The roles of mucus‐forming mucins,peritrophins and peritrophins with mucin domains in the insect midgut

Most insects have a gut lined with a peritrophic membrane (PM) consisting of chitin and proteins, mainly peritrophins that have chitin‐binding domains. The PM is proposed to originate from mucus‐forming mucins (Mf‐mucins), which acquired a chitin‐binding domain that interlocked with chitin, replacing mucus in function. We evaluated the expression of Mf‐mucins and peritrophins by RNA‐sequencing (RNA‐seq) throughout the midgut of four distantly related insects. Mf‐mucins were identified as proteins with high o‐glycosylation and a series of uninterrupted Pro/Thr/Ser residues. The results demonstrate that the mucus layer is widespread in insects, and suggest that insect Mf‐mucins are derived from those found in other animals by the loss of the cysteine knot and von Willebrand domains. The data also support a role of Mf‐mucins in protecting the middle midgut of Musca domestica against acidic buffers. Mf‐mucins may also produce a jelly‐like material associated with the PM that immobilizes digestive enzymes in Spodoptera frugiperda. Peritrophins with a domain similar to Mf‐mucins may be close to the ancestor of peritrophins. Expression data of peritrophins and chitin synthase genes throughout the midgut of M. domestica, S. frugiperda and Tenebrio molitor indicated that peritrophins were incorporated along the PM, according to their preferential sites of formation. Finally, the data support the view that mucus has functions distinct from the PM.     

Plasma carboxypeptidases as regulators of the plasminogen system.

Carboxy-terminal lysine residues on the surface of cells and fibrin bind plasminogen and control its activation. Since plasma contains basic carboxypeptidases, which remove carboxy-terminal lysines from protein substrates, we investigated if these enzymes are involved in the regulation of plasminogen binding sites. Plasma reduced plasminogen binding to cells, and this effect could be ascribed to the activity of the plasma carboxypeptidases. Purified carboxypeptidase N, which is constitutively active, and plasma carboxypeptidase B, which circulates as a zymogen, were both capable of significantly reducing plasminogen binding to cells. Dose titration experiments verified that plasma concentrations of either carboxypeptidase were sufficient to maximally affect plasminogen binding to cells. Furthermore, plasma carboxypeptidase B, but not carboxypeptidase N, reduced the rate of whole blood clot lysis induced by tissue-type plasminogen activator. These findings establish that plasma carboxypeptidases can modulate plasminogen binding to cells and control the rate of fibrinolysis. These functions delineate a novel role for the plasma carboxypeptidases in the regulation of the plasminogen system.     

Gut-specific genes from the black fly Simulium vittatum encoding trypsin-like and carboxypeptidase-like proteins

In haematophagous insects digestion of the blood meal provides nutrients for survival and essential components for egg production. We have isolated and partially characterized two gut-specific genes from the black fly Simulium vittatum. Sequence analysis revealed that both are highly similar to digestive proteases, one to trypsins and the other to carboxypeptidases. RNA blot analysis indicates that the expression of these two genes is regulated in a sexspecific manner; when fed the same sucrose-based diet, expression in males is substantially lower than in females. In females, expression of both genes is strongly induced by a blood meal. At 6 h after the blood meal the trypsin-like gene product was immunolocalized to the midgut epithelium and to the outer layers of the peritrophic matrix.     

cpbAg1 encodes an active carboxypeptidase B expressed in the midgut of Anopheles gambiae

We previously used differential display to identify several Anopheles gambiae genes, whose expression in the mosquito midgut was regulated upon ingestion of Plasmodium falciparum. Here, we report the characterization of one of these genes, cpbAg1, which codes for the first zinc-carboxypeptidase B identified in An. gambiae and in any insect. Expression of cpbAg1 in baculovirus gave rise to an active enzyme, and determination of the N-terminal amino acids confirmed that CPBAg1 contains a signal peptide and a pro-peptide, typical features of digestive zinc carboxypeptidases. cpbAg1 mRNA was mainly produced in the mosquito midgut, where it accumulated in unfed females and was rapidly down-regulated upon blood feeding. Annotation of the An. gambiae genome predicts twenty-three sequences coding for zinc-carboxypeptidases of which only two (cpbAg1 and cpbAg2) are expressed at a significant level in the mosquito midgut.     

Cloning and characterization of a gut-specific cathepsin L from the aphid Aphis gossypii

We have characterized proteinase activities in gut extracts from the cotton-melon aphid (Aphis gossypii Glover), an insect feeding strictly on protein-poor phloem. The major, if not exclusive, intestinal proteinases of this aphid are of the cysteine type. A cDNA has been cloned from a gut library and codes for the cysteine proteinase AgCatL, a cathepsin L-like cysteine proteinase. The AgCatL protein shows high sequence similarity with mammalian and some arthropod cathepsin L-like proteinases, but can be reliably distinguished from the secreted (digestive) proteinases identified in other arthropods. AgCatL is widely expressed in aphid intestinal cells. Immunolocalization of AgCatL showed an intense signal at the level of the anterior 'stomach' part of the midgut, and especially at intracellular localization. Although the precise role of AgCatL in aphid midgut physiology is still unclear, this enzyme could be involved in the processing of exogenous ingested polypeptides.     

Cowpea bruchid Callosobruchus maculatus uses a three-component strategy to overcome a plant defensive cysteine protease inhibitor

The soybean cysteine protease inhibitor, soyacystatin N (scN), negatively impacts growth and development of the cowpea bruchid, Callosobruchus maculatus[Koiwa et al. (1998) Plant J 14: 371-379]. However, the developmental delay and feeding inhibition caused by dietary scN occurred only during the early developmental stages (the 1st, 2nd and 3rd instars) of the cowpea bruchid. The 4th instar larvae reared on scN diet (adapted) exhibited rates of feeding and development which were comparable to those feeding on an scN-free diet (unadapted) prior to pupation. Total gut proteolytic capacity at this larval stage significantly increased in the scN-adapted insects. The elevated enzymatic activity was attributed to a differential expression of insect gut cysteine proteases (representing the major digestive enzymes), and of aspartic proteases. scN degradation by the gut extract was observed only in adapted bruchids, and this activity appeared to be a combined effect of scN-induced cysteine and aspartic proteases. Thirty cDNAs encoding cathepsin L-like cysteine proteases were isolated from insect guts, and they were differentially regulated by dietary scN. Our results suggest that the cowpea bruchid adapts to the challenge of scN by qualitative and quantitative remodelling of its digestive protease complement, and by activating scN-degrading protease activity.     

Releasing Digestive Enzymes by Brain Peptides in the Larvae of Pieris brassicae L. (Lepidoptera: Pieridae)

Neuropeptides synthesized by insect neurosecretory cells are engaged in several physiological process like immunity, feeding and reproduction so they deserve to be undertaken in subsequent experiments for discovery and manipulation regarding applied objectives. Due to importance of insect ability to utilize plant tissues, the present study was conducted to extract brain peptides in the fifth instar larvae of Pieris brassicae via High performance liquid chromatography and to identify their role in induction of digestive enzyme syntheses. Five peaks were determined and named as fractions 1, 2, 3, 4 and 5. The midguts of starved fifth larval instars were treated by each fraction of brain extract in vitro. The highest activities of α-amylase and lipase were found in the midgut treated by fractions 4 and 1, respectively while the fraction number 5 imposed the highest activities of glucosidases. Fraction 2 led to the highest activities of trypsin, chymotrypsin and elastase as the given serine proteinases as well as the two exopeptidases namely amino- and carboxypeptidases. Moreover, midgut treatments by different concentrations of TLCK (N-p-Tosyl-l-lysine methyl ester hydrochloride), TPCK (Tosyl phenylalanyl chloromethyl ketone), AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride) and Acarbose significantly decreased the activities of α-amylase, trypsin, chymotrypsin and elastase by the calculated IC₅₀ values of 0.287, 0.439, 0.288 and 0.361 mM, respectively. The findings demonstrated that different neuropeptides would be engaged in enzymatic release of P. brassicae hence, manipulation of peptide receptors can change physiological process of insects and opens a selective strategy to pest control.

     

Bowman‐Birk inhibitor affects pathways associated with energy metabolism in Drosophila melanogaster

Bowman‐Birk inhibitor (BBI) is toxic when fed to certain insects, including the fruit fly, Drosophila melanogaster. Dietary BBI has been demonstrated to slow growth and increase insect mortality by inhibiting the digestive enzymes trypsin and chymotrypsin, resulting in a reduced supply of amino acids. In mammals, BBI influences cellular energy metabolism. Therefore, we tested the hypothesis that dietary BBI affects energy‐associated pathways in the D. melanogaster midgut. Through microarray and metabolomic analyses, we show that dietary BBI affects energy utilization pathways in the midgut cells of D. melanogaster. In addition, ultrastructure studies indicate that microvilli are significantly shortened in BBI‐fed larvae. These data provide further insights into the complex cellular response of insects to dietary protease inhibitors.     

Digestion and assimilation of proline-containing peptides by rat intestinal brush border membrane carboxypeptidases. Role of the combined action of angiotensin-converting enzyme and carboxypeptidase P.

Two intestinal brush border membrane carboxypeptidases were found to participate in the sequential digestion of proline-containing peptides representing a novel mechanism of hydrolysis from the COOH terminus. NH2-blocked prolyl tripeptides were rapidly hydrolyzed by either brush border membrane angiotensin converting enzyme (ACE, dipeptidyl carboxypeptidase, E.C. 3.4.15.1) or carboxypeptidase P (E.C.3.4.12-) depending on the position of the proline residue. Furthermore, these two enzymes were found to participate in a concerted manner to sequentially degrade larger proline-containing pentapeptides from the COOH terminus. A brush border membrane associated neutral endopeptidase also participated in the hydrolysis of the prolyl pentapeptides. During in vivo intestinal perfusion, the NH2-blocked prolyl peptides were degraded and their constituent amino acids efficiently absorbed by the intestine. Furthermore, hydrolysis and absorption of these peptides could be dramatically suppressed by low concentrations of captopril, a specific inhibitor of ACE. These studies show that prolyl peptides are efficiently and sequentially hydrolyzed from the COOH terminus by the combined action of ACE and carboxypeptidase P, and that these enzymes may play an important role in the digestion and assimilation of proline-containing peptides.     

Molecular cloning and characterization of an endogenous digestive β‐glucosidase from the midgut of the fungus‐growing termite Macrotermes barneyi

β‐glucosidase from the midgut of the fungus‐growing termite Macrotermes barneyi was first cloned and characterized to gain a better understanding of cellulolytic systems in fungus‐growing termites. β‐glucosidase activity was proven to present primarily in the midgut of M. barneyi and two β‐glucosidases were partially purified from the midgut. Based on the N‐terminus sequence of one of the β‐glucosidases, a full‐length cDNA fragment of 1708 bp was obtained. This sequence encodes a 493 amino acid protein belonging to glycoside hydrolase family 1. Quantitative real‐time PCR analysis proved that the β‐glucosidase gene was primarily expressed in the midgut. β‐glucosidase was expressed heterologously and biochemically characterized. Results indicate that β‐glucosidase is an endogenous, midgut‐origin termite digestive enzyme. It may have applications in understanding the mechanism of lignocellulose degradation in fungus‐growing termites.     

Human mast cell carboxypeptidase. Purification and characterization.

A carboxypeptidase activity was recently identified in highly purified human lung mast cells and dispersed mast cells from skin. Using affinity chromatography with potato-tuber carboxypeptidase inhibitor as ligand, mast cell carboxypeptidase was purified to homogeneity from whole skin extracts. The purified enzyme yielded a single staining band of approximately 34,500 D on SDS-PAGE. Carboxypeptidase enzyme content estimated by determination of specific activity, was 0.5, 5, and 16 micrograms/10(6) mast cells from neonatal foreskin, adult facial skin, and adult foreskin, respectively. Human mast cell carboxypeptidase resembled bovine carboxypeptidase A with respect to hydrolysis of synthetic dipeptides and angiotensin I, but was distinguished from carboxypeptidase A in its inability to hydrolyze des-Arg9 bradykinin. The amino acid composition of human mast cell carboxypeptidase was similar to the composition of rat mast cell carboxypeptidase. The amino-terminal amino acid sequence of mast cell carboxypeptidase demonstrated 65% positional identity with human pancreatic carboxypeptidase B, but only 19% with human carboxypeptidase A. Thus, human mast cell carboxypeptidase is a novel member of the protein family of zinc-containing carboxypeptidases, in that it is functionally similar but not identical to bovine carboxypeptidase A, but has structural similarity to bovine and human pancreatic carboxypeptidase B.     

RNA interference and dietary inhibitors induce a similar compensation response in Tribolium castaneum larvae

Tribolium castaneum is a major agriculture pest damaging stored grains and cereal products. The T. castaneum genome contains 26 cysteine peptidase genes, mostly cathepsins L and B, and seven have a major role in digestion. We targeted the expression of the most highly expressed cathepsin L gene on chromosome 10, TC011001, by RNA interference (RNAi), using double‐stranded RNA (dsRNA) constructs of different regions of the gene (3′, middle, 5′ and entire coding regions). RNA sequencing and quantitation (RNA‐seq) was used to evaluate knockdown and specificity amongst the treatments. Overall, target gene expression decreased in all treatment groups, but was more severe and specific in dsRNA targeting the 3′ and entire coding regions, encoding the proteolytic active site in the enzyme. Additional cysteine cathepsin genes also were down‐regulated (off‐target effects), but some were up‐regulated in response to RNAi treatment. Notably, some serine peptidase genes were increased in expression, especially in dsRNA targeting 5′ and middle regions, and the response was similar to the effects of dietary cysteine protease inhibitors. We manually annotated these serine peptidase genes to gain insight into function and relevance to the RNAi study. The data indicate that T. castaneum larvae compensate for the loss of digestive peptidase activity in the larval gut, regardless of the mechanism of disruption.     

Gene duplication and subsequent functional diversification of sucrose hydrolase in Papilio xuthus

Sucrose is the main product of photosynthesis in plants, providing a rich carbon and energy source for the physiological growth and development of insects. In a previous study, we identified a novel sucrose hydrolase (SUH) in the larval midgut of moths. Intriguingly, there are two copies of Suh, namely Suh1 and Suh2, in several species of butterflies. However, the biochemical characteristics of SUHs in butterflies remain unclear. In this study, we found that this duplication and subsequent diversification produced two Suh genes in Papilio xuthus. These two PxSuh genes were significantly divergent in terms of their expression pattern and enzyme properties. PxSuh messenger RNA expression was highest during the larval stage, reduced in the prepupal and pupal stages and, for PxSuh1, slightly increased again in the adult. The observed levels of PxSuh2 were overall below those of PxSuh1 amongst the development stages examined. Compared with PxSUH2, which has maintained the original gene function of maltose hydrolysis, PxSUH1 exhibits substrate specificity for sucrose with an optimum enzyme activity occurring at an alkaline pH. The data show that PxSuh1 is evolutionarily adapted for effective functioning in an alkaline digestive system. Furthermore, we find that functional diversification of Suh facilitates P. xuthus to digestive carbohydrate of host plants. Thus, our findings offer new insights into the ecological and evolutionary adaptation of digestive enzymes in butterflies.