Discovery of a Novel and Rich Source of Gluten-Degrading Microbial Enzymes in the Oral Cavity

• Published in: PloS one Vol. 5; no. 10; p. e13264
• Main Authors: Helmerhorst, Eva J., Zamakhchari, Maram, Schuppan, Detlef, Oppenheim, Frank G.
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 11.10.2010; Public Library of Science (PLoS)
• Subjects: Amino Acid Sequence; Amino acids; Antigenic determinants; Autoimmune diseases; Bacteria; Biochemistry; Biodegradation; Biology; Celiac disease; Chromatography, High Pressure Liquid; Degradation; Dental plaque; Dentistry; Diet; Enzymes; Enzymes - chemistry; Enzymes - metabolism; Epitopes; Gastroenterology; Gastroenterology and Hepatology/Small Intestine; Gliadin; Gliadin - metabolism; Glutamine; Gluten; Glutens - metabolism; Health risks; Histocompatibility antigen HLA; Hydrogen-Ion Concentration; Hydrolysis; Immune response; Immunogenicity; Inflammation; Ingestion; Intestine; Isoelectric Focusing; Lymphocytes; Lymphocytes T; Microbiology/Applied Microbiology; Microbiology/Medical Microbiology; Microorganisms; Molecular Sequence Data; Mouth - microbiology; Nutrition/Food Allergies; Oral cavity; Paranitroanilide; Peptides; pH effects; Proline; Proteases; Proteins; Proteolysis; Rodents; Sequence Homology, Amino Acid; Small intestine; Spectrometry, Mass, Electrospray Ionization; Substrates; T cells; Tandem Mass Spectrometry; United States > US; Massachusetts
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0013264

Celiac disease is a T cell mediated-inflammatory enteropathy caused by the ingestion of gluten in genetically predisposed individuals carrying HLA-DQ2 or HLA-DQ8. The immunogenic gliadin epitopes, containing multiple glutamine and proline residues, are largely resistant to degradation by gastric and intestinal proteases. Salivary microorganisms however exhibit glutamine endoprotease activity, discovered towards glutamine- and proline-rich salivary proteins. The aim was to explore if gliadins can serve as substrates for oral microbial enzymes. Proteolytic activity in suspended dental plaque was studied towards a) gliadin-derived paranitroanilide(pNA)-linked synthetic enzyme substrates b) a mixture of natural gliadins and c) synthetic highly immunogenic gliadin peptides (33-mer of α2-gliadin and 26-mer of γ-gliadin). In addition, gliadin zymography was conducted to obtain the approximate molecular weights and pH activity profiles of the gliadin-degrading oral enzymes and liquid iso-electric focusing was performed to establish overall enzyme iso-electric points. Plaque bacteria efficiently hydrolyzed Z-YPQ-pNA, Z-QQP-pNA, Z-PPF-pNA and Z-PFP-pNA, with Z-YPQ-pNA being most rapidly cleaved. Gliadin immunogenic domains were extensively degraded in the presence of oral bacteria. Gliadin zymography revealed that prominent enzymes exhibit molecular weights >70 kD and are active over a broad pH range from 3 to 10. Liquid iso-electric focusing indicated that most gliadin-degrading enzymes are acidic in nature with iso-electric points between 2.5 and 4.0. This is the first reported evidence for gluten-degrading microorganisms associated with the upper gastro-intestinal tract. Such microorganisms may play a hitherto unappreciated role in the digestion of dietary gluten and thus protection from celiac disease in subjects at risk.