Abstract
Detoxification of gluten immunogenic epitopes is a promising strategy for the treatment of celiac disease. Our previous studies have shown that these epitopes can be degraded in vitro by subtilisin enzymes derived from Rothia mucilaginosa, a natural microbial colonizer of the oral cavity. The challenge is that the enzyme is not optimally active under acidic conditions as encountered in the stomach. We therefore aimed to protect and maintain subtilisin-A enzyme activity by exploring two pharmaceutical modification techniques: PEGylation and Polylactic glycolic acid (PLGA) microencapsulation. PEGylation of subtilisin-A (Sub-A) was performed by attaching methoxypolyethylene glycol (mPEG, 5 kDa). The PEGylation protected subtilisin-A from autolysis at neutral pH. The PEGylated Sub-A (Sub-A-mPEG) was further encapsulated by PLGA. The microencapsulated Sub-A-mPEG-PLGA showed significantly increased protection against acid exposure in vitro. In vivo, gluten immunogenic epitopes were decreased by 60% in the stomach of mice fed with chow containing Sub-A-mPEG-PLGA (0.2 mg Sub-A/g chow) (n = 9) compared to 31.9% in mice fed with chow containing unmodified Sub-A (n = 9). These results show that the developed pharmaceutical modification can protect Sub-A from auto-digestion as well as from acid inactivation, thus rendering the enzyme more effective for applications in vivo.
Highlights
Gluten proteins are found in wheat, barley and rye, and trigger celiac disease (CeD), a chronic inflammatory immune-mediated disease affecting the small intestine[1]
Subtilisin Carlsberg is produced by Bacillus licheniformis[20] and comprises two alpha-helices and a large beta-sheet structure. Given their food-grade status, subtilisins hold promise as digestive aides for gluten degradation, but similar to bacterial prolyl endopeptidase (PEP) their activity is dramatically reduced under acidic conditions, as prevail in the stomach
Sub-A-mPEG-Polylactic glycolic acid (PLGA) was fully active after the acid www.nature.com/scientificreports challenge (Fig. 4B). These results indicate that the PLGA coating had protected the encapsulated Sub-A-mPEG enzyme from denaturation during the acid exposure
Summary
Gluten proteins are found in wheat, barley and rye, and trigger celiac disease (CeD), a chronic inflammatory immune-mediated disease affecting the small intestine[1]. Rothia mucilaginosa as well as Rothia aeria exert a high gluten-degrading activity at pH 7.0 Both are capable to thoroughly cleave gluten after glutamine-proline-glutamine (QPQ) and leucine-proline-tyrosine (LPY) sequences which are abundant in immunogenic gluten domains[16,17,18]. The subtilisin enzymes, many being food-grade such as the ones from Bacillus species, could potentially benefit CeD patients if they could more effectively abolish gluten immunogenic epitopes during gastro-duodenal transit in vivo[19]. Subtilisin Carlsberg (subtilisin-A or Sub-A, 274 amino acids) is produced by Bacillus licheniformis[20] and comprises two alpha-helices and a large beta-sheet structure Given their food-grade status, subtilisins hold promise as digestive aides for gluten degradation, but similar to bacterial PEP their activity is dramatically reduced under acidic conditions, as prevail in the stomach. Stability and therapeutic potency of proteins can be improved through the application of pharmaceutical modification and/or enteric coating techniques that permit the drug’s release in the target area of interest, e.g., the small intestine[21,22]
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