Biosynthesis of Astacus protease,a digestive enzyme from crayfish

Summary For the first time, the site of biosynthesis of a well characterized invertebrate digestive enzyme is localized. The enzyme chosen, Astacus protease, is a zinc-metalfoenzyme occuring in high concentration in the gastric fluid of the freshwater crayfish Astacus astacus. Enzyme production was stimulated in adult crayfish either by feeding or by removal of the gastric fluid. Immunohistochemistry, cytology and investigation with radioactive tracers demonstrate that in the hours following stimulation, new enzyme was produced in the F-cells of the midgut gland and subsequently discharged into the midgut gland lumen. The enzyme was then accumulated and stored extracellularly in the cardiac stomach in active form. The mechanism of enzyme production observed in Astacus differs considerably from vertebrates suggesting an alternative model for synthesis and storage of digestive enzymes.Supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to V.S./R.Z. (Sto 75/10) and to W.S./R.Z. (Sto 185/1-1)     

Endogenous production of endo-β-1,4-glucanase by decapod crustaceans

The potential ability to produce cellulase enzymes endogenously was examined in decapods crustaceans including the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes, the amphibious freshwater crab Austrothelphusa transversa, the terrestrial hermit crab, Coenobita variabilis the parastacid crayfish Euastacus, and the crayfish Cherax destructor. The midgut gland of both G. natalis and D. hirtipes contained substantial total cellulase activities and activities of the cellulase enzymes endo-β-1,4-glucanase and β-glucosidase. With the exception of total cellulase and β-glucosidase from D. hirtipes, the enzyme activities within the midgut gland were higher than those within the digestive juice. Hence, the enzyme activities appear to reside predominantly within midgut gland, providing indirect evidence for endogenous synthesis of cellulase enzymes by this tissue. A 900 bp cDNA fragment encoding a portion of the endo-β-1,4-glucanase amino acid sequence was amplified by RT-PCR using RNA isolated from the midgut gland of C. destructor, Euastacus, A. transversa and C. variabilis. This provided direct evidence for the endogenous production of endo-β-1,4-glucanase. The 900 bp fragment was also amplified from genomic DNA isolated from the skeletal muscle of G. natalis and D. hirtipes, clearly indicating that the gene encoding endo-β-1,4-glucanase is also present in these two species. As this group of evolutionary diverse crustacean species possesses and expresses the endo-β-1,4-glucanase gene it is likely that decapod crustaceans generally produce cellulases endogenously and are able to digest cellulose.     

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.     

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.     

A field study on the physiology of digestion in the Antarctic krill, Euphausia superba, with special regard to chitinolytic enzymes

Endo- and exochitinase activities were determined in the stomachand midgut gland of the Antarctic krill, Euphausia superba.along a transect west of the Antarctic Peninsula. Activitieswere compared with the digestive enzymes protease, cellulase(1,4-ß-D-glucanase) and laminarinase (1,3-ß-D-glucanase)The chlorophyll and protein contents in the surface water ofthe corresponding stations were determined. Enzyme activitieswere characterized by high individual and spatial variations.Chitinolytic activity in the stomach correlated well with alldigestive enzymes investigated. In the midgut gland, a correlationwith cellulase and laminannase was evident. The amount of chlorophylla and phytoplankton protein in the surface water was not correlatedwith enzyme activity. Specific enzyme activity was higher inthe stomach than in the midgut gland. showing individual ratiosfor each enzyme. Elevated endochitinase activity in the stomachsuggests that chitinous food is digested to oligomers in thestomach, while the subsequent degradation to amino sugars occurspredominantly in the midgut gland.     

The metabolism of astaxanthin during the embryonic development of the crayfish Astacus leptodactylus Eschscholtz (Crustacea, Astacidea)

The developing eggs of the crayfish Astacus leptodactylus are found to contain important carotenoid amounts. Free astaxanthin, mainly associated with protein and lutein represent the main pigments occurring in the yolk at the end of the embryonic period. Esterification processes affecting astaxanthin appear concomitantly with the resorption of vitellin reserves, the activation of the digestive gland and the rapid decrease of lutein amounts. Esterified astaxanthin contents increase significantly within the interval between the juvenile stages I and II. Nevertheless, free astaxanthin levels appear insufficient to balance the amounts of the esterified form, scored at the end of the endotrophic period.     

An hypothesis of the mechanism controlling proteolytic digestive enzyme production levels in Stomoxys calcitrans

Flies fed a human blood meal and sacrificed 9 h later were assayed to give information on unfed fly weight, meal weight, total midgut protein, total midgut proteolytic activity, anterior midgut protein, anterior midgut proteolytic activity, posterior midgut protein, and posterior midgut proteolytic activity; correlation coefficients were calculated for all pairings of these parameters. Posterior midgut protein showed a positive correlation with posterior midgut proteolytic activity and on this evidence it is concluded that proteolytic digestive enzyme secretion in the midgut of Stomoxys calcitrans is controlled by a secretogogue mechanism.It is proposed that the only direct stimulus the food supplies in the control of digestive enzyme production is that for digestive enzyme release from the production cells. It is also proposed that the basis of the secretogogue mechanism is that digestive enzymes are produced in direct proportion to the quantities of amino-acids available for their synthesis and that this is a consequence of the quantities of amino acids released from the food during digestion.     

alpha2-Macroglobulin from hemolymph of the freshwater crayfish Astacus astacus.

1. A high mol. wt proteinase inhibitor has been purified from the haemolymph of the freshwater crayfish Astacus astacus. 2. The protein is a disulphide-bonded dimer (Mr 390,000) of two identical polypeptide chains (Mr 185,000). 3. The inhibitor displays a broad specificity and protects trypsin from inhibition by soybean trypsin inhibitor and thus is similar to vertebrate alpha 2-macroglobulin. 4. The alpha 2-macroglobulin-like inhibitor from Astacus interacts with bovine trypsin in an equimolar stoichiometry thereby decreasing tryptic hydrolysis of N-benzoyl-L-arginine-ethylester to 50% residual activity. In contrast, the activity of Astacus protease, a digestive zinc proteinase from crayfish toward succinyl-alanyl-alanyl-alanyl-4-nitroanilide is inhibited almost completely. 5. Sensitivity of the inhibitor to methylamine and autolytic cleavage suggests the presence of an internal thioester bond. 6. The N-terminal amino acid sequence of Astacus alpha 2-macroglobulin is strongly related to the alpha 2-macroglobulins from Pacifastacus leniusculus (91% identity) and from the lobster Homarus americanus (72% identity). In contrast, only 25% of the residues are identical with the alpha 2-macroglobulin from the horseshoe crab Limulus polyphemus. There is also a faint similarity to human complement protein C3 and human alpha 2-macroglobulin.     

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.     

意大利蜜蜂胚后发育过程中中肠上皮组织细胞的更替

中肠是昆虫消化、 吸收营养物质的主要部位。本研究通过形态解剖、 BrdU免疫组织化学和原位末端转移酶标记(TUNEL)细胞凋亡检测等技术, 对意大利蜜蜂Apis mellifera ligustica中肠胚后发育过程中细胞的增殖和凋亡模式进行了比较研究。结果表明：意大利蜜蜂幼虫发育早期, 中肠的增加主要来自于上皮细胞的分裂以及再生细胞的增殖。在变态发育期间, 中肠上皮经历了广泛的重组, 由再生细胞重新形成的蛹上皮替代了幼虫上皮。再生细胞在蜜蜂中肠的整个发育阶段始终存在, 为中肠的生长和更替提供了主要的细胞来源。本研究为昆虫组织细胞自噬和凋亡机制的研究提供一定的证据。     

黑水虻消化道形态及组织结构的观察

本文比较了不同发育阶段黑水虻Hermetia illucens消化道的形态学差异,掌握了幼虫消化系统的组织学特征。利用体视镜观察黑水虻5龄幼虫、预蛹及成虫的消化道形态,利用光学显微镜和扫描电镜观察幼虫消化道各段(前肠、中肠、后肠)的显微及超微结构。结果表明：黑水虻幼虫及预蛹的消化道均由前肠(食道和前胃)、中肠及后肠组成,从幼虫到成虫,消化道的长度不断缩短。与幼虫和预蛹相比,成虫消化道形态变化明显,前胃消失,出现了嗉囊及胃盲囊,中肠进一步缩短,后肠分化为回肠、结肠和直肠。组织学观察结果显示,幼虫的唾液腺开口于口腔,由膨大的管状腺体和腺管组成。食道由特化为角质刺突的内膜层及发达的肌层组成,其末端延伸至前胃。前胃膨大为球状,包括三层组织结构。根据上皮细胞形态的差异,中肠可分为四个区段。后肠薄,肠腔内褶丰富,肠壁可见数量较多的杆状细菌。马氏管开口于中、后肠交界处,包括4支盲管,管内壁密布微绒毛。黑水虻消化道形态随发育阶段的变化,反映了各阶段摄食及消化生理的差异。幼虫消化道各段具有各自典型的组织学特征,其前、中、后肠可能分别承担了食物接纳与初步消化、消化与吸收以及重吸收功能。本研究结果为进一步了...     

Crayfish carboxypeptidase. Affinity chromatography, characterization and amino-terminal sequence.

In an effort to trace the evolutionary history of the pancreatic metalloexopeptidases, carboxypeptidase has been isolated from the cardia of the crayfish Astacus fluviatilis. The isolation procedure included affinity chromatography on a column of potato carboxypeptidase inhibitor covalently linked to Sepharose. Approximately 25 mg of pure enzyme can be obtained by the present procedure from 50 ml of cardia fluid. The pure enzyme resembles bovine carboxypeptidase B in specificity and is inhibited both by 3-phenyllactate and by 6-aminohexanoate. The pH optimum of activity is about pH 6.5, and the isoelectric point,pH 4.0. Inhibition by typical metal chelating agents (i.e. ethylenediamine tetraacetate and 1,10-phenanthroline) and neutron activation analysis indicate that, like the mammalian enzyme, crayfish carboxypepetidase is a zinc metalloenzyme. The purified enzyme migrates as a single band in cellulose acetate, disc gel and sodium dodecylsulfate gel electrophoresis. The amino acid composition is similar to that of pancreatic carboxypeptidases except for a higher content of acidic amino acid residues. The amino acid sequence of the first 19 amino-terminal residues reveals significant homology to that of pancreatic carboxypeptidases A and B.     

Effect of different diets on digestive enzyme activities,in vitro digestibility,and midgut gland structure in juvenile crayfish,Cherax quadricarinatus

This study investigated the effects of food quality on digestive enzyme activities, in vitro protein digestibility and histological traits of the midgut gland in juvenile crayfish Cherax quadricarinatus. Animals of a wide weight range were fed different diets: two commercial diets with high or low lipid content (high lipid and low lipid, respectively) and were compared with a reference diet (RF) previously formulated for this species. Proteinase, lipase and amylase activities were significantly influenced by diet and weight. Specific trypsin activity was significantly higher for crayfish fed with the HL diet. Trypsin activity depended on diet and weight. Protein digestibility showed that HL was the most digestible diet and RF the least. The weight of the animals did not affect protein digestibility. Structural disorganization, hypertrophy of B‐cells and presence of large vacuoles in R‐cells were mainly observed in juveniles fed with HL, indicative of malnutrition. Thus, our data suggest that the HL diet would not be the most appropriate for C. quadricarinatus, while RF diet would be more convenient for culture of this species.     

粉尘螨消化系统的形态学观察

光镜下观察了粉尘螨Dermatophagoides farinae消化系统结构,其组成包括：口前腔、前肠、中肠、后肠、肛门和唾液腺。口前腔由颚体围绕而成;前肠包括一个肌肉的咽和食道,食道从脑中穿过;中肠分为前中肠（包括一对盲肠）和后中肠,中肠的上皮细胞呈现多种形态; 后肠包括相对大的结肠和狭窄的直肠;消化腺为不规则形,位于脑前方。本文阐述了消化道的分支情况、显微结构及细胞形态。     

UDPglucosyltransferase and its kinetic fluorimetric assay

A rapid, kinetic assay for UDPglucosyltransferase has been developed using 1-naphthol as substrate. It is based on the continuous fluorimetric monitoring of 1-naphthyl glucoside formation during the reaction at physiological pH. The conjugate is easily distinguished from aglycone, since their fluorimetric properties differ. Glucoside biosynthesis in vitro by microsomal preparations isolated from the gut and fat body of cockroaches Periplaneta americana and Leucophaea maderae, and from the green gland and hepatopancreas of the crayfish Astacus astacus, has been demonstrated. The effects of buffer, pH, MgCl2, UDP-glucuronic acid, UDP-N-acetylglucosamine, sodium cholate and sonication on the enzyme activity have been assessed. The kinetic parameters of 1-naphthol and UDP-glucose have also been determined.     

Activation of pro-astacin. Immunological and model peptide studies on the processing of immature astacin, a zinc-endopeptidase from the crayfish Astacus astacus.

To contribute knowledge of the processing and activation of invertebrate proteolytic enzymes, we studied the metalloprotease astacin, a digestive enzyme from the freshwater crayfish Astacus astacus (decapod crustacean). It is the prototype of the protein family of astacins, members of which occur in organisms from bacteria to man and are involved in a variety of physiological reactions. According to its genomic structure, astacin is produced as a zymogen [Geier, G., Jacob, E., St?cker, W. & Zwilling, R. (1997) Arch. Biochem. Biophys. 337, 300-307]. To localize and follow the processing of pro-astacin in different parts of the digestive tract, we synthesized two peptides covering the pro part of pro-astacin and raised antibodies against them. In addition, antiserum against the whole active astacin was produced. Using immunohistochemical investigation, we detected pro-astacin in the F cells of the hepatopancreas and all the way into the tubular lumen and the collecting ducts of this gland. Immunoblot assays revealed only active astacin, and never pro-astacin, present in the cardiac stomach. We conclude from these studies that astacin is secreted into the lumen of the hepatopancreatic tubules in its pro form and is activated on its way to the stomach. To investigate which of the two endopeptidases found in the digestive tract of crayfish, astacin or trypsin, is responsible for cleaving the propeptide from pro-astacin, we synthesized different peptides that mimick the activation site. MS analysis of the cleavage products of astacin and trypsin showed that astacin is capable of catalyzing its own activation. Any contribution of trypsin would require the successive action of an aminopeptidase. Substituting glycine for arginine at position -1 of the activation site does not prevent astacin activity. As most members of the astacin protein family have basic amino-acid residues in this position, in these cases also astacin-specific cleavage would be possible.     

The effect of adipokinetic hormones on the activity of digestive enzymes

The role of the adipokinetic hormone (AKH) in the control of protease, amylase and lipase activities is examined using the cockroach Periplaneta americana and the fruit fly Drosophila melanogaster as model species. The effects of Peram‐CAH‐I and ‐II on the activity of cockroach digestive enzymes in the gastric caeca and midgut are measured both in vivo and in vitro. The results show the activity of proteases, amylases and lipases in both parts of the gut: amylase activity is higher in the gastric caeca than in the midgut; lipase activity presents the opposite trend; and protease activity is similar in both organs. The applied hormones stimulate the activity of all digestive enzymes, although this stimulation is not uniform; AKHs affect enzymes selectively, and in some cases unequally, in the gastric caeca and midgut. No substantial differences between Peram‐CAH‐I and ‐II stimulation are recorded. The in vitro results demonstrate that AKH stimulates digestive enzyme activity directly. In agreement with the cockroach results, enzymatic activity in D. melanogaster larvae producing nonfunctional AKH is lower than that in the larvae with ectopically expressed Akh gene, where enzyme activity reaches or even exceeds that of the controls. Overall, the results demonstrate the active role of AKHs in the stimulation of digestive enzyme activity in insects.     

Peritrophic membrane role in enhancing digestive efficiency. Theoretical and experimental models

The peritrophic membrane (PM) is an anatomical structure surrounding the food bolus in most insects. Rejecting the idea that PM has evolved from coating mucus to play the same protective role as it, novel functions were proposed and experimentally tested. The theoretical principles underlying the digestive enzyme recycling mechanism were described and used to develop an algorithm to calculate enzyme distributions along the midgut and to infer secretory and absorptive sites. The activity of a Spodoptera frugiperda microvillar aminopeptidase decreases by 50% if placed in the presence of midgut contents. S. frugiperda trypsin preparations placed into dialysis bags in stirred and unstirred media have activities of 210 and 160%, respectively, over the activities of samples in a test tube. The ectoperitrophic fluid (EF) present in the midgut caeca of Rhynchosciara americana may be collected. If the enzymes restricted to this fluid are assayed in the presence of PM contents (PMC) their activities decrease by at least 58%. The lack of PM caused by calcofluor feeding impairs growth due to an increase in the metabolic cost associated with the conversion of food into body mass. This probably results from an increase in digestive enzyme excretion and useless homeostatic attempt to reestablish destroyed midgut gradients. The experimental models support the view that PM enhances digestive efficiency by: (a) prevention of non-specific binding of undigested material onto cell surface; (b) prevention of excretion by allowing enzyme recycling powered by an ectoperitrophic counterflux of fluid; (c) removal from inside PM of the oligomeric molecules that may inhibit the enzymes involved in initial digestion; (d) restriction of oligomer hydrolases to ectoperitrophic space (ECS) to avoid probable partial inhibition by non-dispersed undigested food. Finally, PM functions are discussed regarding insects feeding on any diet.     

Inhibition of digestive enzyme release by neuropeptides in larvae of Opisina arenosella (Lepidoptera: Cryptophasidae)

Leucokinins are a group of structurally related neuropeptides stimulating gut motility and fluid secretion by Malpighian tubule in insects. For studying effect of neuropeptides on digestive enzyme release, empty midgut tubes of larvae of Opisina arenosella ligated at both ends with hair were incubated with Leucokinins (LK I-VIII), LK analogues and Leucopyrokinin (LPK) in a bioassay apparatus at 37 degrees C for 30 min. The lumen contents were subsequently analyzed for digestive enzyme levels. The neuropeptides LK III, FFSWG amide, 122 A[1] WP-2, LPK and 434 [phi2] WP-1 inhibited the release of digestive enzymes, protease and amylase while LK VIII, unique in having tyrosine residue, stimulated protease release. The minimum sequence of amino acids at the C-terminal required for activity of LK peptides was found to be FXSWGamide (X=Asn, His, Ser, or Trp). The N-terminal pyroglutamate residue and proline at the C-terminal may contribute to the inhibitory effect of LPK on digestive enzyme release. The present study reveals for the first time an inhibitory effect for leucokinins and pyrokinin on the release of digestive enzymes from the insect midgut.