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

Abstract

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.

Proteases: Nature’s Destroyers and the Drugs that Stop Them

“…First, libraries of small molecules have been screened for inhibition of specific proteases [6,28]. Screening libraries in a high-throughput assay gives hits quickly.…”

“…Proteases account for ~2% of the human genome with 553 defined members [1,2] and 1-5% of the genomes in infectious organisms such as bacteria, parasites and viruses [3]. Proteases regulate many cellular processes, such as protein degradation [4,5], digestion [6,7] blood coagulation [8], wound healing [9], ovulation [10], embryonic development [11], bone formation [12], neuronal outgrowth [13], cell-cycle regulation [14], immune and inflammatory response [15], angiogenesis [16] and apoptosis [17]. Proteases are viable drug targets because of their role in cellular functions of both mammalian cells and pathogens.…”

Production and characterization of two major Aspergillus oryzae secreted prolyl endopeptidases able to efficiently digest proline-rich peptides of gliadin

“…This enzyme was found to initiate gliadin degradation in the stomach before it reached the intestinal lumen. AN-PEP produced and purified in large amounts was tested in a clinical assay including patients with coeliac disease (Stepniak et al, 2006). (ii) The use of a combination of a prolyl endoprotease (PEP) from Flavobacterium meningosepticum and a glutamine-specific endoprotease B (EP-B2, cysteine-protease) derived from germinating barley seeds Siegel et al, 2006).…”

Designing a Microbial Prolyl Peptidase Delivery System for the Treatment of Celiac Disease

“…Recently a new treatment strategy based upon the possibility of enzyme therapy using prolyl endopeptidases derived from bacteria or fungi has been proposed (Shan et al 2004, Stepniak et al 2006. These enzymes break down the immunodominant peptides by hydrolyzing proline-Xaa bonds.…”

“…Prolyl endopeptidases from Aspergillus niger and Myxococcus xanthus have been described previously (Stepniak et al, 2006;Shan et al, 2004). Four peptidases from L.…”