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
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
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肠道 Caco-2 细胞单层对牛奶酪蛋白进行从顶端到基底外侧的跨上皮转运：基于 MS 的细胞降解 α 和 β 酪蛋白片段定量
DOI：
10.1093/jb/mvy034
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Tsukasa Matsuda
中科院分区：
文献类型：
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作者：
Nao Sakurai;Shunsuke Nishio;Yuka Akiyama;Shinji Miyata;Kenzi Oshima;Daita Nadano;Tsukasa Matsuda
酪蛋白 (CN) 是滋养婴儿的主要乳蛋白，但在某些人群中，它会引起牛奶过敏，这表明抗原性 CN 及其肽通过肠上皮被吸收。使用人肠 Caco-2 单层细胞，研究了 CN 的顶端到基底的跨上皮转运。使用成分特异性抗体的共聚焦显微镜显示，αs1-CN抗原在顶端细胞质处以点状信号的形式被检测到，并在几个小时内以紧密连接水平到达细胞质。这种细胞内 CN 信号比其他抗原、β-乳球蛋白和卵清蛋白更显着，它们部分与早期内体标记蛋白 (EEA1) 共定位，并且在细胞松弛素 D 或叠氮化钠存在下以及在 4°C 低温下减弱。液相色谱结合质谱分析基础侧培养基中的蛋白质部分，以每孔 100-1,000 fmol 的水平鉴定出包括 N 末端区域的 αs1-CB 片段和含有多肽中心部分的 αs2-CN 片段。此外，在基础培养基中低于 10 kDa 的肽级分中鉴定出了 β-CN C 端重叠肽。这些结果表明，CN 被细胞蛋白酶和/或肽酶部分降解，并且免疫活性 CN 片段被转运至细胞单层的基底侧。
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.