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Addressing proteolytic efficiency in enzymatic degradation therapy for celiac disease.

ABSTRACT: Celiac disease is triggered by partially digested gluten proteins. Enzyme therapies that complete protein digestion in vivo could support a gluten-free diet, but the barrier to completeness is high. Current options require enzyme amounts on the same order as the protein meal itself. In this study, we evaluated proteolytic components of the carnivorous pitcher plant (Nepenthes spp.) for use in this context. Remarkably low doses enhance gliadin solubilization rates, and degrade gliadin slurries within the pH and temporal constraints of human gastric digestion. Potencies in excess of 1200:1 (substrate-to-enzyme) are achieved. Digestion generates small peptides through nepenthesin and neprosin, the latter a novel enzyme defining a previously-unknown class of prolyl endoprotease. The digests also exhibit reduced TG2 conversion rates in the immunogenic regions of gliadin, providing a twin mechanism for evading T-cell recognition. When sensitized and dosed with enzyme-treated gliadin, NOD/DQ8 mice did not show intestinal inflammation, when compared to mice challenged with only pepsin-treated gliadin. The low enzyme load needed for effective digestion suggests that gluten detoxification can be achieved in a meal setting, using metered dosing based on meal size. We demonstrate this by showing efficient antigen processing at total substrate-to-enzyme ratios exceeding 12,000:1.

SUBMITTER: Rey M

PROVIDER: S-EPMC4969619 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Addressing proteolytic efficiency in enzymatic degradation therapy for celiac disease.

Rey Martial M Yang Menglin M Lee Linda L Zhang Ye Y Sheff Joey G JG Sensen Christoph W CW Mrazek Hynek H Halada Petr P Man Petr P McCarville Justin L JL Verdu Elena F EF Schriemer David C DC

Scientific reports 20160802

Celiac disease is triggered by partially digested gluten proteins. Enzyme therapies that complete protein digestion in vivo could support a gluten-free diet, but the barrier to completeness is high. Current options require enzyme amounts on the same order as the protein meal itself. In this study, we evaluated proteolytic components of the carnivorous pitcher plant (Nepenthes spp.) for use in this context. Remarkably low doses enhance gliadin solubilization rates, and degrade gliadin slurries wi ...[more]

PMID: 27481162

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Project description:Gluten is known to be the main triggering factor for celiac disease (CeD), an immune-mediated disorder. CeD is therefore managed using a strict and lifelong gluten-free diet (GFD), the only effective treatment available currently. However, the GFD is restrictive. Hence, efforts are being made to explore alternative therapies. Based on their mechanisms of action on various molecular targets involved in the pathogenesis of CeD, these therapies may be classified into one of the following five broad approaches. The first approach focuses on decreasing the immunogenic content of gluten, using strategies like genetically modified wheat, intra-intestinal gluten digestion using glutenases, microwave thermal treatment of hydrated wheat kernels, and gluten pretreatment with either bacterial/ fungal derived endopeptidases or microbial transglutaminase. The second approach involves sequestering gluten in the gut lumen before it is digested into immunogenic peptides and absorbed, using binder drugs like polymer p(HEMA-co-SS), single chain fragment variable (scFv), and anti- gluten antibody AGY. The third approach aims to prevent uptake of digested gluten through intestinal epithelial tight junctions, using a zonulin antagonist. The fourth approach involves tissue transglutaminase (tTG) inhibitors to prevent the enhancement of immunogenicity of digested gluten by the intestinal tTG enzyme. The fifth approach seeks to prevent downstream immune activation after uptake of gluten immunogenic peptides through the intestinal mucosal epithelial layer. Examples include HLA-DQ2 blockers that prevent presentation of gluten derived- antigens by dendritic cells to T cells, immune- tolerizing therapies like the vaccine Nexvax2 and TIMP-Glia, cathepsin inhibitors, immunosuppressants like corticosteroids, azathioprine etc., and anti-cytokine agents targeting TNF-α and interleukin-15. Apart from these approaches, research is being done to evaluate the effectiveness of probiotics/prebiotics, helminth therapy using Necator americanus, low FODMAP diet, and pancreatic enzyme supplementation in CeD symptom control; however, the mechanisms by which they play a beneficial role in CeD are yet to be clearly established. Overall, although many therapies being explored are still in the pre-clinical phase, some like the zonulin antagonist, immune tolerizing therapies and glutenases have reached phase II/III clinical trials. While these potential options appear exciting, currently they may at best be used to supplement rather than supplant the GFD.

Project description:Celiac disease (CD) is a chronic immune-mediated enteropathy induced by dietary gluten in genetically predisposed individuals. Saliva harbors the second highest bacterial load of the gastrointestinal (GI) tract after the colon. We hypothesized that enzymes produced by oral bacteria may be involved in gluten processing in the intestine and susceptibility to celiac disease. The aim of this study was to investigate salivary enzymatic activities and oral microbial profiles in healthy subjects versus patients with classical and refractory CD. Stimulated whole saliva was collected from patients with CD in remission (n = 21) and refractory CD (RCD; n = 8) and was compared to healthy controls (HC; n = 20) and subjects with functional GI complaints (n = 12). Salivary gluten-degrading activities were monitored with the tripeptide substrate Z-Tyr-Pro-Gln-pNA and the α-gliadin-derived immunogenic 33-mer peptide. The oral microbiome was profiled by 16S rRNA-based MiSeq analysis. Salivary glutenase activities were higher in CD patients compared to controls, both before and after normalization for protein concentration or bacterial load. The oral microbiomes of CD and RCD patients showed significant differences from that of healthy subjects, e.g., higher salivary levels of lactobacilli (P < 0.05), which may partly explain the observed higher gluten-degrading activities. While the pathophysiological link between the oral and gut microbiomes in CD needs further exploration, the presented data suggest that oral microbe-derived enzyme activities are elevated in subjects with CD, which may impact gluten processing and the presentation of immunogenic gluten epitopes to the immune system in the small intestine.IMPORTANCE Ingested gluten proteins are the triggers of intestinal inflammation in celiac disease (CD). Certain immunogenic gluten domains are resistant to intestinal proteases but can be hydrolyzed by oral microbial enzymes. Very little is known about the endogenous proteolytic processing of gluten proteins in the oral cavity. Given that this occurs prior to gluten reaching the small intestine, such enzymes are likely to contribute to the composition of the gluten digest that ultimately reaches the small intestine and causes CD. We demonstrated that endogenous salivary protease activities are incomplete, likely liberating peptides from larger gluten proteins. The potentially responsible microbes were identified. The study included refractory CD patients, who have been studied less with regard to CD pathogenesis.

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Addressing proteolytic efficiency in enzymatic degradation therapy for celiac disease.

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Addressing proteolytic efficiency in enzymatic degradation therapy for celiac disease.

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