Digestive enzymes present in crustacean feces as a tool for biochemical, physiological, and ecological studies

The effect of food composition on the digestive system of Penaeus vannamei shrimp was used to determine the suitability of feces for analysis of class, type, composition of digestive proteinases, and whether alterations in the digestive gland are mirrored in feces composition. Enzymes recovered from feces and the midgut gland of white shrimp P. vannamei were used for comparison purposes. Three groups of shrimp were assembled: two groups fed two different brands of commercial feeds (PI and SC) with different content of protein, and the last group fed 50% PI feed and 50% thawed giant squid. Composition of proteinases in the midgut gland and feces were identical, and trypsin and chymotrypsin paralogues were identified in both samples by substrate-electrophoresis. Total proteolytic, trypsin, and chymotrypsin enzyme activities were higher in both samples from organisms fed SC, than in the other two groups. In the hepatopancreas, trypsin activity was ∼30% higher in SC fed group. Final average weights of shrimp were close in three groups, but hepatopancreas weight was 20% higher in the SC group. The degree of protein hydrolysis (DH) in vitro for the SC and PI was evaluated by the pH-stat method, using enzymes from feces and hepatopancreas of each group. The DH of food was no different, but it was affected by enzyme source, hepatopancreas extract (HPE) or feces extract (FE). DH was always higher when FE was the enzyme source than when HPE was the source. The proposed methods for recovery of enzymes from shrimp feces can be applied to other crustaceans. Measurements were sufficiently sensitive to allow quantifying the effects of feed on digestion physiology and other ecological and physiological applications, without the necessity of killing specimens.     

Influence of molting and starvation on the synthesis of proteolytic enzymes in the midgut gland of the white shrimp Penaeus vannamei

We investigated the effect of starvation as a stimulant of the digestive system on digestive proteinase activities in the white shrimp Penaeus vannamei. The starved organisms were sampled periodically according to the molting stage and compared with a continuously fed group. Molting stage was included as an independent variable. Most analyzed variables, except for trypsin, were more affected by starvation than by molting, indicating that starvation is a stimulant that masks the effect of molting and showing that food or alimentary stress is more conspicuous than physiological ones. We found that starvation is a stimulant that surpasses the effect of molting, and because it affects the activity of digestive proteinases, studies of starving organisms in combination with tools of molecular biology, can be a helpful working model in the understanding of mechanisms of regulation of digestive enzyme activity. In the starved organisms, trypsin and chymotrypsin activities were similar, suggesting dependence of one to the other. Changes in proteolytic activities and the number of protein bands in electrophoresis showed evidence of synthesis regulation in the midgut gland of white shrimp.     

Activity cycles and the control of four digestive proteinases in the posterior midgut of Rhodnius prolixus Stål (Hemiptera: Reduviidae)

Proteinase levels in the posterior midgut of fifth-instar and adult Rhodnius prolixus follow a cyclic pattern after ingestion of the bloodmeal. In the fifth instar, cathepsin B showed two peaks: the first occurred 6 days after ingestion and the second at the time of ecdysis. Cathepsin D, cathepsin B and lysosomal carboxypeptidase B reached maximal levels 6 days after ingestion. At this time the highest levels of these proteinases were found in mated females, the lowest in males and intermediate levels in virgin females. Maximal levels of aminopeptidase occurred later than catheptic enzymes, and the decline, after maximal levels were achieved, was much more gradual.Catheptic-proteinase levels within the posterior midgut in fifth-instar larvae and adults correlated positively with the amount of protein contained in this gut region. This indicates that production of these proteinases is controlled by a secretagogue mechanism. Aminopeptidase levels were controlled in a different manner. The mated state or sex of adults altered the proteinase levels by changing the amount of protein that was passed into the digestive midgut from the crop.     

Sequence and function of lysosomal and digestive cathepsin D-like proteinases of Musca domestica midgut

Musca domestica larvae display in anterior and middle midgut contents, a proteolytic activity with pH optimum of 3.0–3.5 and kinetic properties like cathepsin D. Three cDNAs coding for preprocathepsin D-like proteinases (ppCAD 1, ppCAD 2, ppCAD 3) were cloned from a M. domestica midgut cDNA library. The coded protein sequences included the signal peptide, propeptide and mature enzyme that has all conserved catalytic and substrate binding residues found in bovine lysosomal cathepsin D. Nevertheless, ppCAD 2 and ppCAD 3 lack the characteristic proline loop and glycosylation sites. A comparison among the sequences of cathepsin D-like enzymes from some vertebrates and those found in M. domestica and in the genomes of Aedes aegypti, Drosophila melanogaster, Tribolium castaneum, and Bombyx mori showed that only flies have enzymes lacking the proline loop (as defined by the motif: DxPxPx(G/A)P), thus resembling vertebrate pepsin. ppCAD 3 should correspond to the digestive cathepsin D-like proteinase (CAD) found in enzyme assays because: (1) it seems to be the most expressed CAD, based on the frequency of ESTs found. (2) The mRNA for CAD 3 is expressed only in the anterior and proximal middle midgut. (3) Recombinant procathepsin D-like proteinase (pCAD 3), after auto-activation has a pH optimum of 2.5–3.0 that is close to the luminal pH of M. domestica midgut. (4) Immunoblots of proteins from different tissues revealed with anti-pCAD 3 serum were positive only in samples of anterior and middle midgut tissue and contents. (5) CAD 3 is localized with immunogold inside secretory vesicles and around microvilli in anterior and middle midgut cells. The data support the view that on adapting to deal with a bacteria-rich food in an acid midgut region, M. domestica digestive CAD resulted from the same archetypical gene as the intracellular cathepsin D, paralleling what happened with vertebrates. The lack of the proline loop may be somehow associated with the extracellular role of both pepsin and digestive CAD 3.     

Recombinant expression,characterization and expressional analysis of clam <Emphasis Type="Italic">Meretrix meretrix</Emphasis> cathepsin B,an enzyme involved in nutrient digestion

Cathepsin B is one of the most important proteolytic enzymes involved in the nutrient metabolism of clam Meretrix meretrix. The recombinant fusion protein GST-MmeCB (rGST-MmeCB) was obtained at a high level from Escherichia coli and identified using LC-ESI-MS/MS. The GST tag was cleaved from rGST-MmeCB, and the resulting recombinant MmeCB (rMmeCB) was able to degrade the selective substrate carbobenzoxy-l-arginyl-l-arginyl-7-amino-4-trifluoromethylcoumarin (Z-Arg-Arg-AFC) in vitro. The kinetic parameters of the rMmeCB were calculated as follows: K _m, V_max and k _cat are 6.11 μM, 0.0174 μM min⁻¹ and 277.57 s⁻¹, respectively. Rabbit anti-rGST-MmeCB polyclonal antibodies was prepared and used to analyze the tissue distribution of MmeCB protein in M. meretrix. The results showed that the highest level of cathepsin B was found in the digestive gland and moderate levels were found in gill and mantle. Similar expression patterns were found at the mRNA level as detected by real time PCR. Further analysis showed that starvation caused a slight increase in MmeCB protein synthesis in the digestive gland, while refeeding after starvation caused an apparent increase in MmeCB synthesis in digestive gland, gill and mantle. Real time PCR analysis showed that MmeCB mRNA in digestive gland was significantly up-regulated by starvation and returned to normal level after the starved clams were refed. Together, these results indicated that cathepsin B is probably involved in the nutrient digestion of M. meretrix.     

Digestive proteolytic activity in the pistachio green stink bug,Brachynema germari Kolenati (Hemiptera: Pentatomidae)

Proteolytic activity in the digestive system of the pistachio green stink bug, Brachynema germari, was investigated. The maximum total proteolytic activity in the midgut extract was observed at pH 5, suggesting the presence of cysteine proteases. Hydrolyzing the specific substrates for cysteine proteases revealed the presence of cathepsin B and cathepsin L activities in the midgut extract. The presence of cysteine proteases was confirmed by their noticeable inhibition and activation due to specific inhibitors and activators, respectively. The significant inhibition of chymotryptic activity by the inhibitors showed the presence of chymotrypsin in the midgut. No considerable tryptic activity was observed in the midgut extract. There was no detectable total proteolytic activity in the salivary gland extract. Tryptic activity of the salivary gland extract was also inhibited by the specific inhibitors. The substrates for cysteine proteases were also slightly hydrolyzed by the salivary gland extract. Zymogram analysis showed at least one distinct band due to cysteine protease activity in the midgut extract, and the cysteine protease inhibitor caused almost complete disappearance of the band. Cathepsin B and L activities were mainly detected in midgut divisions m₁ and m₃, respectively, and maximum chymotrypsin and trypsin activities were observed in m₃. In general, the results revealed the significant presence of cathepsin B, cathepsin L, and chymotrypsin proteases in the midgut extract. The major proteolytic activity in the salivary glands seems to be conducted by trypsin-like proteases.     

The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut

Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coli, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAL3) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut. Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut. CAL3 hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C26S) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 Å, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents.     

Characterization of an acidic proteinase from the posterior midgut of Rhodnius prolixus stål (hemiptera: Reduviidae)

Rhodnius prolixus Stål contained cathepsin D in the posterior midgut to breakdown ingested blood proteins. Cathepsin D can be separated from cathepsin B, a second endoprotein contained within midgut preparations, using DEAE ion exchange chromatography. Maximum haemoglobin hydrolysis occurred at pH 2.8 and this substrate was more rapidly hydrolyzed than albumin. Identification of the partially purified proteinase as cathepsin D was based on the pH optimum, substrate preference and inhibition of haemoglobin hydrolysis by pepstatin, diazoacetyl norleucine methyl ester and serum. The presence of cathepsin D as an extracellular digestive proteinase is consistent with the presence of cathepsin B and lysosomal carboxypeptidase B, that have also been detected in the posterior midgut of R. prolixus and other blood sucking Hemiptera.     

Ultrastructure and immunolocalization of digestive enzymes in the midgut of Podisus nigrispinus (Heteroptera: Pentatomidae)

The predatory stinkbug Podisus nigrispinus has been utilized in biological control programs. Its midgut is anatomically divided into anterior, middle and posterior regions, which play different roles in the digestive process. We describe the midgut ultrastructure and the secretion of digestive enzymes in the midgut of P. nigrispinus. Midguts were analyzed with transmission electron microscopy and the digestive enzymes amylase, cathepsin L, aminopeptidase and α-glucosidase were immunolocalized. The ultrastructural features of the digestive cells in the anterior, middle and posterior midgut regions suggest that they play a role in digestive enzyme synthesis, ion and nutrient absorption, storage and excretion. The digestive enzymes have different distribution along the midgut regions of the predator P. nigrispinus. Amylase, aminopeptidase and α-glucosidase occur in three midgut regions, whereas cathepsin L occurs in the middle and posterior midgut regions. The anterior midgut region of P. nigrispinus seems to play a role in water absorption, the middle midgut may be involved in nutrient absorption and the posterior midgut region is responsible for water transport to the midgut lumen.     

A chymotrypsin-like serine protease cDNA involved in food protein digestion in the common cutworm, Spodoptera litura: Cloning, characterization, developmental and induced expression patterns, and localization

A full-length cDNA (Slctlp2) encoding a chymotrypsin-like serine protease was cloned from Spodoptera litura. This cDNA encoded a putative serine protease with a predicted molecular mass of 30.6 kDa, which contained a serine protease catalytic motif GDSGGPL. Temporal and spatial expression of Slctlp2 mRNA and protein detected by Northern blotting, RT-PCR, qPCR and Western blotting analyses revealed that both Slctlp2 mRNA and protein were mainly present in the foregut and midgut of the 5th and 6th instar larvae during the feeding stages. In situ hybridization and immunohistochemistry confirmed that both Slctlp2 mRNA and protein were predominately present in the midgut. Expression of the gene was not induced by bacterial infection. Juvenile hormone III induced the gene expression, while 20-hydroxyecdysone had no impact on the expression. The expression of Slctlp2 mRNA and protein was down-regulated by starvation but up-regulated by re-feeding. The SlCTLP2 protein was detected in the lumen residues of the anterior, middle and posterior midgut and feces of the feeding 6th instar larvae, suggesting that it was secreted from the epithelium into the lumen of the gut. The results suggest that this Slctlp2 gene may be involved in digestive process of food proteins during the feeding stages of the larval development.     

Ultrastructural localisation of cathepsin B in the midgut of Rhodnius prolixus Stål ( Hemiptera : Reduviidae) during blood digestion

The artificial substrate N-benzoyl-dl-arginine-β-naphthylamine, was used to localise cathepsin B in midgut cells of the haematophagous insect, Rhodnius prolixus Stål (Hemiptera : Reduviidae), during blood digestion. Cathepsin B was localised primarily in the lysosomes of cells from all 3 midgut regions and in Golgi vesicles of the digestive intestinal regions, but not in association with any other cellular structures. The timing of localisation correlated with previously described cycles of endoproteinase activity and with known ultrastructural modifications to the midgut cells. Secretory vesicles, which originated from the Golgi complexes, were present only in the intestinal regions, and in the anterior intestine, they showed a strong positive correlation (r = 0.939, P = 0.01) with post-feeding cathepsin B activity. Cathepsin B plays a major role in primary extracellular digestion of blood proteins, and is active in the midgut lumen and lysosomes rather than in association with the microvilli.     

Long-Term Starvation and Posterior Feeding Effects on Biochemical and Physiological Responses of Midgut Gland of Cherax quadricarinatus Juveniles (Parastacidae)

We investigated the effect of long-term starvation and posterior feeding on energetic reserves, oxidative stress, digestive enzymes, and histology of C. quadricarinatus midgut gland. The crayfish (6.27 g) were randomly assigned to one of three feeding protocols: continuous feeding throughout 80 day, continuous starvation until 80 day, and continuous starvation throughout 50 day and then feeding for the following 30 days. Juveniles from each protocol were weighed, and sacrificed at day 15, 30, 50 or 80. The lipids, glycogen, reduced glutathione (GSH), soluble protein, lipid peroxidation (TBARS), protein oxidation (PO), catalase (CAT), lipase and proteinase activities, and histology were measured on midgut gland. Starved crayfish had a lower hepatosomatic index, number of molts, specific growth rate, lipids, glycogen, and GSH levels than fed animals at all assay times. The starvation did not affect the soluble protein, TBARS, PO levels and CAT. In starved juveniles the lipase activity decreased as starvation time increased, whereas proteinase activity decreased only at day 80. The histological analysis of the starved animals showed several signs of structural alterations. After 30 days of feeding, the starved-feeding animals exhibited a striking recovery of hepatosomatic index, number of molts, lipids and glycogen, GSH, lipase activity and midgut gland structure.     

Characterization and comparison of digestive proteinases of the Cortez swimming crab, Callinectes bellicosus, and the arched swimming crab, Callinectes arcuatus

Abstract. Protease activity in the midgut gland, gastric chamber, and gastric juice from the crabs Callinectes bellicosus and Callinectes arcuatus was characterized by several methods, confirming that the composition of digestive proteases is the same in the gastric juice and the midgut gland. Gastric juice was suitable for the identification and characterization of the proteinases trypsin and chymotrypsin. Such enzymes were presented as isotrypsins and isochymotrypsins. Proteinase composition evaluated by SDS-PAGE and substrate-SDS-PAGE showed differences between species, but not between gender. Proteinases were thermostable at 40°–50°C for 1 h and showed maximum activity at pH 6–8, making the use of digestive proteinases for evaluations of protein digestibility by the pHstat method possible. We propose using gastric juice as a source of digestive enzymes for in vitro studies of enzymes in digestibility assays and characterization procedures.     

The effects of three metabolic inhibitors on digestive proteinase production in Rhodnius prolixus Stål (Hemiptera: Reduviidae)

Mated female Rhodnius prolixus were fed citrated rabbit blood containing cordycepin, cycloheximide and 5-fluorouracil. The effect of these compounds on cathepsin B, cathepsin D and aminopeptidase levels in the posterior midgut was determined. Cycloheximide and 5-fluorouracil decreased the levels of all three proteinases in a dose-dependent manner. Cordycepin decreased the levels of cathepsin B and D but did not alter the aminopeptidase levels in a similar manner. These results indicate that after ingestion of a blood meal, production of the extracellular catheptic enzymes is dependent on both RNA and protein synthesis while production of the intracellular aminopeptidase depends on protein synthesis alone. The effects of these compounds on proteinase activity cycles also supports this conclusion.     

Inhibition of bone resrption by selctive inactivators of cysteine proteinases

Incativators of cystein proteinases (CPs) were tested as inhibitors of bone resorption in vitro and in vivo. The following four CP inactivators were tested: Ep453, the membrane-permeant produrg of Ep475, a compound with low membrane pereability which inhibits cathepsins B, L, S, H, and calpain; Ep453, the membrane-permeant prodrug of Ep475; CA074, a compound with low membrane permeability which selectivly inactives cathepsin B; and CA07Me, the membrane-permanent prodrug of CA074. The test systems consisted of (1) monitoring the release of radioisotope from prelabelled mousecalvarial explants and (2) assessing the extene of bone resorption in and isolated osteoclast assay using confocal laser microscopy. Ep453, Ep475, and CA074Me inhebited both stimulated and basal bone resorption in vitro while CA074 WASA without effect; The inhibition was reversible and dose dependent. None of the inhibitors affected protein synthesis, DNA synthesis, the PTH-enhanced secretion of β-glucuronised, and N-acetyle-β-glucosaminidase, or the spontaneous release of lactate dehyrogenese. Ep453, Ep475, and CA074Me does-dependently inhibited the resorption activity of isolated areat osteoclassts cultured on bone slices with a maximal effect at 50 μM. The munber of resorption pits and their mean volume was reduced, whilest the mean administration subcutaneously at a dose of 60 μg/g body weight inhibited bone resorption in vivo as measured by an in vivo/in vitro assay, by about 20%. This study demonestration that cathepsins B,L, and/or S are involved in bone resorpotion in vitro and in vivo. Whilest cathepsin L and/or S act extracellularly, and possibly intractually, cathepsin B mediate its effects intracellularly perpheps through the activation of other proteinases involved in subsosteoclastic collagen degradition.