Three serine proteases from the latex of Euphorbia cyparissias

Three serine-centred proteolytic enzymes, euphorbains y-1, ?2 and ?3, were isolated from the latex of Euphorbia cyparissias. These proteases have different specific activities to azocoll and CBZ glycine p-nitrophenyl ester. The pIs and M_rs of y-1, ?2 and ?3 are 5.2 and 67 000, 5.2 and 33 000, and 6.3 and 67 000, respectively. The enzymes, which are glycoproteins, are immunologically distinct from euphorbain 1, but clearly related to that enzyme in amino acid composition.     

Purification and biochemical characterization of a highly active cysteine protease ervatamin A from the latex of Ervatamia coronaria

Ervatamia coronaria, a flowering plant (family Apocynaceae) indigenous to India, has medicinally important applications. A search for biochemical constituents of the latex of the plant yielded at least three thiol proteases with distinctly different properties. One of them, a highly active protease (ervatamin A), was purified to homogeneity by ion exchange and gel filtration chromatography. The enzyme exhibited high proteolytic activity toward natural substrates and amidolytic activity toward synthetic substrates. The pH and temperature optima for proteolytic activity were 8–8.5 and 50–55°C, respectively. Proteolytic activity of the enzyme was strongly inhibited by thiol-specific inhibitors. The estimated molecular mass of the enzyme was 27.6 kDa. The extinction coefficient (1% 280) of the enzyme was estimated as 21.9, and the protein molecule consists of 8 tryptophan, 11 tyrosine and 7 cysteine residues. Isoelectric point of the purified enzyme was 8.37. Polyclonal antibodies raised against the pure enzyme gave a single precipitin line in Ouchterlony's double immunodiffusion and a typical color in ELISA. The N-terminal sequence of the enzyme showed conserved amino acid residues to other plant cysteine proteases. Ervatamin A shows high activity in relation to the other thiol proteases isolated from the same source.     

Two new cysteine endopeptidases obtained from the latex of Araujia hortorum fruits

Two new endopeptidases were purified to homogeneity from the latex of Araujia hortorum fruits by a simple purification procedure involving ultracentrifugation and ion exchange chromatography. Molecular weights of araujiain h II and araujiain h III were 23,718 and 23546 (mass spectrometry), respectively. The isoelectric point of araujiain h II was 8.9, whereas araujiain h III had a pI higher than 9.3. Maximum proteolytic activity on caseine was reached at pH 8.0-9.0 for both endopeptidases, which were irreversibly inhibited by iodoacetate and E-64, suggesting they belong to the cysteine protease family. Esterolytic activity was determined on N--CBZ-amino acid-p-nitrophenyl esters, and the highest k _cat/K _m values for the both enzymes were obtained with the glutamine derivative. The N-terminal sequences of araujiain h II and araujiain h III showed a high degree of homology with other plant cysteine endopeptidases.     

Induction of cysteine and serine proteases during xylogenesis in Zinnia elegans

The terminal process of xylogenesis, autolysis, is essential for the formation of a tubular system for conduction of water and solutes throughout the whole plant. Several hydrolase types are implicated in autolysis responsible for the breakdown of cytoplasm. Here, we characterize p48h-17 cDNA from in vitro tracheary elements (TEs) of Zinnia elegans which encodes a preproprotein similar to papain. The putative mature protein, a cysteine protease, has a molecular mass of 22,699 Da with a pI of 5.7. DNA gel blot analysis indicated that p48h-17 is likely encoded by one or two genes. The p48h-17 mRNA accumulated markedly in in vitro differentiating TEs, whereas it appeared not to be induced in response to senescence and wounding in the leaves or H₂O₂ challenge in the cultured mesophyll cells. In stems, the expression of the p48h-17 gene was preferentially associated with differentiating xylem. Activity gel assays demonstrated that a cysteine and a serine protease, which had apparent molecular masses of 20 kDa and 60 kDa, respectively, were markedly induced during in vitro TE differentiation. The cysteine protease activity was also preferentially present in the xylem of Zinnia stems. Transient expression of the p48h-17 cDNA in tobacco protoplasts resulted in the production of a 20 kDa cysteine protease. Taken together, the results indicate that the p48h-17 gene appears to be preferentially associated with xylogenesis, and both the cysteine and serine proteases might be involved in autolysis during xylogenesis.     

Silicon-augmented resistance of plants to herbivorous insects: a review

Silicon (Si) is one of the most abundant elements in the earth's crust, although its essentiality in plant growth is not clearly established. However, the importance of Si as an element that is particularly beneficial for plants under a range of abiotic and biotic stresses is now beyond doubt. This paper reviews progress in exploring the benefits at two‐ and three‐trophic levels and the underlying mechanism of Si in enhancing the resistance of host plants to herbivorous insects. Numerous studies have shown an enhanced resistance of plants to insect herbivores including folivores, borers, and phloem and xylem feeders. Silicon may act directly on insect herbivores leading to a reduction in insect performance and plant damage. Various indirect effects may also be caused, for example, by delaying herbivore establishment and thus an increased chance of exposure to natural enemies, adverse weather events or control measures that target exposed insects. A further indirect effect of Si may be to increase tolerance of plants to abiotic stresses, notably water stress, which can in turn lead to a reduction in insect numbers and plant damage. There are two mechanisms by which Si is likely to increase resistance to herbivore feeding. Increased physical resistance (constitutive), based on solid amorphous silica, has long been considered the major mechanism of Si‐mediated defences of plants, although there is recent evidence for induced physical defence. Physical resistance involves reduced digestibility and/or increased hardness and abrasiveness of plant tissues because of silica deposition, mainly as opaline phytoliths, in various tissues, including epidermal silica cells. Further, there is now evidence that soluble Si is involved in induced chemical defences to insect herbivore attack through the enhanced production of defensive enzymes or possibly the enhanced release of plant volatiles. However, only two studies have tested for the effect of Si on an insect herbivore and third trophic level effects on the herbivore's predators and parasitoids. One study showed no effect of Si on natural enemies, but the methods used were not favourable for the detection of semiochemical‐mediated effects. Work recently commenced in Australia is methodologically and conceptually more advanced and an effect of Si on the plants' ability to generate an induced response by acting at the third trophic level was observed. This paper provides the first overview of Si in insect herbivore resistance studies, and highlights novel, recent hypotheses and findings in this area of research. Finally, we make suggestions for future research efforts in the use of Si to enhance plant resistance to insect herbivores.     

Evolutionary dynamics of specialisation in herbivorous stick insects

Understanding the evolutionary dynamics underlying herbivorous insect mega‐diversity requires investigating the ability of insects to shift and adapt to different host plants. Feeding experiments with nine related stick insect species revealed that insects retain the ability to use ancestral host plants after shifting to novel hosts, with host plant shifts generating fundamental feeding niche expansions. These expansions were, however, not accompanied by expansions of the realised feeding niches, as species on novel hosts are generally ecologically specialised. For shifts from angiosperm to chemically challenging conifer hosts, generalist fundamental feeding niches even evolved jointly with strong host plant specialisation, indicating that host plant specialisation is not driven by constraints imposed by plant chemistry. By coupling analyses of plant chemical compounds, fundamental and ecological feeding niches in multiple insect species, we provide novel insights into the evolutionary dynamics of host range expansion and contraction in herbivorous insects.     

A unique latex protein, MLX56, defends mulberry trees from insects

The mulberry (Morus spp.)-silkworm (Bombyx mori) relationship has been a well-known plant-herbivore interaction for thousands of years. Recently, we found that mulberry leaves defend against insect herbivory by latex ingredients. Here we report that a 56-kDa (394 amino acid) defense protein in mulberry latex designated mulatexin (MLX56) with an extensin domain, two hevein-like chitin-binding domains, and an inactive chitinase-like domain provides mulberry trees with strong insect resistance. MLX56 is toxic to lepidopteran caterpillars, including the cabbage armyworm, Mamestra brassicae and the Eri silkworm, Samia ricini, at 0.01% concentration in a wet diet, suggesting that MLX56 is applicable for plant protection. MLX56 is highly resistant to protease digestion, and has a strong chitin-binding activity. Interestingly, MLX56 showed no toxicity to B. mori, suggesting that the mulberry specialist has developed adaptation to the mulberry defense. Our results show that defensive proteins in plant latex play key roles in mulberry-insect interactions, and probably also in other plant-insect interactions. Our results further suggest that plant latexes analogous to animal venom contain a treasury of applicable defense proteins and chemicals that has evolved through inter-specific interactions.     

Midgut cysteine protease-inhibiting activity in Trichoplusia ni protects the peritrophic membrane from degradation by plant cysteine proteases

The action of plant cysteine proteases on the midgut peritrophic membrane (PM) of a polyphagous herbivorous lepidopteran, Trichoplusia ni, was studied. Proteins in PMs isolated from T. ni larvae were confirmed to be highly resistant to the serine proteinases trypsin and chymotrypsin, but were susceptible to degradation by plant cysteine proteases, which is consistent with the known molecular and biochemical characteristics of the T. ni PM proteins. However, the PM proteins were not degraded by plant cysteine proteases in larvae or in the presence of larval midgut fluid in vitro. With further biochemical analysis, cysteine protease-inhibiting activity was identified in the midgut fluid of T. ni larvae. The cysteine protease-inhibiting activity was heat resistant and active in the tested pH range from 6.0 to 10.0, but could be suppressed by thiol reducing reagents or reduced by treatment with catalase. In addition to T. ni, cysteine protease-inhibiting activity was also identified from two other polyphagous Lepidoptera species, Helicoverpa zea and Heliothis virescens. In conclusion, results from this study uncovered that herbivorous insects may counteract the attack of plant cysteine proteases on the PM by inhibiting the potentially insecticidal cysteine proteases from plants in the digestive tract. However, the biochemical identity of the cysteine protease-inhibiting activity in midgut fluid has yet to be identified.     

Differential expression of cysteine proteases in developmental stages of the parasitic ciliate Ichthyophthirius multifiliis

An expressed sequence tag database of the freshwater fish parasite, Ichthyophthirius multifiliis (Ciliophora) was analyzed to seek for proteases potentially involved in the invasion and degradation of host tissues during infection. The translation of the database revealed two cathepsin L cysteine proteases (Icp1 and Icp2) of the C1A peptidase subfamily. The analysis of Icp1 and Icp2 sequences suggested that both proteases would be synthesized as preproproteins, with a mature domain of 27.9 and 22.8 kDa, respectively. Their expression level was determined in the trophont parasitic stage, in the tomont reproductive stage, and in the theront infective stage by real-time RT-PCR. ICP1 and ICP2 were significantly upregulated in trophont and theront stages in comparison with the tomont stage. Mature peptides of Icp1 and Icp2 were identified in crude extracts of I. multifiliis trophonts by LC-MS/MS. Zymograms showed three to seven activity bands at the optimum pH of cathepsin L cysteine proteases. Two bands displaying cysteine protease activity were identified by inhibition with E-64. They represented the major proteolytic activity of the trophont stage at pH 5-7, suggesting that cysteine proteases play an important role in the infection process.     

Excretory bladder: the source of cysteine proteases in Paragonimus westermani metacercariae

The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine proteases were distributed at the linings of excretory bladder and excretory concretions of the metacercariae. It was suggested that the excretory epithelium of P. westermani undertake the secretory function of metacercarial cysteine proteases, in addition to its role as a route for eliminating waste products.     

植食性昆虫唾液效应子和激发子的研究进展

植食性昆虫与寄主植物通过协同进化形成了复杂的防御和反防御机制.本文系统综述了昆虫唾液效应子和激发子在植物与昆虫互作中的作用及机理.昆虫取食中释放的唾液激发子被植物识别而激活植物早期免疫反应,昆虫也能从口腔分泌效应子到植物体内抑制免疫;抗性植物则利用抗性(R)蛋白识别昆虫无毒效应子,启动效应子诱导的免疫反应,而昆虫又进化...     

Inhibition of cysteine proteases by a natural biflavone: behavioral evaluation of fukugetin as papain and cruzain inhibitor

Cruzain is the major cysteine protease of Trypanosoma cruzi, the infectious agent responsible for Chagas disease, and cruzain inhibitors display considerable antitrypanosomal activity. In the present work we elucidated crystallographic data of fukugetin, a biflavone isolated from Garcinia brasiliensis, and investigated the role of this molecule as cysteine protease inhibitor. The kinetic analyses demonstrated that fukugetin inhibited cruzain and papain by a slow reversible type inhibition with K_I of 1.1 and 13.4 µM, respectively. However, cruzain inhibition was about 12 times faster than papain inhibition. Lineweaver–Burk plots demonstrated partial competitive inhibition for cruzain and hyperbolic mixed-type inhibition for papain. Furthermore, the docking results showed that the biflavone binds to ring C′ in the S2 pocket and to ring C in the S3 pocket through hydrophobic interactions and hydrogen bonds. Finally, fukugetin also presented inhibitory activity on proteases of the T. cruzi extract, with IC₅₀ of 7 µM.     

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.     

Recognition of host-specific chemical stimulants in two sympatric host races of the pea aphid Acyrthosiphon pisum

Abstract.  1. In ecological speciation , adaptation to variation in the external environment provides the crucial push that starts the process of genetic divergence and eventually leads to speciation. This emphasis on the role of ecological specialisation in speciation events has brought with it a renewed interest in its proximate mechanisms in recently diverged groups such as host races. Here, the proximate mechanisms of feeding specialisation are investigated in two host races of the pea aphid Acyrthosiphon pisum .
2. Using alfalfa and clover extracts, enclosed in diet chambers or applied on whole plants, it is shown that feeding specialisation depends on recognition of stimulants specific to the host plant, not on deterrents or toxins specific to the non-host plants.
3. Because pea aphids mate on their host plant, feeding specialisation leads to de facto assortative mating. This study suggests that behavioural recognition of host-specific chemicals, rather than avoidance of deterrents or/and plant toxins, contributes to gene flow restriction between the alfalfa and clover host races.     

A Kunitz trypsin inhibitor gene family from trembling aspen (Populus tremuloides Michx.): cloning,functional expression,and induction by wounding and herbivory

Three Kunitz trypsin inhibitor genes were isolated from trembling aspen (Populus tremuloides) by PCR and cDNA screening. Based on sequence similarity, they were grouped into two classes. Southern blots showed complex banding patterns and a high level of restriction fragment polymorphism between different aspen genotypes, suggesting that these trypsin inhibitors are members of a large, rapidly evolving gene family. One of the trypsin inhibitor genes, PtTI2. was over-expressed in Escherichia coli and its product shown to inhibit bovine trypsin in vitro. Both classes of PtTI genes are induced by wounding and herbivory, permitting rapid adaptive responses to herbivore pressure. The response appears to be mediated by an octadecanoid-based signaling pathway, as methyl jasmonate treatments induced the trypsin inhibitors. Wound-induced accumulation of trypsin inhibitor protein was also observed by western blot analysis. The pattern of expression, the apparent rapid evolution of TI genes, and the in vitro trypsin inhibitory activity are consistent with a role in herbivore defense. This work establishes the presence of a functional protein-based inducible defense system in trembling aspen.     

Evolution of latex and its constituent defensive chemistry in milkweeds (Asclepias): a phylogenetic test of plant defense escalation

A tremendous diversity of plants exude sticky and toxic latex upon tissue damage, and its production has been widely studied as a defensive adaptation against insect herbivores. Here, we address variation in latex production and its constituent chemical properties (cardenolides and cysteine proteases) in 53 milkweeds [Asclepias spp. (Apocynaceae)], employing a phylogenetic approach to test macroevolutionary hypotheses of defense evolution. Species were highly variable for all three traits, and they showed little evidence for strong phylogenetic conservatism. Latex production and the constituent chemical defenses are thus evolutionarily labile and may evolve rapidly. Nonetheless, in phylogenetically independent analyses, we show that the three traits show some correlations (and thus share a correlated evolutionary history), including a positive correlation between latex exudation and cysteine protease activity. Conversely, latex exudation and cysteine protease activity both showed a trade‐off with cardenolide concentrations in latex. We also tested whether these traits have increased in their phenotypic values as the milkweeds diversified, as predicted by plant defense escalation theory. Alternative methods of testing this prediction gave conflicting results – there was an overall negative correlation between amount of evolutionary change and amount of latex exudation; however, ancestral state reconstructions indicated that most speciation events were associated with increases in latex. We conclude by (i) summarizing the evidence of milkweed latex itself as a multivariate defense including the amount exuded and toxin concentrations within, (ii) assessing the coordinated evolution of latex traits and how this fits with our previous notion of ‘plant defense syndromes’, and finally, (iii) proposing a novel hypothesis that includes an ‘evolving community of herbivores’ that may promote the escalation or decline of particular defensive strategies as plant lineages diversify.     

Protein aggregation in Escherichia coli: role of proteases

Protein aggregation is involved in several human diseases, and presumed to be an important process in protein quality control. In bacteria, aggregation of proteins occurs during stress conditions, such as heat shock. We studied the protein aggregates of Escherichia coli during heat shock. Our results demonstrate that the concentration and diversity of proteins in the aggregates depend on the availability of proteases. Aggregates obtained from mutants in the Lon (La) protease contain three times more protein than wild-type aggregates and show the broadest protein diversity. The results support the assumption that protein aggregates are formed from partially unfolded proteins that were not refolded by chaperones or degraded by proteases.     

Novel proteases from the genome of the carnivorous plant Drosera capensis: Structural prediction and comparative analysis

In his 1875 monograph on insectivorous plants, Darwin described the feeding reactions of Drosera flypaper traps and predicted that their secretions contained a “ferment” similar to mammalian pepsin, an aspartic protease. Here we report a high‐quality draft genome sequence for the cape sundew, Drosera capensis, the first genome of a carnivorous plant from order Caryophyllales, which also includes the Venus flytrap (Dionaea) and the tropical pitcher plants (Nepenthes). This species was selected in part for its hardiness and ease of cultivation, making it an excellent model organism for further investigations of plant carnivory. Analysis of predicted protein sequences yields genes encoding proteases homologous to those found in other plants, some of which display sequence and structural features that suggest novel functionalities. Because the sequence similarity to proteins of known structure is in most cases too low for traditional homology modeling, 3D structures of representative proteases are predicted using comparative modeling with all‐atom refinement. Although the overall folds and active residues for these proteins are conserved, we find structural and sequence differences consistent with a diversity of substrate recognition patterns. Finally, we predict differences in substrate specificities using in silico experiments, providing targets for structure/function studies of novel enzymes with biological and technological significance. Proteins 2016; 84:1517–1533. © 2016 Wiley Periodicals, Inc.     

Isolation and characterization of a cysteine protease from the latex of Araujia hortorum fruits

A new protease (araujiain h l) was purified to mass spectroscopy homogeneity from the latex of Araujia hortorum Fourn. (Asclepiadaceae) fruits by ultracentrifugation and ion exchange chromatography. The enzyme has a molecular mass of 24,031 (mass spectrometry) and an isoelectric point higher than 9.3. The optimum pH range for casein hydrolysis was 8.0–9.5. The enzyme showed remarkable caseinolytic activity at high temperatures, although its thermal stability decayed rapidly. The proteinase was activated by thiol compounds and inhibited by common thiol-blocking reagents, particularly E-64 and HgCl₂, suggesting the enzyme belongs to the cysteine protease family. The concentration of active sites as determined by titration with E-64 was 3.3 M. When assayed on N--CBZ-amino acid-p-nitrophenyl esters, the enzyme showed higher preference for the glutamine derivative, followed by those of alanine, asparagine, glycine, and leucine, in decreasing order. Partial homology (36–48%) with other plant cysteine proteinases was observed in an internal fragment obtained by Protease V8 treatment.