D.P. Zhuzhikov

Digestive Proteinases of Yellow Mealworm (Tenebrio molitor) Larvae: Purification and Characterization of a Trypsin-Like Proteinase

A new trypsin-like proteinase was purified to homogeneity from the posterior midgut of Tenebrio molitor larvae by ion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Superdex-75. The isolated enzyme had molecular mass of 25.5 kD and pI7.4. The enzyme was also characterized by temperature optimum at 55°C, pH optimum at 8.5, and Km value of 0.04 mM (for hydrolysis of Bz-Arg-pNA). According to inhibitor analysis the enzyme is a trypsin-like serine proteinase stable within the pH range of 5.0–9.5. The enzyme hydrolyzes peptide bonds formed by Arg or Lys residues in the P1 position with a preference for relatively long peptide substrates. The N- terminal amino acid sequence, IVGGSSISISSVPXQIXLQY, shares 50–72% identity with other insect trypsin-like proteinases, and 44–50% identity to mammalian trypsins. The isolated enzyme is sensitive to inhibition by plant proteinase inhibitors and it can serve as a suitable target for control of digestion in this stored product pest.

DIGESTIVE PROTEOLYSIS ORGANIZATION IN TWO CLOSELY RELATED TENEBRIONID BEETLES: RED FLOUR BEETLE (Tribolium castaneum) AND CONFUSED FLOUR BEETLE (Tribolium confusum)

The spectra of Tribolium castaneum and T. confusum larval digestive peptidases were characterized with respect to the spatial organization of protein digestion in the midgut. The pH of midgut contents in both species increased from 5.6-6.0 in the anterior to 7.0-7.5 in the posterior midgut. However, the pH optimum of the total proteolytic activity of the gut extract from either insect was pH 4.1. Approximately 80% of the total proteolytic activity was in the anterior and 20% in the posterior midgut of either insect when evaluated in buffers simulating the pH and reducing conditions characteristic for each midgut section. The general peptidase activity of gut extracts from either insect in pH 5.6 buffer was mostly due to cysteine peptidases. In the weakly alkaline conditions of the posterior midgut, the serine peptidase contribution was 31 and 41% in T. castaneum and T. confusum, respectively. A postelectrophoretic peptidase activity assay with gelatin also revealed the important contribution of cysteine peptidases in protein digestion in both Tribolium species. The use of a postelectrophoretic activity assay with p-nitroanilide substrates and specific inhibitors revealed a set of cysteine and serine endopeptidases, 8 and 10 for T. castaneum, and 7 and 9 for T. confusum, respectively. Serine peptidases included trypsin-, chymotrypsin-, and elastase-like enzymes, the latter being for the first time reported in Tenebrionid insects. These data support a complex system of protein digestion in the Tribolium midgut with the fundamental role of cysteine peptidases.

Cysteine digestive peptidases function as post-glutamine cleaving enzymes in tenebrionid stored-product pests.

The major storage proteins in cereals, prolamins, have an abundance of the amino acids glutamine and proline. Storage pests need specific digestive enzymes to efficiently hydrolyze these storage proteins. Therefore, post-glutamine cleaving peptidases (PGP) were isolated from the midgut of the stored-product pest, Tenebrio molitor (yellow mealworm). Three distinct PGP activities were found in the anterior and posterior midgut using the highly-specific chromogenic peptide substrate N-benzyloxycarbonyl-L-Ala-L-Ala-L-Gln p-nitroanilide. PGP peptidases were characterized according to gel elution times, activity profiles in buffers of different pH, electrophoretic mobility under native conditions, and inhibitor sensitivity. The results indicate that PGP activity is due to cysteine and not serine chymotrypsin-like peptidases from the T. molitor larvae midgut. We propose that the evolutionary conservation of cysteine peptidases in the complement of digestive peptidases of tenebrionid stored-product beetles is due not only to the adaptation of insects to plants rich in serine peptidase inhibitors, but also to accommodate the need to efficiently cleave major dietary proteins rich in glutamine.

Properties of post-proline cleaving enzymes from Tenebrio molitor

Two post-proline cleaving peptidases PPCP1 and PPCP2 with molecular masses of 101 and 63 kDa, respectively, hydrolyzing Z-AlaAlaPro-pNA were isolated for the first time from the larval midgut of the yellow mealworm Tenebrio molitor and characterized. PPCP1 was active only in acidic media, with a maximum at pH 5.6, whereas PPCP2, both in acidic and alkaline media with a maximum at pH 7.9. Using inhibitory analysis, both PPCP1 and PPCP2 were shown to belong to serine peptidases. The data obtained indicate that a Cys residue is located close to the PPCP2 substrate binding site. Z-Pro-prolinal, a specific inhibitor of prolyl oligopeptidases, completely inhibited PPCP2 and partially PPCP1. The substrate specificities of the isolated enzymes were studied. Z-Ala-Ala-Pro-pNA was the best substrate for PPCP1, and Z-Ala-Pro-pNA, for PPCP2. The combination of the properties allows characterization of PPCP2 as a proplyl oligopeptidase.

Complex of digestive proteinases of Galleria mellonella Caterpillars: composition, properties, and limited proteolysis of Bacillus thuringiensis 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.

Toxin-binding proteins isolated from yellow mealworm Tenebrio molitor and wax moth Galleria mellonella

A 67-kDa protein that can specifically bind the activated Cry9A endotoxin under ligand-blotting conditions was purified from midgut epithelium apical membranes of wax moth Galleria mellonella by affinity chromatography. N-Terminal amino acid sequencing enabled identification of this protein as aminopeptidase N. In similar experiments, 66- and 58-kDa proteins specific to endotoxin Cry3A were isolated from the midgut epithelium apical membranes of Tenebrio molitor larvae. Mass spectrometry showed close similarity of the 58-kDa protein to the Tenebrio molitor α-amylase.

Dipeptidyl peptidase 4 – An important digestive peptidase in Tenebrio molitor larvae

Dipeptidyl peptidase 4 (DPP 4) is a proline specific serine peptidase that plays an important role in different regulatory processes in mammals. In this report, we isolated and characterized a unique secreted digestive DPP 4 from the anterior midgut of a stored product pest, Tenebrio molitor larvae (TmDPP 4), with a biological function different than that of the well-studied mammalian DPP 4. The sequence of the purified enzyme was confirmed by mass-spectrometry, and was identical to the translated RNA sequence found in a gut EST database. The purified peptidase was characterized according to its localization in the midgut, and substrate specificity and inhibitor sensitivity were compared with those of human recombinant DPP 4 (rhDPP 4). The T. molitor enzyme was localized mainly in the anterior midgut of the larvae, and 81% of the activity was found in the fraction of soluble gut contents, while human DPP 4 is a membrane enzyme. TmDPP 4 was stable in the pH range 5.0-9.0, with an optimum activity at pH 7.9, similar to human DPP 4. Only specific inhibitors of serine peptidases, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, suppressed TmDPP 4 activity, and the specific dipeptidyl peptidase inhibitor vildagliptin was most potent. The highest rate of TmDPP 4 hydrolysis was found for the synthetic substrate Arg-Pro-pNA, while Ala-Pro-pNA was a better substrate for rhDPP 4. Related to its function in the insect midgut, TmDPP 4 efficiently hydrolyzed the wheat storage proteins gliadins, which are major dietary proteins of T. molitor.

Midgut proteinases of the cockroachNauphoeta cinerea

The study of properties of proteolytic enzymes in midgut of imago of the cockroachNauphoeta cinerea Oliv. Has been carried out. It is shown that the total proteolytic activity of digestive proteases, measured with azocasein as substrate, is maximal at pH 11.5 both in the anterior and in the posterior parts of the midgut. The predominant part of this activity (67%) was present in the posterior part. Fractionation of preparation from the posterior part on a column with Sephadex G-50 and subsequent analysis of the activity in the obtained fractions using specificp-nitroanilide substrates and effects of activators and inhibitors of active center have allowed revealing three types of activity of serine proteinases and one cysteine proteinase. No activity of aspartic and metalloproteinases were detected. Among serine proteinases, one trypsin-like, one unusual SHdependent serine, one chymotrypsin-like, and not less than two enzymes hydrolyzing specific substrate of subtilisin were established. The fractionation of the preparation from the anterior part has allowed revealing only three proteinases that were similar by their properties to cysteine, SHdependent serine, and chymotrypsin-like ones in the posterior part of midgut. Their activity was lower in the anterior, than in the posterior part of the midgut. The probable causes of the low proteolytic activity in the anterior part of the midgut are discussed.

Prolidase is a critical enzyme for complete gliadin digestion in Tenebrio molitor larvae

Prolidase is a proline-specific metallopeptidase that cleaves imidodipeptides with C-terminal Pro residue. Prolidase was purified and characterized from the Tenebrio molitor larval midgut. The enzyme was localized in the soluble fraction of posterior midgut tissues, corresponding to a predicted cytoplasmic localization of prolidase according to the structure of the mRNA transcript. Expression of genes encoding prolidase and the major digestive proline-specific peptidase (PSP)-dipeptidyl peptidase 4-were similar. The pH optimum of T. molitor prolidase was 7.5, and the enzyme was inhibited by Z-Pro, indicating that it belongs to type I prolidases. In mammals, prolidase is particularly important in the catabolism of a proline-rich protein-collagen. We propose that T. molitor larval prolidase is a critical enzyme for the final stages of digestion of dietary proline-rich gliadins, providing hydrolysis of imidodipeptides in the cytoplasm of midgut epithelial cells. We propose that the products of hydrolysis are absorbed from the luminal contents by peptide transporters, which we have annotated in the T. molitor larval gut transcriptome. The origin of prolidase substrates in the insect midgut is discussed in the context of overall success of grain feeding insects.

Study of Inhibitors of Proteinases in the Midgut Anterior Part of the Cockroach Nauphoeta cinerea

The study of proteinase inhibitors in the midgut of the omnivorous cockroach Nauphoeta cinerea was carried out under conditions excluding their food origin. One trypsin inhibitor of molecular mass of 8.0 kDa and three subtilisin inhibitors of molecular masses of 13.0, 8.0, and 4.5 kDa were found in the protein preparations, using Sephadex G-50 fractionation. 94% of the activity of the both inhibitor types were located in the anterior midgut part. Using a high performance liquid chromatography on Mono Q column, the preparation of trypsin inhibitor was purified 120 times. Its isoelectric point was to 4.3. The inhibitor lost a part of its activity both under acidic and, especially, under alkaline conditions and was completely inactivated at pH 10. The studied inhibitors inhibited effectively activities of trypsin-like and subtilisin-like proteinases from the cockroach posterior midgut part. The possible physiological role of the proteinase inhibitors and, particularly, their participation in regulation of digestion in the midgut of N. cinerea are discussed.