different isoforms of genes from different triatomine species have been identified encoding cathepsin B, L and D and serine carboxypeptidase [18][19][20][21][22]. At the same time Borges et al. [23] have demonstrated a significant increase of cathepsin D activity in Rhodnius prolixus infected with T. cruzi. However, despite the numerous studies concerning the Triatominae, many questions about their digestive system and its interactions with the parasite are still unanswered. For example, it is quite possible that there are further types of luminal digestive enzymes waiting to be discovered in triatomines. A recent transcriptome analysis of the R. prolixus intestine has revealed many genes related to digestive physiology including digestive enzymes lacking some amino acid residues important for enzymatic activity [24]. Furthermore, it would be very important to know whether or not microbiota plays a role in the digestion of triatomines and how strong the influence of T. cruzi on the digestive system might be. It would also be interesting ...

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“…Porém, há outra classe importante de proteases produzidas por essa lepidóptera, as cisteíno proteases. Algumas isoenzimas de cisteino proteases foram reconhecidas e seus genes sequenciados em Tenebrio molitor (Linnaeus, 1758) (Cristofoletti et al, 2005).…”

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Caracterização enzimática de isoformas de cisteíno protease de Anticarsia gemmatalis (Hübner, 1818)

et al. 2011

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RESUMOIsoformas de cisteíno protease obtidas do intestino médio de lagartas de 5º instar de Anticarsia gemmatalis (Hübner, 1818) foram caracterizadas. A isoforma solúvel foi chamada de Fração Solúvel enquanto a isoforma ligada à membrana celular, de Fração Insolúvel. As maiores atividades foram observadas em pH 3,6 a 45° C para a Fração Solúvel e pH 4,6 a 50° C para a Fração Insolúvel. Ao analisar o efeito de modificadores químicos, a Fração Solúvel mostrou-se insensível à aprotinina e E-64, porém teve sua atividade aumentada pela adição de EDTA e levemente inibida pela adição de íons Ca 2+ , mostrando se tratar de enzimas independentes de íons metálicos para sua atividade. A Fração Insolúvel também se mostrou insensível à aprotinina, porém teve sua atividade parcialmente inibida por E-64. A adição de EDTA levou a uma redução nos valores de atividade, demonstrando a necessidade de íons metálicos para a atividade dessas enzimas, porém não se trata de enzimas cálcio-dependentes, uma vez que sua atividade foi reduzida com a adição desse íon. Os valores de K M app e V máx app foram, respectivamente, 0,6398 mM e 42,556 nM s -1 para Fração Solúvel e 0,0413 mM e 10,854 nM s -1 para Fração Insolúvel. Esses resultados fornecem evidências da presença de cisteíno protease solúvel e ligada à membrana celular do intestino de lagartas de A. gemmatalis. O conhecimento e a caracterização das principais classes de proteases presentes no trato digestivo da lagarta da soja, bem como a interação dessas enzimas com inibidores de protease têm uma importante consequência aos programas de melhoramento de soja.Termos para indexação: Proteases digestivas, Lepidoptera, controle de pragas, inibição de proteases. ABSTRACTCysteine protease isoforms obtained from the midgut of 5 th instar Anticarsia gemmatalis (Hübner, 1818) larvae were characterized. The soluble enzyme isoform was called Soluble Fraction and cell membrane-bound enzyme isoform, the Insoluble Fraction. The highest activities were observed at pH 3.6 and 45° C for the Soluble Fraction and pH 4.6 and 50° C for the Insoluble Fraction. Analyzing the effect of chemical modifiers, the Soluble Fraction was shown to be insensitive to aprotinin and E-64, although its activity was increased by the addition of EDTA and slightly inhibited by the addition of Ca 2+ , showing to be enzymes independent of metal ions to its activity. The Insoluble Fraction was also insensitive to aprotinin, but its activity was partially inhibited by E-64. The addition of EDTA reduced the activity values, demonstrating the need for metal ions for the activity of these enzymes, but they are not calcium-dependent enzymes, since their activity was reduced with the addition of this ion. The values of K M app and V max app were, respectively, 0.6398 mM and 42.556 nM s -1 for Soluble Fraction and 0.0413 mM and 10.854 nM s -1 for Insoluble Fraction. These results provide evidence for the presence of soluble and cell membrane-bound cysteine proteases in the gut of A. gemmatalis larvae. The knowledge and characterizatio...

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The cathepsin L-like proteinases from the midgut of Tenebrio molitor larvae: Sequence, properties, immunocytochemical localization and function

Cited by 89 publications

References 49 publications

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

Pathways of Insect Protein Digestion: Triatominae (Kissing Bugs)

Caracterização enzimática de isoformas de cisteíno protease de Anticarsia gemmatalis (Hübner, 1818)

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