Prolyl Endoprotease Research Articles

Characterization of a prolyl endoprotease from Eurygaster integriceps puton (Sunn pest) infested wheat

Sunn pest, Eurygaster integriceps, Puton, infested and uninfested wheat seeds were obtained from the International Center for Agriculture Research in the Dry Areas (ICARDA), Aleppo, Syria, with the primary objective to identify the type of enzyme deposited by the Sunn pest on the wheat responsible for the gluten degradation. Enzyme levels were extremely low due to the enzyme being secreted by the insect in localized areas on the seed. Only extract from the infested wheat contained glutenase activity. Anion exchange, Cu(2+) sepharose, and gel filtration chromatography were used to partially purify and enrich protein samples from both infested wheat and uninfested wheat. An SDS-gluten assay was used to show gluten specificity while a commercially available chromogenic proline peptide, benzyloxycarbonyl-Gly-Pro-p-nitroanalide (ZGPpNA), was utilized to identify fractions containing the active proline specific enzyme activity and to determine Michaelis-Menten kinetics. Despite low levels of enzyme on the infested wheat, the enzyme was partially purified and enriched exhibiting a specific activity of 4.5 U/mg of total protein for gluten in a SDS gluten assay (1 U of enzyme activity was defined as the decrease in gel height in millimeters in 1 h) and exhibited a high-affinity Km of 65 microM for ZGPpNA, cleaving at the carboxy terminus of the proline residue. The enzyme exhibited optimal activity between pH 8 and 10.0 at temperatures between 20 degrees and 35 degrees C. The enzyme was identified to be a prolyl endoprotease.

379 Can Prolyl Endoprotease Enzyme Treatment Mitigate the Toxic Effect of Gluten in Coeliac Patients?

379 Can Prolyl Endoprotease Enzyme Treatment Mitigate the Toxic Effect of Gluten in Coeliac Patients?

Identification of N‐glycosylation in prolyl endoprotease from Aspergillus niger and evaluation of the enzyme for its possible application in proteomics

An acidic prolyl endoprotease from Aspergillus niger was isolated from the commercial product Brewers Clarex to evaluate its possible application in proteomics. The chromatographic purification yielded a single protein band in sodium dodecyl sulfate polyacrylamide gel electrophoresis providing an apparent molecular mass of 63 kDa and a broad peak (m/z 58,061) in linear matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) indicating the glycoprotein nature of the enzyme. Indeed, a colorimetric assessment with phenol and sulfuric acid showed the presence of neutral sugars (9% of weight). The subsequent treatment with N-glycosidase F released a variety of high-mannose type N-glycans, which were successfully detected using MALDI-TOF MS. MALDI-TOF/TOF tandem MS analysis of glycopeptides from a tryptic digest of prolyl endoprotease unraveled the identity of the N-glycosylation site in the primary structure. The data obtained also show that the enzyme is present in its processed form, i.e. without putative signal and propeptide parts. Spectrophotometric measurements demonstrated optimal activity at pH 4.0-4.5 and also high thermostability for the cleavage at the C-terminal part of proline residues. In-solution digestion of standard proteins (12-200 kDa) allowed to evaluate the cleavage specificity. The enzyme acts upon proline and alanine residues, but there is an additional minor cleavage at some other residues like Gly, Leu, Arg, Ser and Tyr. The digestion of a honeybee peptide comprising six proline residues (apidaecin 1A) led to the detection of specific peptides terminated by proline as it was confirmed by MALDI postsource decay analysis.

Efficient degradation of gluten by a prolyl endoprotease in a gastrointestinal model: implications for coeliac disease

Background:Coeliac disease is caused by an immune response to gluten. As gluten proteins are proline rich they are resistant to enzymatic digestion in the gastrointestinal tract, a property that probably...

Aspergillus niger prolyl endoprotease as a treatment for celiac disease

Aspergillus niger prolyl endoprotease as a treatment for celiac disease

Highly efficient gluten degradation with a newly identified prolyl endoprotease: implications for celiac disease

Celiac disease is a T cell-driven intolerance to wheat gluten. The gluten-derived T cell epitopes are proline-rich and thereby highly resistant to proteolytic degradation within the gastrointestinal tract. Oral supplementation with prolyl oligopeptidases has therefore been proposed as a potential therapeutic approach. The enzymes studied, however, have limitations as they are irreversibly inactivated by pepsin and acidic pH, both present in the stomach. As a consequence, these enzymes will fail to degrade gluten before it reaches the small intestine, the site where gluten induces inflammatory T cell responses that lead to celiac disease. We have now determined the usefulness of a newly identified prolyl endoprotease from Aspergillus niger for this purpose. Gluten and its peptic/tryptic digest were treated with prolyl endoprotease, and the destruction of the T cell epitopes was tested using mass spectrometry, T cell proliferation assays, ELISA, reverse-phase HPLC, SDS-PAGE, and Western blotting. We observed that the A. niger prolyl endoprotease works optimally at 4-5 pH, remains stable at 2 pH, and is completely resistant to digestion with pepsin. Moreover, the A. niger-derived enzyme efficiently degraded all tested T cell stimulatory peptides as well as intact gluten molecules. On average, the endoprotease from A. niger degraded gluten peptides 60 times faster than a prolyl oligopeptidase. Together these results indicate that the enzyme from A. niger efficiently degrades gluten proteins. Future studies are required to determine if the prolyl endoprotease can be used as an oral supplement to reduce gluten intake in patients.