Cathepsin B from the white shrimp Litopenaeus vannamei: cDNA sequence analysis, tissues-specific expression and biological activity

Cathepsin B is a cystein proteinase scarcely studied in crustaceans. Its function has not been clearly described in shrimp species belonging to the sub-order Dendrobranchiata, which includes the white shrimp Litopenaeus vannamei and other species from the Penaeidae family. Studies on vertebrates suggest that these lysosomal enzymes intracellularly hydrolize protein, as other cystein proteinases. However, the expression of the gene encoding the shrimp cathepsin B in the midgut gland was affected by starvation in a similar way as other digestive proteinases which extracellularly hydrolyze food protein. In this study the white shrimp L. vannamei cathepsin B (LvCathB) cDNA was sequenced, and characterized. Its gene expression was evaluated in various shrimp tissues, and changes in the mRNA amounts were compared with those observed on other digestive proteinases from the midgut gland during starvation. By using qRT-PCR it was found that LvCathB is expressed in most shrimp tissues except in pleopods and eye stalk. Changes on LvCathB mRNA during starvation suggest that the enzyme participates during intracellular protein hydrolysis but also, after food ingestion, it participates in hydrolyzing food proteins extracellularly as confirmed by the high activity levels we found in the gastric juice and midgut gland of the white shrimp.     

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.     

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.     

Digestive proteinases of red shrimp Pleoticus muelleri (Decapoda, Penaeoidea): partial characterization and relationship with molting

The present study describes the activity and some characteristics of proteinases in the hepatopancreas of red shrimp Pleoticus muelleri during the different stages of the molting cycle. Proteolytic activity was highest between pH 7.5 and 8. The hepatopancreatic protein content in the premolt stage was higher than in the other stages of the molting cycle (P<0.05). No significant differences were found in total proteolytic activity in the hepatopancreas when comparing molting stages. The proteolytic activity of the P. muelleri hepatopancreas enzyme preparations is the main responsibility of serine proteinases. TLCK, a trypsin inhibitor, reduced azocasein hydrolysis between 26% (intermolt) and 37% (premolt). TPCK, a chymotrypsin inhibitor, did not decrease hydrolytic activity, except for in postmolt. Low trypsin and chymotrypsin activities were found during intermolt, and increased in postmolt. The electrophoretogram of the enzyme extracts shows 12 bands of activity during intermolt (from 16.6 to 53.1 kDa). Some fractions were not detected in the postmolt and premolt stages. Three low molecular weight trypsin forms (17.4, 19.1 and 20 kDa) were found in all molting stages. One band of chymotrypsin (21.9 kDa) was observed in all molting stages. High molecular mass active bands (66-205 kDa) could not be characterized with inhibitors. Comparison of the protease-specific activity of the hepatopancreas of some species indicated a relationship between digestive enzyme activity and feeding habits of the shrimp. Omnivorous shrimp, such as Penaeus vannamei (syn: Litopenaeus vannamei) and Penaeus monodon, showed higher protease activity than the carnivorous shrimp, Penaeus californiensis (syn: Farfantepenaeus californiensis) and P. muelleri. In fact, the enzymatic activity in the hepatopancreas of P. muelleri showed variations in relation to feeding habit and molting cycle.     

Digestive proteinases of larvae of the corn earworm, Heliothis zea: characterization, distribution, and dietary relationships.

Proteinases and peptidases from the intestinal tract of fifth-instar larvae of Heliothis (= Helicoverpa) zea (Boddie) (Lepidoptera:Noctuidae) were characterized based on their substrate specificity, tissue of origin, and pH optimum. Activity corresponding to trypsin, chymotrypsin, carboxypeptidases A and B, and leucine aminopeptidase was detected in regurgitated fluids, midgut contents, and midgut wall. High levels of proteinase activity were detected in whole midgut homogenates, with much lower levels being observed in foregut and salivary gland homogenates. In addition, enzyme levels were determined from midgut lumen contents, midgut wall homogenates, and regurgitated fluids. Proteinase activities were highest in the regurgitated fluids and midgut lumen contents, with the exception of leucine aminopeptidase activity, which was found primarily in the midgut wall. Larvae fed their natural diet of soybean leaves had digestive proteinase levels that were similar to those of larvae fed artificial diet. No major differences in midgut proteinase activity were detected between larvae reared under axenic or xenic conditions, indicating that the larvae are capable of digesting proteins in the absence of gut microorganisms. The effect of pH on the activity of each proteinase was studied. The pH optima for the major proteinases were determined to be pH 8.0-8.5 for trypsin, when tosyl-L-arginine methyl ester was used as the substrate; and pH 7.5-8.0 for chymotrypsin, when benzoyl-L-tyrosine ethyl ester was used as the substrate.     

The effects of dietary supplementation with butyrate and polyhydroxybutyrate on the digestive capacity and intestinal morphology of Pacific White Shrimp (Litopenaeus vannamei)

This study evaluated dietary supplementation with sodium butyrate and polyhydroxybutyrate (PHB) on the enzymatic activity, in vitro nutrient digestibility, and intestinal morphology of Pacific white shrimp (Litopenaeus vannamei). The treatments were: shrimp fed diet supplemented with 2% butyrate, shrimp fed diet supplemented with 2% PHB, and shrimp fed unsupplemented diet. Shrimp fed with PHB-supplemented diet showed higher values of intestinal protease, trypsin, chymotrypsin, and amylase. Shrimp fed with butyrate-supplemented diet showed higher intestinal lipase than unsupplemented shrimp. Butyrate increased hepatopancreatic protease, trypsin, and chymotrypsin activity and in vitro protein digestibility. Shrimp fed with PHB-supplemented diet and butyrate had higher digestibility of polysaccharides and lipids. Shrimp fed with PHB-supplemented diet presented increase in the length, width, and perimeter of intestinal villi. Animals fed with both diets retained overall integrity of their intestinal mucous membrane. The findings show that dietary supplementation with PHB and butyrate can alter intestinal morphology and could improve the digestive capacity of L. vannamei.     

Biochemical characterization of digestive proteases and carbohydrases of the carob moth,Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae)

Digestive proteinases and carbohydrases of Ectomyelois ceratoniae (Zeller) larvae were investigated using appropriate substrates and inhibitors. Midgut pH in larvae was determined to be slightly alkaline. Midgut extracts showed optimum activity for proteolysis of hemoglobin at pH 9–12. Midgut proteinases also hydrolyzed the synthetic substrates of trypsin, chymotrypsin, and elastase at pH 8–11. Maximum digestive α-amylase activity was also observed at pH 8–11. However, optimum activity for α- and β-glucosidase occurred at pH 5–8. Alpha- and β-galactosidases optimum activities occurred at pH 5 and pH 6, respectively. Inhibitors of serine proteases were effective on midgut serine proteases (trypsin and chymotrypsin proteases). Zymogram analyses revealed at least five bands of total proteolytic activity in the larval midgut. Protease-specific zymogram analyses revealed at least four, two, and one isozymes for trypsin-, chymotrypsin-, and elastase-like activities respectively. Two α-amylase isozymes were found in the midgut of fifth instar larvae and in the whole bodies of 1st through 5th instar larvae. Zymogram studies also revealed the presence of one and two bands of activity for β- and α-glucosidase, respectively. Recycling of α-amylase and proteases in the larval midgut was not complete. At least one isozyme of trypsin, chymotrypsin, elastase, and α-amylase were not recycled and were observed in the larval hindgut.     

Distribution of digestive proteinases in the alimentary tract of the european corn borer Ostrinia nubilalis (lepidoptera: Pyralidae)

The distribution of digestive proteinases in either the anterior and posterior midgut or between the midgut epithelium and ectoperitrophic and endo-peritrophic spaces in the midgut were examined in the European corn borer, Ostrinia nubilalis. Trypsin, chymotrypsin, elastase, and aminopeptidase activities were the same in the anterior and posterior halves of the midgut. Of the total aminopeptidase activity, 95% was located in the midgut epithelium, and 90% of the trypsin, 97% of chymotrypsin, and 93% of the elastase activity were found in the midgut lumen. Trypsin, measured by hydrolysis of benzoyl-L-arginine ethyl ester, and chymotrypsin levels were significantly higher in the ectoperitrophic space compared to the endoperitrophic space. Digestion in the midgut is proposed to be sequential with tryptic digestion occurring in the endoperitrophic space. Ingested protein is digested further in the ectoperitrophic space by the action of elastase, chymotrypsin, and a second trypsin. Final digestion occurs by an intracellular aminopeptidase. © 1995 Wiley-Liss, Inc.     

Enzymatic Response of the Eucalypt Defoliator Thyrinteina arnobia (Stoll) (Lepidoptera: Geometridae) to a Bis-Benzamidine Proteinase Inhibitor

Ingestion of proteinase inhibitors leads to hyperproduction of digestive proteinases, limiting the bioavailability of essential amino acids for protein synthesis, which affects insect growth and development. However, the effects of proteinase inhibitors on digestive enzymes can lead to an adaptive response by the insect. In here, we assessed the biochemical response of midgut proteinases from the eucalypt defoliator Thyrinteina arnobia (Stoll) to different concentrations of berenil, a bis-benzamidine proteinase inhibitor, on eucalyptus. Eucalyptus leaves were immersed in berenil solutions at different concentrations and fed to larvae of T. arnobia. Mortality was assessed daily. The proteolytic activity in the midgut of T. arnobia was assessed after feeding on plants sprayed with aqueous solutions of berenil, fed to fifth instars of T. arnobia for 48?h before midgut removal for enzymatic assays. Larvae of T. arnobia were able to overcome the effects of the lowest berenil concentrations by increasing their trypsin-like activity, but not as berenil concentration increased, despite the fact that the highest berenil concentration resulted in overproduction of trypsin-like proteinases. Berenil also prevented the increase of the cysteine proteinases activity in response to trypsin inhibition.     

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.     

Digestive peptidases in Tenebrio molitor and possibility of use to treat celiac disease

Digestion in Tenebrio molitor larvae occurs in the midgut, where there is a sharp pH gradient from 5.6 in the anterior midgut (AM) to 7.9 in the posterior midgut (PM). Accordingly, digestive enzymes are compartmentalized to the AM or PM. Enzymes in the AM are soluble and have acidic or neutral pH optima, while PM enzymes have alkaline pH optima. The main peptidases in the AM are cysteine endopeptidases presented by two to six subfractions of anionic proteins. The major activity belongs to cathepsin L, which has been purified and characterized. Serine post‐proline cleaving peptidase with pH optimum 5.3 was also found in the AM. Typical serine digestive endopeptidases, trypsin‐like and chymotrypsin‐like, are compartmentalized to the PM. Trypsin‐like activity is due to one cationic and three anionic proteinases. Chymotrypsin‐like activity consists of one cationic and four anionic proteinases, four with an extended binding site. The major cationic trypsin and chymotrypsin have been purified and thoroughly characterized. The predicted amino acid sequences are available for purified cathepsin L, trypsin and chymotrypsin. Additional sequences for putative digestive cathepsins L, trypsins and chymotrypsins are available, implying multigene families for these enzymes. Exopeptidases are found in the PM and are presented by a single membrane aminopeptidase N‐like peptidase and carboxypeptidase A, although multiple cDNAs for carboxypeptidase A were found in the AM, but not in the PM. The possibility of the use of two endopeptidases from the AM – cathepsin L and post‐proline cleaving peptidase – in the treatment of celiac disease is discussed.     

Substrate specificity in the control of digestive enzymes in larvae of the black carpet beetle

When starved larvae of the black carpet beetle, Attagenus megatoma, were fed selected diets, increases in proteolytic, trypsin, and chymotrypsin activity were correlated with total midgut protein and not with the amount of food consumed. Although larvae initially consumed more of a starch diet than of 2 diets that contained added protein, total protease activity in these larvae was minimal. Starch-fed larvae and larvae fed a casein-sucrose diet had a consistently higher level of sucrase activity than larvae fed an all-casein diet. These total results support a secretagogue mechanism for control of digestive enzyme synthesis in insects. In addition, the absence of parallel stimulation of different digestive enzymes by a single substrate (starch) indicated nutrient class specificity in the control of inducible midgut enzymes in this species.     

The effect of dietary Panax ginseng polysaccharide extract on the immune responses in white shrimp, Litopenaeus vannamei

The immunostimulatory effects of orally administered Panax ginseng root or its polysaccharides (GSP) in white shrimp, Litopenaeus vannamei, were investigated in this study. Shrimp were fed a diet containing 0.4 g kg^?1 GSP over a period of 84 days, during which the activities of total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), acid phosphatase (ACP), and alkaline phosphatase (AKP), as well as malondialdehyde (MDA) content, and expressions of cytosolic superoxide dismutase (cyt-SOD), CAT, GSH-Px, and peroxiredoxin (Prx) genes were determined in various tissues of the shrimp. Results showed that the shrimp fed the GSP diet had significantly increased ACP and AKP activities in the gills. The GSP-fed shrimp also displayed significantly increased T-SOD and GSH-Px activities in the gills and hepatopancreas of the shrimp; meanwhile there was enhanced CAT activity in the gills, but decreased MDA content in the gills, hepatopancreas and muscle. The mRNA expressions of cyt-SOD, CAT, GSH-Px and Prx were significantly elevated in the gills and hepatopancreas of the shrimp fed the GSP diet for 84 days, compared with that of the control. Therefore, GSP can be used as an immunostimulant for shrimp through dietary administration to increase immune enzyme activity and modify expression of immune genes in shrimp.     

Adaptation of Helicoverpa armigera (Lepidoptera: Noctuidae) to a proteinase inhibitor expressed in transgenic tobacco

A giant taro proteinase inhibitor (GTPI) cDNA was expressed in transgenic tobacco using three different gene constructs. The highest expression level obtained was ca. 0.3% of total soluble protein when the cDNA was driven by the Arabidopsis rbcS ats1 promoter. Repeated feeding trials with Helicoverpa armigera larvae fed on clonally derived T₀ and T₁ plants expressing GTPI demonstrated that, relative to those fed on control plants, some growth inhibition (22–40%) occurs, but there was no increase in larval mortality. Proteinase activities of larvae fed on GTPI-expressing tobacco or GTPI-containing diet were examined to monitor the spectrum of digestive proteinases in the midgut. Total proteinase activity was reduced by 13%, but GTPI-insensitive proteinase activity was increased by up to 17%. Trypsin was inhibited by 58%, but chymotrypsin and elastase were increased by 26% and 16% respectively. These results point to an adaptive mechanism in this insect that elevates the levels of other classes of proteinases to compensate for the trypsin activity inhibited by dietary proteinase inhibitors.     

Protein digestion in larvae of the red oak borer Enaphalodes rufulus

Abstract In the Ozark Mountains of the U.S.A., the red oak borer Enaphalodes rufulus contributes to the destruction of red oaks. To understand nutrient digestion in E. rufulus larvae, digestive proteinases are compared in both larvae fed heartwood phloem and those transferred to artificial diet. The pH of gut extracts is approximately 6.3 in the midgut and foregut and decreases to 5.5 in the hindgut region. The hydrolysis of casein by midgut extracts from E. rufulus larvae fed either artificial diet or phloem from tree sections increases in buffers greater than pH 6.19, with maximum hydrolysis being observed at pH 10.1. Casein zymogram analysis reveals two major proteinase activities in larval midgut extracts of diet‐fed larvae, with molecular masses of approximately 25 and 40–60 kDa, whereas phloem‐fed larvae have proteinase activities corresponding to 40, 45, 60, 80 and >100 kDa. Substrate analysis indicates at least one major trypsin‐like activity in both gut extracts with a molecular mass of >100 kDa, but two chymotrypsin‐like activities of approximately 25 and >200 kDa are found only in diet‐fed larvae. Inhibitors of serine proteinases are most effective in reducing the general proteolytic activity of midgut extracts from larvae fed either food source. The data indicate that serine proteinase inhibitors have the potential to reduce E. rufulus larval damage to oaks. In particular, transgenic technologies incoporating trypsin inhibitors may be effective in reducing protein digestion in phloem‐feeding larvae.     

Identification of six chymotrypsin cDNAs from larval midguts of Helicoverpa zea and Agrotis ipsilon feeding on the soybean (Kunitz) trypsin inhibitor

Lepidopteran insects like Helicoverpa zea and Agrotis ipsilon produce STI-insensitive trypsins in the midgut following ingestion of dietary plant proteinase inhibitors like STI [Broadway, R. M., J. Insect Physiol. 43(9) (1997) 855-874]. In this paper, the effects of dietary STI on a related family of midgut serine proteinases, the chymotrypsins, were investigated. STI-insensitive midgut chymotrypsins were detected in larvae of H. zea and A. ipsilon feeding on diets containing 1% STI while STI-sensitive chymotrypsins were present in larvae feeding on diets containing 0% STI. These chymotrypsins were unaffected by TPCK, a diagnostic inhibitor of mammalian chymotrypsins but were fully inhibited by chymostatin. Four midgut cDNA libraries were constructed from larvae of each species fed either 0% STI or 1% STI diets. Six full-length cDNAs(1) encoding diverse preprochymotrypsins were isolated (three from H. zea and three from A. ipsilon) with certain sequence motifs that set them apart from their mammalian counterparts. Northern blots showed that some chymotrypsin mRNA were detected at higher levels while others were down-regulated when comparing insects reared on 0% STI and 1% STI diets. Southern hybridizations suggested that (like mammals) both species contained several chymotrypsin genes. A full-length chymotrypsin gene(1) from H. zea was sequenced for the first time and the presence of four introns was deduced. A first time comparison of 5' upstream regions(1) from three chymotrypsin genes and two trypsin genes of A. ipsilon indicated the presence of putative TATA boxes and regulatory elements. However a lack of consensus motifs in these upstream regions suggested the likelihood of multiple trans factors for regulation of genes encoding digestive proteinases and a complex response mechanism linked to ingestion of proteinase inhibitors.     

Complex of digestive proteinases of <Emphasis Type="Italic">Galleria mellonella</Emphasis> caterpillars. Composition,properties, and limited proteolysis of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> endotoxins

The complex of digestive proteinases in caterpillars of the greater wax moth Galleria mellonella was studied. Using chromogenic substrates and inhibitor analysis, it was found that serine proteinases play a key role in this complex. Three anionic and two cationic forms of trypsin and one anionic and one cationic form of chymotrypsin were identified by zymography in the midgut extract of G. mellonella. The most active trypsin was purified to electrophoretic homogeneity, and its N-terminal amino acid sequence was shown to be identical to that of mature trypsin from Plodia interpunctella. Midgut extract from G. mellonella was capable of processing Cry-proteins from Bacillus thuringiensis ssp. galleriae. Enzymes with tryptic and chymotryptic activities participate in this process, and activation of protoxin Cry9A is not the rate-limiting stage in the toxic action of this protein on the greater wax moth.     

Proteinase, amylase, and proteinase-inhibitor activities in the gut of six cockroach species

Representative species, two from each of the cockroach families Blattidae, Blattellidae, and Blaberidae, have similar morphology of the digestive tract but differ in the physiology of digestion. The pH of crop and along the midgut varies in different species from 5.9 to 9.0 and the redox parameter from 10.1 to 12.9. Activities of proteinases and amylases in comparable gut regions differ among the species up to 100 times. Proteolytic activity is high in the midgut and moderate in the crop of Blattidae; in the other species, it is very low in the crop and increases to a moderate level in the posterior half of midgut (PM). The level of amylolytic activity is similar in the examined gut compartments of Blattidae and Blattellidae but low in the PM of Blaberidae. Blaberidae are also characterized by a high potential of the salivary glands, crop, and midgut to inhibit subtilisin, trypsin, and chymotrypsin. Inhibition of these proteinases by the extracts of salivary glands and gut is several orders of magnitude lower and often undetectable in the representatives of Blattidae and Blattellidae.