Gliadin degradation in vitro and in vivo by Rothia aeria bacteria and pharmaceutically modified subtilisin-A enzyme
Darwish, Ghassan M.
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INTRODUCTION: Foods enter the oral cavity and mix with saliva. Some foods are not well tolerated, for instance, gluten proteins in individuals suffering from celiac disease (CD). Celiac disease is a chronic immune-mediated inflammation of the duodenum, triggered by gliadin component of gluten contained in wheat, barley and rye. In previous studies we showed that oral Rothia bacteria can degrade gliadin in vitro. The objective of this study was to gain more insights into the role of Rothia bacteria and subtilisin-A enzyme on gliadin digestion in vivo, with the ultimate goal to find new therapeutic options for CD. MATERIALS AND METHODS: Part I: Rothia bacterial proteins were analyzed for enzyme activity and subjected to LC-ESI-MS/MS. For in vivo, mice chow was prepared with and without R. aeria. Gliadin epitope abolishment was assessed in the mice stomach contents (n=9/group) by ELISA. Part II: Subtilisin-A was dissolved in various solutions, temperatures and incubation time to assess enzyme activity by using enzyme substrate (Suc-AAPF-pNA). Part III: PEGylation of subtilisin-A (Sub-A) was performed by mixing Sub-A with methoxy-polyethylene glycol (mPEG) and further encapsulated by polylactic-glycolic acid (PLGA). The activity of the modified enzyme to detoxify the immunogenic gliadin epitopes was evaluated at pH3.0. RESULTS: Part I: R. aeria gliadin-degrading enzyme was found to be a member of the subtilisin family. In vivo, gliadins immunogenic epitopes were reduced by 32.6%. Part II: Sub-A dissolved at pH1.5 showed a band of 27kDa, while it only showed bands below 10kDa when dissolved at pH7.0, suggesting auto-proteolysis. The enzyme activity was completely lost at temperatures exceeding 60°C and also reduced 4-fold after 6hr incubation at 37°C. Part III: PEGylation protected Sub-A from autolysis. The microencapsulated Sub-A-mPEG-PLGA showed significantly increased protection against acid exposure in vitro. In vivo, gliadin immunogenic epitopes were decreased by 60% in the stomach of the mice fed with chow containing Sub-A-mPEG-PLGA. CONCLUSION: The results provide proof for the contribution of oral Rothia bacteria to gliadin digestion and pharmaceutical modification can protect Sub-A from auto-digestion as well as from acidic insults, thus rendering the usefulness of coated subtilisins as a digestive aid for gluten degradation.