title: The Unusual Resistance of Avian Defensin AvBD7 to Proteolytic Enzymes Preserves Its Antibacterial Activity.
abstract: Defensins are frontline peptides of mucosal immunity in the animal kingdom, including birds. Their resistance to proteolysis and their ensuing ability to maintain antimicrobial potential remains questionable and was therefore investigated. We have shown by bottom-up mass spectrometry analysis of protein extracts that both avian beta-defensins AvBD2 and AvBD7 were ubiquitously distributed along the chicken gut. Cathepsin B was found by immunoblotting in jejunum, ileum, caecum, and caecal tonsils, while cathepsins K, L, and S were merely identified in caecal tonsils. Hydrolysis product of AvBD2 and AvBD7 incubated with a panel of proteases was analysed by RP-HPLC, mass spectrometry and antimicrobial assays. AvBD2 and AvBD7 were resistant to serine proteases and to cathepsins D and H. Conversely cysteine cathepsins B, K, L, and S degraded AvBD2 and abolished its antibacterial activity. Only cathepsin K cleaved AvBD7 and released Ile4-AvBD7, a N-terminal truncated natural peptidoform of AvBD7 that displayed antibacterial activity. Besides the 3-stranded antiparallel beta-sheet typical of beta-defensins, structural analysis of AvBD7 by two-dimensional NMR spectroscopy highlighted the restricted accessibility of the C-terminus embedded by the N-terminal region and gave a formal evidence of a salt bridge (Asp9-Arg12) that could account for proteolysis resistance. The differential susceptibility of avian defensins to proteolysis opens intriguing questions about a distinctive role in the mucosal immunity against pathogen invasion.
authors: ISP, INRA, Université François Rabelais de Tours, UMR 1282, Nouzilly, France.
authors: Geoffrey
authors: G
authors: Bailleul
authors: CEPR, INSERM, Université François Rabelais, UMR 1100, Tours, France.
authors: Amanda
authors: A
authors: Kravtzoff
authors: ISP, INRA, Université François Rabelais de Tours, UMR 1282, Nouzilly, France.
authors: Alix
authors: A
authors: Joulin-Giet
authors: CEPR, INSERM, Université François Rabelais, UMR 1100, Tours, France.
authors: Fabien
authors: F
authors: Lecaille
authors: PRC, INRA, Université François Rabelais de Tours, UMR 85, UMR CNRS 7247, Plate-forme d'Analyse Intégrative des Biomolécules, Laboratoire de Spectrométrie de Masse, Nouzilly, France.
authors: Valérie
authors: V
authors: Labas
authors: CBM, CNRS, UPR 4301, Orléans, France.
authors: Hervé
authors: H
authors: Meudal
authors: CBM, CNRS, UPR 4301, Orléans, France.|Collegium Sciences et Techniques, Université d'Orléans, Orléans, France.
authors: Karine
authors: K
authors: Loth
authors: ISP, INRA, Université François Rabelais de Tours, UMR 1282, Nouzilly, France.|PRC, INRA, Université François Rabelais de Tours, UMR 85, UMR CNRS 7247, Plate-forme d'Analyse Intégrative des Biomolécules, Laboratoire de Spectrométrie de Masse, Nouzilly, France.
authors: Ana-Paula
authors: AP
authors: Teixeira-Gomes
authors: ISP, INRA, Université François Rabelais de Tours, UMR 1282, Nouzilly, France.
authors: Florence B
authors: FB
authors: Gilbert
authors: Plate-forme de Protéomique "PISSARO" de l'IRIB, Université de Rouen, CNRS, UMR 6270, Mont-Saint Aignan, France.
authors: Laurent
authors: L
authors: Coquet
authors: Plate-forme de Protéomique "PISSARO" de l'IRIB, Université de Rouen, CNRS, UMR 6270, Mont-Saint Aignan, France.
authors: Thierry
authors: T
authors: Jouenne
authors: Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, British Columbia, Canada.
authors: Dieter
authors: D
authors: Brömme
authors: ISP, INRA, Université François Rabelais de Tours, UMR 1282, Nouzilly, France.
authors: Catherine
authors: C
authors: Schouler
authors: CBM, CNRS, UPR 4301, Orléans, France.
authors: Céline
authors: C
authors: Landon
authors: CEPR, INSERM, Université François Rabelais, UMR 1100, Tours, France.
authors: Gilles
authors: G
authors: Lalmanach
authors: ISP, INRA, Université François Rabelais de Tours, UMR 1282, Nouzilly, France.
authors: Anne-Christine
authors: AC
authors: Lalmanach
mesh_terms: D000818:Animals; D002401:Cathepsin B / Q000378:metabolism; D002402:Cathepsin D / Q000378:metabolism; D056657:Cathepsin K / Q000378:metabolism; D056668:Cathepsin L / Q000378:metabolism; D002403:Cathepsins / Q000378:metabolism; D002645:Chickens / Q000276:immunology*; D002918:Chymotrypsin / Q000737:chemistry; D006868:Hydrolysis; D007413:Intestinal Mucosa / Q000378:metabolism; D019272:Leukocyte Elastase / Q000378:metabolism; D013058:Mass Spectrometry; D008968:Molecular Conformation; D014066:Palatine Tonsil / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D059748:Proteolysis; D014357:Trypsin / Q000737:chemistry; D023083:beta-Defensins / Q000378:metabolism*
chemical_list: D023083:beta-Defensins; D002403:Cathepsins; D010447:Peptide Hydrolases; D002918:Chymotrypsin; D019272:Leukocyte Elastase; D014357:Trypsin; D002401:Cathepsin B; D056668:Cathepsin L; C028217:cathepsin S; D056657:Cathepsin K; D002402:Cathepsin D