Celiac.com 12/26/2016 - Could gluten-degrading enzymes offer a better future for celiac patients? Rothia mucilaginosa is an oral microbial colonizer that can break down proline- and glutamine-rich proteins present in wheat, barley, and rye that contain the immunogenic sequences that drive celiac disease. A team of researchers recently set out to isolate and identify the enzymes and evaluate their potential as novel enzyme therapeutics for celiac disease.
They first extracted and separated membrane-associated R. mucilaginosa proteins using DEAE chromatography. They tracked enzyme activities using paranitroanilide-derivatized and fluorescence resonance energy transfer (FRET) peptide substrates, and by gliadin zymography. They determined epitope elimination in R5 and G12 ELISAs. They identified gliadin-degrading Rothia enzymes by LC-ESI-MS/MS as hypothetical proteins ROTMU0001_0241 (C6R5V9_9MICC), ROTMU0001_0243 (C6R5W1_9MICC), and ROTMU0001_240 (C6R5V8_9MICC).
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 celiac disease. To do this, the team cleaved succinyl-Ala-Ala-Pro-Phe-paranitroanilide, 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.
They found that 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.
Rothia and food-grade Bacillus subtilisins show promise for development as enzyme therapies for celiac disease.