Identification and partial characterization of digestive proteinases from Triatoma phyllosoma pallidipennis stål (hemiptera: Reduviidae)

doi:10.1016/0305-0491(81)90006-7

Comparative Biochemistry and Physiology Part B: Comparative Biochemistry

Volume 70, Issue 4, 1981, Pages 713-717

https://doi.org/10.1016/0305-0491(81)90006-7 Get rights and content

Abstract

1.
1. The digestive midgut of Triatoma phyllosoma pallidipennis contains cathepsin B, lysosomal carboxypeptidase B, aminopeptidase and an unnamed proteinase that hydrolyzes haemoglobin at an optimum of pH 3.8.
2.
2. Aminopeptidase activity is located intracellularly in the gut wall while the other proteinase are located extracellularly in the gut lumen.
3.
3. These digestive proteinases are detected in all instars of the insect.
4.
4. This set of proteinases used for digesting blood is similar to that in Rhodnius prolixus and appears to be characteristic of the blood feeding Hemiptera.

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Cathepsin B and other thiol proteinases
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Cathepsin D and other carboxyl proteinases

There are more references available in the full text version of this article.

Cited by (24)

Effects of low temperatures on the survival and development of Callosobruchus chinensis (L.) (Coleoptera: Bruchidae) under different storage durations
2017, Journal of Asia-Pacific Entomology
Citation Excerpt :
Another, ionic activity which includes changes in oxygen solubility, increment of hydrogen ions concentration, and pH levels as a function of low temperature. Slight change in pH can bring huge variation in the enzyme activities of insects (Houseman and Downe, 1981). Insects developed after imbalanced chemical and physiological processes have to spend more time for optimum development.
Azuki bean weevil, Callosobruchus chinensis L. is a cosmopolitan pest of stored grain legumes. Larvae cause huge losses of energy and seeds in the storage. This study assessed the effects of short-term exposure to low-temperatures (4, 0, and − 4 °C) under seven durations of storage (1, 5, 10, 15, 20, 25, and 30 days) on the survival, development, and adult performance of C. chinensis in laboratory. The effects were studied on three life stages (egg, larva [2–3 instar], and pupa) of C. chinensis. The results showed that low-temperature treatments have significant effects on the life variables of C. chinensis. Among the stages, egg and pupa stages were most sensitive to low-temperature. The very low survival rates of eggs at − 4 °C, no pupa stages of C. chinensis survived after 10 days storage. Cold exposure at − 4 °C for < 10 days not only increased egg and pupa mortality, but the negative effects also transmitted to the larvae and pupa developed from those eggs (64.3 days) and least adults were emerged with minimum longevity. Increasing the duration of cold exposure further reduced survival rates, e.g. about 20% of the eggs survived after 15 days exposure at − 4 °C, but < 30% of them survived after 25 days treatment at 4 and 0 °C. Low-temperature treatments have also affected on the adult performances such as adult weight. However, there were not any negative effects of low-temperatures on the seed germination. The life variables of C. chinensis are discussed in terms of targeting particular susceptibilities to low-temperatures in different durations of storage as an alternative to chemical treatments.
Serine carboxypeptidases of Triatoma brasiliensis (Hemiptera, Reduviidae): Sequence characterization, expression pattern and activity localization
2014, Journal of Insect Physiology
Citation Excerpt :
There are some examples of carboxypeptidase B-like enzymes, but they seem to be less active and not well described (Terra and Ferreira, 1994, 2005). In triatomines, the digestive enzymes from R. prolixus (Garcia and Guimarães, 1979; Houseman and Downe, 1981a, 1983) and T. p. pallidipennis (Houseman and Downe, 1981b) were partially characterized, classified initially as carboxypeptidase A or B. Terra and Ferreira (1994) considered these enzymes as cysteine carboxypeptidases, as they were activated by DTT, inhibited by IAA and not affected by EDTA. So far, from insects only few SCPs have been characterized.
Using specific oligonucleotides, 5′- and 3′-RACE and sequencing, two cDNAs encoding serine carboxypeptidases (tbscp-1 and tbscp-2) from the midgut of the blood sucking heteropteran Triatoma brasiliensis were identified. Both cDNAs with an open reading frame of 1389 bp, encode serine carboxypeptidase precursors of 463 amino acid residues, which possess a signal peptide cleavage site after Ala19. Analysis of tbscp-1 and tbscp-2 genomic DNA showed an absence of introns in both sequences and the presence of a further intron-free SCP encoding gene (tbscp-2b). By reverse transcription polymerase chain reaction (RT-PCR), tbscp-1 and tbscp-2 transcript abundance was found similarly in fifth instar nymphs at different days after feeding (daf), high in the posterior midgut (small intestine), lower in the anterior midgut (stomach) and fat body and almost undetectable in the salivary glands. In the anterior, middle and posterior regions of the small intestine at 5 daf the transcript abundance of both genes was almost identical. Also in adult female and male insects at 5 daf both genes showed the strongest signal in the posterior midgut. Molecular modeling suggested that TBSCP-1 has carboxypeptidase D activity; activities against Hippuryl-Phenylalanine and Hippuryl-Arginine were also located at the posterior midgut, both were induced after blood feeding. Treatment of the posterior midgut extracts with the serine protease inhibitor PMSF strongly reduced carboxypeptidase activity. These findings suggest that triatomines might use serine carboxypeptidases, which are usually found in lysosomes, as digestive enzymes in the posterior midgut lumen, from which TBSCP-1 and TBSCP-2 are possible candidates to fulfill this function.
Intestinal aspartate proteases TiCatD and TiCatD2 of the haematophagous bug Triatoma infestans (Reduviidae): Sequence characterisation, expression pattern and characterisation of proteolytic activity
2012, Insect Biochemistry and Molecular Biology
Citation Excerpt :
The pH value of the midgut lumen of mosquitoes and lice is alkaline; the midgut lumen of triatomines is suggested to be acidic (Schaub, 2009). The acidic proteases cathepsin B and D, lysosomal carboxypeptidase and aminopeptidase have been characterised in the small intestine of R. prolixus, Triatoma phyllosoma pallidipennis and T. infestans by biochemical approaches using synthetic substrates and specific inhibitors (Garcia and Garcia, 1977; Houseman, 1978; Houseman and Downe, 1980, 1981a, b, 1983a; Kollien et al., 2004). Since the development of intestinal bacterial symbionts and Trypanosoma cruzi, the aetiological agent of Chagas disease which is transmitted by reduviid bugs, is affected by the nutritional stage and availability of digestion products in the gut lumen (Kollien and Schaub, 2000; Schaub, 2009; Garcia et al., 2010), understanding the digestive mechanisms provides important insights into vector-parasite/microbe interactions.
Two aspartate protease encoding complementary deoxyribonucleic acids (cDNA) were characterised from the small intestine (posterior midgut) of Triatoma infestans and the corresponding genes were named TiCatD and TiCatD2. The deduced 390 and 393 amino acid sequences of both enzymes contain two regions characteristic for cathepsin D proteases and the conserved catalytic aspartate residues forming the catalytic dyad, but only TiCatD2 possesses an entire C-terminal proline loop. The amino acid sequences of TiCatD and TiCatD2 show 51–58% similarity to other insect cathepsin D-like proteases and, respectively, 88 and 58% similarity to the aspartate protease ASP25 from T. infestans available in the GenBank database. In phylogenetic analysis, TiCatD and ASP25 clearly separate from cathepsin D-like sequences of other insects, TiCatD2 groups with cathepsin D-like proteases with proline loop. The activity of purified TiCatD and TiCatD2 was highest between pH 2 and 4, respectively, and hence, deviate from the pH values of the lumen of the small intestine, which varied in correlation with the time after feeding between pH 5.2 and 6.7 as determined by means of micro pH electrodes. Both cathepsins, TiCatD and TiCatD2, were purified from the lumen of the small intestine using pepstatin affinity chromatography and identified by nanoLC-ESI-MS/MS analysis as those encoded by the cDNAs. The proteolytic activity of the purified enzymes is highest at pH 3 and the respective genes are expressed in the both regions of the midgut, stomach (anterior midgut) and small intestine, not in the rectum, salivary glands, Malpighian tubules or haemocytes. The temporal expression pattern of both genes in the small intestine after feeding revealed a feeding dependent regulation for TiCatD but not for TiCatD2.
Insecticidal effect of Canavalia ensiformis major urease on nymphs of the milkweed bug Oncopeltus fasciatus and characterization of digestive peptidases
2011, Insect Biochemistry and Molecular Biology
Citation Excerpt :
This wide diversity implies multiple functions of insect enzymes, including involvement in developmental processes besides interactions with food components. Hemipteran proteolytic enzymes are predominantly acidic, belonging to aspartic and cysteine peptidases classes (Houseman, 1978; Houseman and Downe, 1981, 1982a,b, 1983). The role played by these enzymes, usually located intracellularly, in extracellular protein digestion in insects (Cristofoletti et al., 2003; Houseman et al., 1984; Terra and Ferreira, 1994) is probably due to evolution and adaptation (Cristofoletti et al., 2003; Houseman et al., 1985).
Jackbean (Canavalia ensiformis) ureases are entomotoxic upon the release of internal peptides by insect’s digestive enzymes. Here we studied the digestive peptidases of Oncopeltus fasciatus (milkweed bug) and its susceptibility to jackbean urease (JBU). O. fasciatus nymphs fed urease showed a mortality rate higher than 80% after two weeks. Homogenates of midguts dissected from fourth instars were used to perform proteolytic activity assays. The homogenates hydrolyzed JBU in vitro, yielding a fragment similar in size to known entomotoxic peptides. The major proteolytic activity at pH 4.0 upon protein substrates was blocked by specific inhibitors of aspartic and cysteine peptidases, but not significantly affected by inhibitors of metallopeptidases or serine peptidases. The optimal activity upon N-Cbz-Phe-Arg-MCA was at pH 5.0, with complete blockage by E-64 in all pH tested. Optimal activity upon Abz–AIAFFSRQ–EDDnp (a substrate for aspartic peptidases) was detected at pH 5.0, with partial inhibition by Pepstatin A in the pH range 2–8. Fluorogenic substrates corresponding to the N- and C-terminal regions flanking a known entomotoxic peptide within urease sequence were also tested. While the midgut homogenate did not hydrolyze the N-terminal peptide, it cleaved the C-terminal peptide maximally at pH 4.0–5.0, and this activity was inhibited by E-64 (10 μM). The midgut homogenate was submitted to ion-exchange chromatography followed by gel filtration. A 22 kDa active fraction was obtained, resolved in SDS-PAGE (12%), the corresponding band was in-gel digested by trypsin, the peptides were analyzed by mass spectrometry, retrieving a cathepsin L protein. The purified cathepsin L was shown to have at least two possible cleavage sites within the urease sequence, and might be able to release a known insecticidal peptide in a single or cascade event. The results suggest that susceptibility of O. fasciatus nymphs to jackbean urease is, like in other insect models, due mostly to limited proteolysis of ingested protein and subsequent release of entomotoxic peptide(s) by cathepsin-like digestive enzymes.
Partial purification and characterization of Helicoverpa armigera (Lepidoptera: Noctuidae) active aminopeptidase secreted in midgut
2010, Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology
We report the partial purification to apparent homogeneity of a soluble aminopeptidase (EC 3.4.11.1) from midgut of Helicoverpa armigera larvae, which preferentially degraded Leucine p-nitroanilide (LpNA). After midgut isolation, extraction and precipitation of soluble proteins with acetone, proteins were purified in two consecutive steps including gel filtration and ion-exchange chromatographies. Aminopeptidase activity was increased 8.95 fold after gel filtration chromatography. The purified enzyme appeared as single band with a molecular mass of ∼ 112 kDa in SDS-PAGE, with a pH optimum of 7.0. Zymogram analysis revealed two enzymatically active proteinases using LpNA as substrate. The optimal temperature of aminopeptidase activity was 50–60 °C. The enzyme was characterized as metalloprotease as it was strongly inhibited by 1,10 phenanthroline. Strong inhibition was also being observed using the specific aminopeptidase inhibitor bestatin. Heavy metal ions, EDTA and cysteine strongly inhibited the enzyme, while Ca^+ 2, Mn^+ 2 and Mg^+ 2 somewhat stimulated aminopeptidase activity. Besides LpNA, the purified aminopeptidase also cleaved with decreasing activity ApNA, VpNA and BApNA. Study could be helpful to understand the mechanism of action of N-terminal degrading enzymes and also important is to further study the differential interaction of Bacillus thuringiensis cry insecticidal toxin with midgut receptor of insects.
Partial purification and characterization of midgut leucyl aminopeptidase of Morimus funereus (Coleoptera: Cerambycidae) larvae
2003, Comparative Biochemistry and Physiology - B Biochemistry and Molecular Biology
Exopeptidases of Morimus funereus larvae were partially purified and characterized. Specific leucyl aminopeptidase (LAP) activity was increased eight-fold by gel filtration of the crude midgut extract. The partially purified LAP had a molecular mass greater than 100 kDa with pH optima from 7.0–9.0 and no strict substrate specificity. M. funereus LAP preferentially hydrolyzed p-nitroanilides with hydrophobic amino acids in the active site, with a K_m for leucine-p-nitroanilide of 0.21 mM. Zymogram analysis of an electropherogram obtained by native polyacrylamide gel electrophoresis revealed four enzymatically active proteinases using leucine-p-nitroanilide and methionine-p-nitroanilide as substrates and two enzymatically active proteinases using lysine-p-nitroanilide as a substrate. Although the optimal temperature of LAP activity was 40 °C, the enzyme was active over a broad temperature range from 2 to 60 °C. Among a number of inhibitors tested, heavy metals and 1,10-phenanthroline completely inhibited the enzyme, while methanol, ethanol and EGTA stimulated somewhat LAP activity.

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Comparative Biochemistry and Physiology Part B: Comparative Biochemistry

Abstract

References (16)

A new assay of cathepsin B1 and other thiol proteinases

Analyt. Biochem.

A simple colorimetric method for the determination of protein

Analyt. Biochem.

The preparation and properties of two chromogenic substrates for trypsin

Archs Biochem. Biophys.

Endoproteinase activity in the posterior midgut of Rhodnius prolixus Stål (Hemiptera: Reduviidae)

Insect Biochem.

The control of protease synthesis in the intestine of adults of Rhodnius prolixus

J. Insect Physiol.

Colorimetric procedure for amino acids

Cathepsin B and other thiol proteinases

Cathepsin D and other carboxyl proteinases

Cited by (24)

Effects of low temperatures on the survival and development of Callosobruchus chinensis (L.) (Coleoptera: Bruchidae) under different storage durations

Serine carboxypeptidases of Triatoma brasiliensis (Hemiptera, Reduviidae): Sequence characterization, expression pattern and activity localization

Intestinal aspartate proteases TiCatD and TiCatD2 of the haematophagous bug Triatoma infestans (Reduviidae): Sequence characterisation, expression pattern and characterisation of proteolytic activity

Insecticidal effect of Canavalia ensiformis major urease on nymphs of the milkweed bug Oncopeltus fasciatus and characterization of digestive peptidases

Partial purification and characterization of Helicoverpa armigera (Lepidoptera: Noctuidae) active aminopeptidase secreted in midgut

Partial purification and characterization of midgut leucyl aminopeptidase of Morimus funereus (Coleoptera: Cerambycidae) larvae

General paperIdentification and partial characterization of digestive proteinases from Triatoma phyllosoma pallidipennis stål (hemiptera: Reduviidae)

Abstract

Analyt. Biochem.

Analyt. Biochem.

Archs Biochem. Biophys.

Insect Biochem.

J. Insect Physiol.

Cathepsin B and other thiol proteinases

Cathepsin D and other carboxyl proteinases

General paper
Identification and partial characterization of digestive proteinases from Triatoma phyllosoma pallidipennis stål (hemiptera: Reduviidae)