title: Identification of Pseudolysin (lasB) as an Aciduric Gluten-Degrading Enzyme with High Therapeutic Potential for Celiac Disease.
abstract: OBJECTIVE
  Immunogenic gluten proteins implicated in celiac disease (CD) largely resist degradation by human digestive enzymes. Here we pursued the isolation of gluten-degrading organisms from human feces, aiming at bacteria that would digest gluten under acidic conditions, as prevails in the stomach.
  
  
  METHODS
  Bacteria with gluten-degrading activities were isolated using selective gluten agar plates at pH 4.0 and 7.0. Proteins in concentrated bacterial cell sonicates were separated by diethylaminoethanol chromatography. Enzyme activity was monitored with chromogenic substrates and gliadin zymography. Elimination of major immunogenic gluten epitopes was studied with R5 and G12 enzyme-linked immunosorbent assays.
  
  
  RESULTS
  Gliadin-degrading enzyme activities were observed for 43 fecal isolates, displaying activities in the ~150-200 and <50 kDa regions. The active strains were identified as Pseudomonas aeruginosa. Gliadin degradation in gel was observed from pH 2.0 to 7.0. Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis identified the enzyme as pseudolysin (lasB), a metalloprotease belonging to the thermolysin (M4) family proteases. Its electrophoretic mobility in SDS-polyacrylamide gel electrophoresis and gliadin zymogram gels was similar to that of a commercial lasB preparation, with tendency of oligomerization. Pseudolysin eliminated epitopes recognized by the R5 antibody, while those detected by the G12 antibody remained intact, despite destruction of the nearby major T-cell epitope QPQLPY.
  
  
  CONCLUSIONS
  Pseudolysin was identified as an enzyme cleaving gluten effectively at extremely low as well as near-neutral pH values. The potential to degrade gluten during gastric transport opens possibilities for its application as a novel therapeutic agent for the treatment of CD.
authors.affiliation: Department of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston, Massachusetts, USA.
authors.forename: Guoxian
authors.initials: G
authors.lastname: Wei
authors.affiliation: Department of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston, Massachusetts, USA.
authors.forename: Na
authors.initials: N
authors.lastname: Tian
authors.affiliation: Department of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston, Massachusetts, USA.
authors.forename: Adriana C
authors.initials: AC
authors.lastname: Valery
authors.affiliation: Department of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston, Massachusetts, USA.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhong
authors.affiliation: 1] Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA [2] Institute of Translational Immunology and Research Center for Immunology (FZI), University Medical Center, Johannes-Gutenberg-University, Mainz, Germany.
authors.forename: Detlef
authors.initials: D
authors.lastname: Schuppan
authors.affiliation: Department of Molecular and Cell Biology, Henry M. Goldman School of Dental Medicine, Boston, Massachusetts, USA.
authors.forename: Eva J
authors.initials: EJ
authors.lastname: Helmerhorst
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002446:Celiac Disease; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005243:Feces / Q000201:enzymology* / Q000382:microbiology; D005903:Gliadin / Q000378:metabolism*; D005983:Glutens / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000378:metabolism*; D064307:Microbiota / Q000502:physiology*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D005983:Glutens; D005903:Gliadin; D008666:Metalloendopeptidases; C043859:pseudolysin, Pseudomonas aeruginosa