Characterization of a Glutenin-Specific Serine Proteinase of Sunn Bug Eurygaster integricepts Put.

Abstract

Glutenin hydrolyzing proteinases (GHPs) have been purified, by affinity chromatography, from wheat seeds damaged by the Sunn bug Eurygaster integriceps (Hemiptera, Scutelleridae). A 28 kDa protein was partially sequenced by mass spectrometry and Edman degradation which showed homology to serine proteases from various insects. Three full length clones were obtained from cDNA isolated from Sunn bug salivary glands using degenerate PCR based on the sequences obtained. The cleavage site of the protease was determined using recombinant and synthetic peptides and shown to be between the consensus hexapeptide and nonapeptide repeat motifs present in the high molecular weight subunits of wheat glutenin (PGQGQQ∧GYYPTSLQQ). Homology models were generated for the three proteinases identified in this study using the high resolution X-ray structure of a crayfish (Pontastacus leptodactylus) trypsin complexed with a peptide inhibitor as template (PDB accession 2F91). The novel specificity of this protease may find applications in both fundamental and applied studies.

Prolyl-specific peptidases for applications in food protein hydrolysis

“…The gel height corresponded inverse linearly to the enzyme activity. Konarev et al (2011) extracted a peptidase from salivary glands of E. integriceps which was able to hydrolyse glutenin. The protein extracts showed glutenin hydrolysis bands in an IEF zymogram.…”

Novel Detection Methods Used in Conjunction with Affinity Chromatography for the Identification and Purification of Hydrolytic Enzymes or Enzyme Inhibitors from Insects and Plants

“…For example, sunn pest is a highly specialized phytophage (in contrast to T. molitor); during co-evolution of this bug with wheat its digestive amylases developed decreased sensitivity to inhibitors from wheat grains that weakened negative role of this proteins (Konarev, 1981. The same can be true for the sunn pest digestive proteinases, insensitive to inhibitors from wheat grain (Konarev et al, 2011;Konarev and Fomicheva, 1991a) and also for proteinases of some other insect pests (Gatehouse, 2011).…”

“…in design of effective inhibitors for improving wheat resistance to pests and limiting proteinase activity in food technologies. Besides, the proteinase cleaves one of epitopes of glutenin defined by Wal et al (1999) as minimal epitope for the HLDQ8 form of celiac disease, so probably it can be used for gluten proteins modification to decrease their toxicity and autoimmune activity for gluten-sensitive people (Konarev et al, 2011).…”

“…the gluten-specific proteinase of sunn pest Eurygaster integriceps Put. and chymotrypsin inhibitor I from potato, Calbiochem), after loading of the sample the affinity gel was washed just with the solvent used in the sample (0.01 M ethanolamine with 0.2 M NaCl and 0.01% Triton X-100 pH 10) and eluted with water/0.01% (v/v) Triton X-100 followed by 0.01M HCl/0.01% (v/v) Triton X-100 (Konarev et al, 2011). Both eluates contained almost pure proteinase.…”

“…For the detection of glutenin-specific proteinase from the salivary glands of sunn pest E. integriceps, a layer of insoluble in acetic acid glutenin was attached to the plastic film as substrate (Konarev et al, 2011).…”