title: Structural analysis and Caco-2 cell permeability of the celiac-toxic A-gliadin peptide 31-55.
abstract: Celiac disease is a chronic enteropathy caused by the ingestion of wheat gliadin and other cereal prolamines. The synthetic peptides 31-43 (P31-43) and 31-49 (P31-49) from A-gliadin are considered to be model peptides for studying innate immunity in celiac disease. Our previous study demonstrated that P31-43 and P31-49 are encrypted within peptide 31-55 (P31-55), which is naturally released from gastropancreatic digestion and is not susceptible to hydrolysis by brush border membrane enzymes. Here, we analyzed the permeability of P31-55 through the epithelial cell layer of confluent Caco-2 cells using high-performance liquid chromatography, mass spectrometry, and fluorescence-activated cell sorting. Twenty-three percent of the P31-55 added to the apical chamber was transported to the basolateral chamber after 4 h of incubation without being degraded by hydrolysis. Treatment of Caco-2 cells with whole gliadin digests extracted from a common wheat cultivar increased the epithelial P31-55 translocation by approximately 35%. Moreover, we observed an atypical chromatographic profile consisting of a double peak. Chromatography using different column temperatures and circular dichroism highlighted the presence of more conformational structures around the amide bond of the two adjacent prolines 38 and 39. These findings confirm that P31-55 is gastrointestinally resistant and is permeable across a Caco-2 monolayer. Moreover, we hypothesize that the various conformations of P31-55 may play a role in the activation of innate immunity.
authors.affiliation: Istituto di Scienze dell'Alimentazione, Consiglio Nazionale delle Ricerche, Avellino, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Iacomino
authors.forename: Olga
authors.initials: O
authors.lastname: Fierro
authors.forename: Sabato
authors.initials: S
authors.lastname: D'Auria
authors.forename: Gianluca
authors.initials: G
authors.lastname: Picariello
authors.forename: Pasquale
authors.initials: P
authors.lastname: Ferranti
authors.forename: Claudia
authors.initials: C
authors.lastname: Liguori
authors.forename: Francesco
authors.initials: F
authors.lastname: Addeo
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Mamone
mesh_terms: D018938:Caco-2 Cells; D002446:Celiac Disease / Q000503:physiopathology; D002463:Cell Membrane Permeability; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004847:Epithelial Cells / Q000378:metabolism*; D005434:Flow Cytometry; D005903:Gliadin / Q000378:metabolism*; D006801:Humans; D007113:Immunity, Innate; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D021381:Protein Transport; D053719:Tandem Mass Spectrometry; D014908:Triticum / Q000737:chemistry
chemical_list: D010446:Peptide Fragments; D005903:Gliadin