Gluten are proline- and glutamine-rich 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 FRET peptide substrates, and by gliadin zymography. Epitope elimination was determined in R5 and G12 ELISA assays. 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 blast search 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-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. The Rothia subtilisins as well as two subtilisins from Bacillus licheniformis, subtilisin A and the food-grade Nattokinase efficiently degraded the immunogenic gliadin-derived 33-mer peptide as well as 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.
- enzyme purification
- drug discovery
- drug action
- drug development
- Copyright © 2016, American Journal of Physiology- Gastrointestinal and Liver Physiology