title: Dephosphorylation reduces passage of ovalbumin antigen through intestinal epithelial Caco-2 cell monolayers.
abstract: Allergenic potential of food proteins is associated with stability to gastric and pancreatic digestive enzymes. However, much attention has not been focused on intracellular digestion of protein antigens during the passage through intestinal epithelia. We report here the degradation and survival of a bis-phosphorylated protein, ovalbumin (OVA), in the course of passage through Caco-2 cell monolayers cultured on porous membrane. SDS-PAGE in combination with phosphoprotein staining showed that OVA, which had passed through the cell layers, was almost intact in its polypeptide chain but partly dephosphorylated. By contrast, quantitative analysis using ELISA indicated that complete dephosphorylation in advance by an alkaline phosphatase markedly reduced the OVA passage. The reduced passage was restored in the presence of cathepsin inhibitors, leupeptin and pepstatin-A. Moreover, the complete dephosphorylation increased susceptibility of OVA to in vitro digestion with cathepsin B, which cleaved near an OVA phosphorylation site, Ser345. The susceptibility of OVA to lysosomal proteases may affect its passage through the intestinal epithelia, leading to determination of allergic sensitization and elicitation in egg allergy.
authors: Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan. tmatsuda@agr.nagoya-u.ac.jp
authors: Takeshi
authors: T
authors: Matsubara
authors: Yuka
authors: Y
authors: Akiyama
authors: Kenzi
authors: K
authors: Oshima
authors: Tetsuya
authors: T
authors: Okajima
authors: Daita
authors: D
authors: Nadano
authors: Tsukasa
authors: T
authors: Matsuda
mesh_terms: D000469:Alkaline Phosphatase; D000818:Animals; D000941:Antigens / Q000378:metabolism; D018938:Caco-2 Cells / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D002403:Cathepsins / Q000037:antagonists & inhibitors; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007413:Intestinal Mucosa / Q000276:immunology / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D010047:Ovalbumin / Q000276:immunology* / Q000378:metabolism*; D010436:Pepstatins / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011480:Protease Inhibitors / Q000494:pharmacology
chemical_list: D000941:Antigens; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D010436:Pepstatins; D011480:Protease Inhibitors; D010047:Ovalbumin; D000469:Alkaline Phosphatase; D002403:Cathepsins; C032854:leupeptin; C031375:pepstatin