Yakov E. Dunaevsky

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.

LOCALIZATION OF A METALLOPROTEINASE AND ITS INHIBITOR IN THE PROTEIN BODIES OF BUCKWHEAT SEEDS

Cotyledons of dry buckwheat (Fagopyrum esculentum Moench) seeds were used to study the cellular localization of a metalloproteinase which performs in vitro the initial limited proteolysis of the main storage protein of the seed, and of its proteinaceous inhibitor. Fractions of complex protein bodies (PB 1) and of the cytoplasm and membrane material (CMM) were obtained by fractionating cotyledons in a mixture of acetone and CCl4. The greater part of the metalloproteinase activity was found to be localized in the PB 1 fraction, with a lesser amount in the CMM fraction, whereas the metalloproteinase inhibitor was localized almost entirely in the PB 1 fraction. The data obtained indicate that the complex protein bodies of dry buckwheat seeds contain the components of the proteolytic system responsible for the initial degradation of the main storage protein — the 13S globulin — of buckwheat seeds, i.e. 13S globulin, the metalloproteinase, and its inhibitor. This confirms that it is possibile for the metalloproteinase to perform a controlled proteolysis of the 13S globulin in vivo. The effect of divalent cations on the degradation of the 13S globulin was also studied. A mechanism is discussed whereby the proteolysis of 13S globulin is initiated by divalent cations released as a result of phytin decationization during seedling growth.

Buckwheat trypsin inhibitor with helical hairpin structure belongs to a new family of plant defence peptides

A new peptide trypsin inhibitor named BWI-2c was obtained from buckwheat (Fagopyrum esculentum) seeds by sequential affinity, ion exchange and reversed-phase chromatography. The peptide was sequenced and found to contain 41 amino acid residues, with four cysteine residues involved in two intramolecular disulfide bonds. Recombinant BWI-2c identical to the natural peptide was produced in Escherichia coli in a form of a cleavable fusion with thioredoxin. The 3D (three-dimensional) structure of the peptide in solution was determined by NMR spectroscopy, revealing two antiparallel α-helices stapled by disulfide bonds. Together with VhTI, a trypsin inhibitor from veronica (Veronica hederifolia), BWI-2c represents a new family of protease inhibitors with an unusual α-helical hairpin fold. The linker sequence between the helices represents the so-called trypsin inhibitory loop responsible for direct binding to the active site of the enzyme that cleaves BWI-2c at the functionally important residue Arg(19). The inhibition constant was determined for BWI-2c against trypsin (1.7×10(-1)0 M), and the peptide was tested on other enzymes, including those from various insect digestive systems, revealing high selectivity to trypsin-like proteases. Structural similarity shared by BWI-2c, VhTI and several other plant defence peptides leads to the acknowledgement of a new widespread family of plant peptides termed α-hairpinins.

Use of buckwheat seed protease inhibitor gene for improvement of tobacco and potato plant resistance to biotic stress.

The possibility to use agrobacterial transformation of leaf discs to produce resistance to bacterial infections in tobacco and potato plants by introduction of a single gene encoding the serine proteinase inhibitor BWI-1a (ISP) from buckwheat seeds is shown. All studied PCR-positive transgenic plants exhibited antibacterial activity in biotests. It was shown that the presence of just a single gene of serine proteinase inhibitor provides sufficient protection at least against two bacterial phytopathogens, Pseudomonas syringae pv. tomato and Clavibacter michiganensis sbsp. michiganensis. The biotest including tobacco plant infection by the white wings butterfly in the green house has also demonstrated the existence of protective effect in transgenic tobacco plants. Significant genotypic variations in the protection efficiency were found between members of different genera of the same family (potato and tobacco) as well as between different lines of the same species. Northern blot analysis of four transgenic potato lines and three tobacco lines transformed by a vector plasmid containing the ISP gene of serine proteinases BWI-1a from buckwheat seeds has shown the presence of the expected size mRNA transcript.

Cationic inhibitors of serine proteinases from buckwheat seeds.

Preparations of low molecular weight protein inhibitors of serine proteinases have been obtained from buckwheat (Fagopyrum esculentum) seeds by chromatography of seed extract on trypsin-Sepharose 4B, Mono-Q, and Mono-S ion exchangers (FPLC regime). Their molecular masses, determined by mass spectrometry, were 5203 (BWI-1c), 5347 (BWI-2c), 7760 (BWI-3c), and 6031 daltons (BWI-4c). All of the inhibitors possess high pH- and thermal stability in the pH range 2-12. In addition to trypsin, BWI-3c and BWI-4c inhibited chymotrypsin and subtilisin-like bacterial proteases. The N-terminal sequences of all of the inhibitors were determined: BWI-1c (23 residues), BWI-2c (33 residues), BWI-3c (18 residues), and BWI-4c (20 residues). In their physicochemical properties and N-terminal amino acid sequences, the buckwheat seed trypsin inhibitors BWI-3c and BWI-4c appear to belong to potato proteinase inhibitor I family.

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.

New protease inhibitors from buckwheat seeds: properties, partial amino acid sequences and possible biological role.

Abstract Preparations of new low molecular weight protein inhibitors of serine proteinases have been obtained from buckwheat Fagopyrum esculentum seeds by chromatography of seed extracts on trypsinSepharose 4B, MonoQ and MonoS ionexchangers. Their molecular masses, determined by mass spectrometry, were equal to 5203 (BWI-1c), 5347 (BWI-2c), 7760 (BWI-3c) and 6031 daltons (BWI-4c). All inhibitors possessed high pHstability in the pH range 212 and thermostability. In addition to trypsin, BWI-3c and BWI-4c inhibitors inhibited chymotrypsin and subtilisinlike proteases. The inhibition constants (K) for trypsin, chymotrypsin and subtilisin by the studied inhibitors were determined. The Nterminal sequences of all inhibitors were established: BWI-1c (23 residues), BWI-2c (33 residues), BWI-3c (18 residues) and BWI-4c (20 residues). According to the physicochemical properties and Nterminal amino acid sequences, buckwheat seed protease inhibitors BWI-3c and BWI-4c are suggested to belong to the potato proteinase inhibitor I family.

Protease inhibitors in buckwheat seeds : Comparison of anionic and cationic inhibitors

Summary Protease inhibitors in buckwheat seeds are separated into two main groups — anionic and cationic inhibitors, according to their behaviour on ion-exchange chromatography. Three anionic inhibitors (BWI-la, BWI-2a and BWI-4a) and two cationic (BWI-2c and BWI-4c) were purified to homogeneity and characterized. Molecular masses of anionic inhibitors were in the range 7.7–9.2 kD and of cationic 6.0 kD. Both anionic and cationic inhibitors were highly pH- and thermostable. All anionic and cationic inhibitors inhibited trypsin. In addition to trypsin BWI-la and BWI-2a inhibited chymotrypsin, however, less effectively. Cationic inhibitor BWI-4c besides trypsin and chymotrypsin inhibited also bacterial subtilisin. Inhibitors BWI-la, BWI-2a, BWI-4a and BWI-2c contain an Arg residue at the reactive site whereas BWI-4c contains a Lys residue. According to determined amino acid sequences anionic inhibitors BWI-1a, BWI-2a and BWI-4a belong to the potato proteinase inhibitor I family.

Isolation and properties of anionic protease inhibitors from buckwheat seeds

Three protease inhibitors (BWI‐1, BWI‐2 and BWI‐4) from buckwheat seeds were purified to homogeneity and characterized. Their molecular masses were 7.7‐9.2 kDa according to gel‐filtration and mass spectrometry. Amino acid analysis revealed a high content of glutamic acid and valine and a low content of isoleucine, aromatic and sulfur‐containing amino acids. Data illustrating the temperature and the pH stability of the inhibitors are presented. Each of the inhibitors formed a inhibitor complex with trypsin in a molar ratio 1:1 and contained an Arg residue at the reactive site. In addition to trypsin, BWI‐1 and BWI‐2 inhibited chymotrypsin, however, less effectively. None of the isolated inhibitors suppressed activity of papain, leukocyte elastase, pepsin and subtilisin.

Fungal inhibitors of proteolytic enzymes: Classification, properties, possible biological roles, and perspectives for practical use

Peptidase inhibitors are ubiquitous regulatory proteins controlling catalytic activity of proteolytic enzymes. Interest in these proteins increased substantially after it became clear that they can be used for therapy of various important diseases including cancer, malaria, and autoimmune and neurodegenerative diseases. In this review we summarize available data on peptidase inhibitors from fungi, emphasizing their properties, biological role, and possible practical applications of these proteins in the future. A number of fungal peptidase inhibitors with unique structure and specificity of action have no sequence homology with other classes of peptidase inhibitors, thus representing new and specific candidates for therapeutic use. The main classifications of inhibitors in current use are considered. Available data on structure, mechanisms and conditions of action, and diversity of functions of peptidase inhibitors of fungi are analyzed. It is mentioned that on one side the unique properties of some inhibitors can be used for selective inhibition of peptidases responsible for initiation and development of pathogenic processes. On the other side, general inhibitory activity of other inhibitors towards peptidases of various catalytic classes might be able to provide efficient defense of transgenic plants against insect pests by overcoming compensatory synthesis of new peptidases by these pests in response to introduction of a fungal inhibitor. Together, the data analyzed in this review reveal that fungal inhibitors extend the spectrum of known peptidase inhibitors potentially suitable for use in medicine and agriculture.