A Food-Grade Enzyme Preparation with Modest Gluten Detoxification Properties

et al. (2009) A Food-Grade Enzyme Preparation with Modest Gluten Detoxification Properties. PLoS ONE 4(7): e6313. doi:10.1371/journal.pone.0006313 A Food-Grade Enzyme Preparation with Modest Gluten Detoxification Properties Jennifer Ehren 0 Belen Moro n 0 Edith Martin 0 Michael T. Bethune 0 Gary M. Gray 0 Chaitan Khosla 0 Hany A. El-Shemy, Cairo University, Egypt 0 1 Department of Chemical Engineering, Stanford University, Stanford, California, United States of America, 2 Department of Chemistry, Stanford University, Stanford, California, United States of America, 3 Department of Biochemistry, Stanford University, Stanford, California, United States of America, 4 Department of Medicine, Stanford University , Stanford, California , United States of America Background and Aims: Celiac sprue is a life-long disease characterized by an intestinal inflammatory response to dietary gluten. A gluten-free diet is an effective treatment for most patients, but accidental ingestion of gluten is common, leading to incomplete recovery or relapse. Food-grade proteases capable of detoxifying moderate quantities of dietary gluten could mitigate this problem. Methods: We evaluated the gluten detoxification properties of two food-grade enzymes, aspergillopepsin (ASP) from Aspergillus niger and dipeptidyl peptidase IV (DPPIV) from Aspergillus oryzae. The ability of each enzyme to hydrolyze gluten was tested against synthetic gluten peptides, a recombinant gluten protein, and simulated gastric digests of whole gluten and whole-wheat bread. Reaction products were analyzed by mass spectrometry, HPLC, ELISA with a monoclonal antibody that recognizes an immunodominant gluten epitope, and a T cell proliferation assay. Results: ASP markedly enhanced gluten digestion relative to pepsin, and cleaved recombinant a2-gliadin at multiple sites in a non-specific manner. When used alone, neither ASP nor DPPIV efficiently cleaved synthetic immunotoxic gluten peptides. This lack of specificity for gluten was especially evident in the presence of casein, a competing dietary protein. However, supplementation of ASP with DPPIV enabled detoxification of moderate amounts of gluten in the presence of excess casein and in whole-wheat bread. ASP was also effective at enhancing the gluten-detoxifying efficacy of cysteine endoprotease EPB2 under simulated gastric conditions. Conclusions: Clinical studies are warranted to evaluate whether a fixed dose ratio combination of ASP and DPPIV can provide near-term relief for celiac patients suffering from inadvertent gluten exposure. Due to its markedly greater hydrolytic activity against gluten than endogenous pepsin, food-grade ASP may also augment the activity of therapeutically relevant doses of glutenases such as EP-B2 and certain prolyl endopeptidases. - Funding: This research was supported by a grant from the National Institutes of Health (DK 063158) to C.K. J.E. was a recipient of an NSF predoctoral fellowship, and a grant from the Celiac Sprue Research Foundation. B.M. was the recipient of a postdoctoral fellowship from Fundacio n Caja Madrid. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: C.K. is a Director & Stockholder in Alvine Pharmaceuticals, a company that is developing an oral enzyme drug for celiac disease. None of the other authors have any financial interests to disclose. . These authors contributed equally to this work. Celiac sprue is an inheritable, life-long disease that is characterized by an inflammatory reaction to dietary gluten in the human small intestine. The hallmark of the disease is a characteristic flattening of intestinal villi along with crypt hypertrophy. As a consequence, there is tremendous loss of surface area and malabsorption of nutrients, vitamins and minerals. If untreated, celiac sprue is associated with complications such as anemia, bone diseases, infertility, neurological problems, cancer and other complications due to persistent inflammation and micronutrient deficiencies. Screening studies predict that approximately 1% of the United States population has the disease, yet only ca. 10% of affected individuals have been diagnosed thus far [1]. At present, the only suitable treatment is strict, life-long exclusion of gluten from the patients diet. Although a large fraction of patients who attempt to follow such a diet still exhibit signs or symptoms of active disease [25], there is no available supplementary therapy for such conditions. A potential cost-effective solution to the aforementioned problem is an oral protease or protease mixture that is composed solely of commercially available food-grade enzymes. In this study we have evaluated the gluten detoxification properties of one such enzyme cocktail. It includes at least two proteases, aspergillopepsin (ASP) from Aspergillus niger and dipeptidyl peptidase IV (DPPIV) from Aspergillus oryzae, both of which are widely used in the foods and feeds, food processing and dietary supplement industries. Our data suggest that, whereas neither enzyme preparation alone is able to detoxify gluten under simulated gastric conditions, a defined dose ratio of the two enzyme preparations is able to detoxify moderate quantities of dietary gluten. Clinical studies are therefore warranted to evaluate the activity of this food-grade enzyme preparation in celiac sprue patients with an inadequate response to a gluten-free diet. Our results also show that, under gastric conditions, ASP augments the efficacy of gluten-detoxifying endoproteases such as the glutamine specific EP-B2 from barley [6]. Identity The Materials Safety Data Sheet of the commercial ASP powder from Bio-Cat Inc. designates this enzyme as aspergillopepsin from A. niger (EC 3.4.23.18). N-terminal sequencing of the major 41 kD protein in this powder yielded an N-terminal sequence of KSAVTT, consistent with its identity as aspergillopepsin A precursor (a.k.a., aspergillopepsin I) having a molecular weight of 41 kD. Mass mapping of tryptic fragments further verified its identity. Proaspergillopepsin A is believed to spontaneously self-activate into the mature enzyme under acidic conditions [7]. The Materials Safety Data Sheet of the commercial Peptidase P powder from Bio-Cat Inc. designates this enzyme as dipeptidyl peptidase IV from A. oryzae (EC 3.4.21.63). Although DPPIV activity could be verified by the chromogenic substrate Gly-PropNA, SDS-PAGE revealed that the enzyme preparation was a complex mixture of proteins that lacked a prominent band with MW higher than 75 kD, as might be expected for this DPPIV [8]. We therefore used an alternate strategy to verify the identity of this serine protease in the commercial enzyme preparation. An inhibition assay was performed with various concentrations of known DPPIV inhibitor, Boc-L-Prolinal. At a concentration of 9 mM inhibitor in the reaction volume, 100% inhibition of DPPIV ( (...truncated)