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Gluten are proline- and glutaminerich proteins present in wheat, barley, and rye and contain the immunogenic sequences that drive celiac disease (CD). Rothia mucilaginosa, an oral microbial colonizer, can cleave these gluten epitopes. The aim was to isolate and identify the enzymes and evaluate their potential as novel enzyme therapeutics for CD. The membrane-associated R. mucilaginosa proteins were extracted and separated by DEAE chromatography. Enzyme activities were monitored with paranitroanilide-derivatized and fluorescence resonance energy transfer (FRET) peptide substrates, and by gliadin zymography. Epitope elimination was determined in R5 and G12 ELISAs. The gliadin-degrading Rothia enzymes were identified by LC-ESI-MS/MS as hypothetical proteins ROTMU0001_0241 (C6R5V9_9MICC), ROTMU0001_0243 (C6R5W1_9MICC), and ROTMU0001_240 (C6R5V8_9MICC). A search with the Basic Local Alignment Search Tool revealed that these are subtilisin-like serine proteases belonging to the peptidase S8 family. Alignment of the major Rothia subtilisins indicated that all contain the catalytic triad with Asp (D), His (H), and Ser (S) in the D-H-S order. They cleaved succinyl-Ala-Ala-Pro-Pheparanitroanilide, a substrate for subtilisin with Pro in the P2 position, as in Tyr-Pro-Gln and Leu-Pro-Tyr in gluten, which are also cleaved. Consistently, FRET substrates of gliadin immunogenic epitopes comprising Xaa-Pro-Xaa motives were rapidly hydrolyzed. The Rothia subtilisins and two subtilisins from Bacillus licheniformis, subtilisin A and the food-grade Nattokinase, efficiently degraded the immunogenic gliadin-derived 33-mer peptide and the immunodominant epitopes recognized by the R5 and G12 antibodies. This study identified Rothia and food-grade Bacillus subtilisins as promising new candidates for enzyme therapeutics in CD. [ABSTRACT FROM AUTHOR]

Background: Gluten proteins, prominent constituents of barley, wheat and rye, cause celiac disease in genetically predisposed subjects. Gluten is notoriously difficult to digest by mammalian proteolytic enzymes and the protease-resistant domains contain multiple immunogenic epitopes. The aim of this study was to identify novel sources of gluten-digesting microbial enzymes from the upper gastro-intestinal tract with the potential to neutralize gluten epitopes. Methodology/Principal Findings: Oral microorganisms with gluten-degrading capacity were obtained by a selective plating strategy using gluten agar. Microbial speciations were carried out by 16S rDNA gene sequencing. Enzyme activities were assessed using gliadin-derived enzymatic substrates, gliadins in solution, gliadin zymography, and 33-mer a-gliadin and 26-mer &agr;-gliadin immunogenic peptides. Fragments of the gliadin peptides were separated by RP-HPLC and structurally characterized by mass spectrometry. Strains with high activity towards gluten were typed as Rothia mucilaginosa and Rothia aeria. Gliadins (250 μg/ml) added to Rothia cell suspensions (OD620 1.2) were degraded by 50% after ∼30 min of incubation. Importantly, the 33-mer and 26-mer immunogenic peptides were also cleaved, primarily C-terminal to Xaa-Pro-Gln (XPQ) and Xaa-Pro-Tyr (XPY). The major gliadin-degrading enzymes produced by the Rothia strains were ∼70-75 kDa in size, and the enzyme expressed by Rothia aeria was active over a wide pH range (pH 3-10). Conclusion/Significance: While the human digestive enzyme system lacks the capacity to cleave immunogenic gluten, such activities are naturally present in the oral microbial enzyme repertoire. The identified bacteria may be exploited for physiologic degradation of harmful gluten peptides. [ABSTRACT FROM AUTHOR]

Rothia mucilaginosa, a natural microbial inhabitant of the oral cavity, cleaves gluten (gliadin) proteins at regions that are resistant to degradation by mammalian enzymes. The aim of this study was to investigate to what extent the R. mucilaginosa cell-associated enzymes abolish gliadin immunogenic properties. Degradation of total gliadins and highly immunogenic gliadin 33-mer or 26-mer peptides was monitored by SDS-PAGE and RP-HPLC, and fragments were sequenced by liquid chromatography and electrospray ionization tandem mass spectrometer (LC-ESI-MS/MS). Peptide deamidation by tissue transglutaminase (TG2), a critical step in rendering the fragments more immunogenic, was assessed by TG2-mediated cross-linking to monodansyl cadaverine (MDC), and by a +1-Da mass difference by LC-ESI-MS. Survival of potential immunogenic gliadin epitopes was determined by use of the R5 antibody-based ELISA. R. mucilaginosa-associated enzymes cleaved gliadins, 33-mer and 26-mer peptides into smaller fragments. TG2-mediated cross-linking showed a perfect inverse relationship with intact 33-mer and 26-mer peptide levels, and major degradation fragments showed a slow rate of MDC cross-linking of 6.18 ± 2.20 AU/min compared with 97.75 ± 10.72 and 84.17 ± 3.25 AU/min for the intact 33-mer and 26-mer, respectively, which was confirmed by reduced TG2-mediated deamidation of the fragments in mass spectrometry. Incubation of gliadins with Rothia cells reduced R5 antibody binding by 20, 82, and 97% after 30 min, 2 h, and 5 h, respectively, which was paralleled by reduced reactivity of enzymetreated 33-mer and 26-mer peptides in the R5 competitive ELISA. Our broad complementary approach to validate gluten degrading activities qualifies R. mucilaginosa-associated enzymes as promising tools to neutralize T cell immunogenic properties for treatment of celiac disease. [ABSTRACT FROM AUTHOR]

Background: Celiac disease is a T cell mediated-inflammatory enteropathy caused by the ingestion of gluten in genetically predisposed individuals carrying HLA-DQ2 or HLA-DQ8. The immunogenic gliadin epitopes, containing multiple glutamine and proline residues, are largely resistant to degradation by gastric and intestinal proteases. Salivary microorganisms however exhibit glutamine endoprotease activity, discovered towards glutamine- and proline-rich salivary proteins. The aim was to explore if gliadins can serve as substrates for oral microbial enzymes. Methodology/Principal Findings: Proteolytic activity in suspended dental plaque was studied towards a) gliadin-derived paranitroanilide(pNA)-linked synthetic enzyme substrates b) a mixture of natural gliadins and c) synthetic highly immunogenic gliadin peptides (33-mer of α2-gliadin and 26-mer of γ-gliadin). In addition, gliadin zymography was conducted to obtain the approximate molecular weights and pH activity profiles of the gliadin-degrading oral enzymes and liquid iso-electric focusing was performed to establish overall enzyme iso-electric points. Plaque bacteria efficiently hydrolyzed Z-YPQ-pNA, Z-QQP-pNA, Z-PPF-pNA and Z-PFP-pNA, with Z-YPQ-pNA being most rapidly cleaved. Gliadin immunogenic domains were extensively degraded in the presence of oral bacteria. Gliadin zymography revealed that prominent enzymes exhibit molecular weights >70 kD and are active over a broad pH range from 3 to 10. Liquid iso-electric focusing indicated that most gliadin-degrading enzymes are acidic in nature with iso-electric points between 2.5 and 4.0. Conclusions/Significance: This is the first reported evidence for gluten-degrading microorganisms associated with the upper gastro-intestinal tract. Such microorganisms may play a hitherto unappreciated role in the digestion of dietary gluten and thus protection from celiac disease in subjects at risk. [ABSTRACT FROM AUTHOR]

Celiac disease is characterized by a chronic immune-mediated inflammation of the small intestine, triggered by gluten contained in wheat, barley, and rye. Rothia aeria, a gram-positive natural colonizer of the oral cavity and the upper digestive tract is able to degrade and detoxify gluten in vitro. The objective of this study was to assess gluten-degrading activity of live and dead R. aeria bacteria in vitro, and to isolate the R. aeria gluten-degrading enzyme. Methods: After an overnight fast, Balb/c mouse were fed a 1 g pellet of standard chow containing 50% wheat (and 4% gliadin) with or without 1.6 × 107 live R. aeria bacteria. After 2 h, in vivo gluten degradation was assessed in gastric contents by SDS-PAGE and immunoblotting, and immunogenic epitope neutralization was assessed with the R5 gliadin ELISA assay. R. aeria enzyme isolation and identification was accomplished by separating proteins in the bacterial cell homogenate by C18 chromatography followed by gliadin zymography and mass spectrometric analysis of excised bands. Results: In mice fed with R. aeria, gliadins and immunogenic epitopes were reduced by 20% and 33%, respectively, as compared to gluten digested in control mice. Killing of R. aeria bacteria in ethanol did not abolish enzyme activity associated with the bacteria. The gluten degrading enzyme was identified as BAV86562.1, here identified as a member of the subtilisin family. Conclusion: This study shows the potential of R. aeria to be used as a first probiotic for gluten digestion in vivo, either as live or dead bacteria, or, alternatively, for using the purified R. aeria enzyme, to benefit the gluten-intolerant patient population. [ABSTRACT FROM AUTHOR]

Celiac disease (CeD) affects about 1% of most world populations. It presents a wide spectrum of clinical manifestations, ranging from minor symptoms to mild or severe malabsorption, and it may be associated with a wide variety of autoimmune diseases. CeD is triggered and maintained by the ingestion of gluten proteins from wheat and related grains. Gluten peptides that resist gastrointestinal digestion are antigenically presented to gluten specific T cells in the intestinal mucosa via HLA-DQ2 or HLA-DQ8, the necessary genetic predisposition for CeD. To date, there is no effective or approved treatment for CeD other than a strict adherence to a gluten-free diet, which is difficult to maintain in professional or social environments. Moreover, many patients with CeD have active disease despite diet adherence due to a high sensitivity to traces of gluten. Therefore, safe pharmacological treatments that complement the gluten-free diet are urgently needed. Oral enzyme therapy, employing gluten-degrading enzymes, is a promising therapeutic approach. A prerequisite is that such enzymes are active under gastro-duodenal conditions, quickly neutralize the T cell activating gluten peptides and are safe for human consumption. Several enzymes including prolyl endopeptidases, cysteine proteases and subtilisins can cleave the human digestion-resistant gluten peptides in vitro and in vivo. Examples are several prolyl endopeptidases from bacterial sources, subtilisins from Rothia bacteria that are natural oral colonizers and synthetic enzymes with optimized gluten-degrading activities. Without exception, these enzymes must cleave the otherwise unusual glutamine and proline-rich domains characteristic of antigenic gluten peptides. Moreover, they should be stable and active in both the acidic environment of the stomach and under near neutral pH in the duodenum. This review focuses on those enzymes that have been characterized and evaluated for the treatment of CeD, discussing their origin and activities, their clinical evaluation and challenges for therapeutic application. Novel developments include strategies like enteric coating and genetic modification to increase enzyme stability in the digestive tract. [ABSTRACT FROM AUTHOR]