Apical-to-basolateral transepithelial transport of cow’s milk caseins by intestinal Caco-2 cell monolayers: MS-based quantitation of cellularly degraded α- and β-casein fragments

Casein (CN) is the major milk protein to nourish infants but, in certain population, it causes cow's milk allergy, indicating the uptake of antigenic CN and their peptides through the intestinal epithelium. Using human intestinal Caco-2 cell monolayers, the apical-to-basal transepithelial transport of CN was investigated. Confocal microscopy using component-specific antibodies showed that αs1-CN antigens became detectable as punctate signals at the apical-side cytoplasm and reached to the cytoplasm at a tight-junction level within a few hours. Such intracellular CN signals were more remarkable than those of the other antigens, β-lactoglobulin and ovalbumin, colocalized in part with an early endosome marker protein (EEA1) and decreased in the presence of cytochalasin D or sodium azide and also at lowered temperature at 4°C. Liquid chromatography coupled with mass spectroscopy analysis of the protein fraction in the basal-side medium identified the αs1-CB fragment including the N-terminal region and the αs2-CN fragment containing the central part of polypeptide at 100-1,000 fmol per well levels. Moreover, β-CN C-terminal overlapping peptides were identified in the peptide fraction below 10 kDa of the basal medium. These results suggest that CNs are partially degraded by cellular proteases and/or peptidases and immunologically active CN fragments are transported to basal side of the cell monolayers.