title: Ineffective degradation of immunogenic gluten epitopes by currently available digestive enzyme supplements.
abstract: BACKGROUND
  Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP).
  
  
  METHODS
  Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays.
  
  
  RESULTS
  The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data.
  
  
  CONCLUSIONS
  Currently available digestive enzyme supplements are ineffective in degrading immunogenic gluten epitopes.
authors.affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, The Netherlands.
authors.forename: George
authors.initials: G
authors.lastname: Janssen
authors.affiliation: DSM Food Specialties, Delft, The Netherlands.
authors.forename: Chantal
authors.initials: C
authors.lastname: Christis
authors.affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, The Netherlands.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Kooy-Winkelaar
authors.affiliation: DSM Food Specialties, Delft, The Netherlands.
authors.forename: Luppo
authors.initials: L
authors.lastname: Edens
authors.affiliation: DSM Food Specialties, South Bend, United States of America.
authors.forename: Drew
authors.initials: D
authors.lastname: Smith
authors.affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, The Netherlands.
authors.forename: Peter
authors.initials: P
authors.lastname: van Veelen
authors.affiliation: Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, Leiden, The Netherlands.
authors.forename: Frits
authors.initials: F
authors.lastname: Koning
mesh_terms: D002478:Cells, Cultured; D019587:Dietary Supplements; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry* / Q000276:immunology; D005903:Gliadin / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000276:immunology; D059748:Proteolysis
chemical_list: D018984:Epitopes, T-Lymphocyte; D005903:Gliadin; D010447:Peptide Hydrolases