DataSheet3_Salinity-Dependent Shift in the Localization of Three Peptide Transporters along the Intestine of the Mozambique Tilapia (Oreochromis mossambicus).DOCX

The peptide transporter (PepT) systems are well-known for their importance to protein absorption in all vertebrate species. These symporters use H+ gradient at the apical membrane of the intestinal epithelial cells to mediate the absorption of small peptides. In fish, the intestine is a multifunctional organ, involved in osmoregulation, acid-base regulation, and nutrient absorption. Therefore, we expected environmental stimuli to affect peptide absorption. We examined the effect of three environmental factors; salinity, pH and feeding, on the expression, activity and localization of three PepT transporters (PepT1a, PepT1b, PepT2) along the intestine of the Mozambique tilapia (Oreochromis mossambicus). Quantitative real time PCR (qPCR) analysis demonstrated that the two PepT1 variants are typical to the proximal intestinal section while PepT2 is typical to the distal intestinal sections. Immunofluorescence analysis with custom-made antibodies supported the qPCR results, localized both transporters on the apical membrane of enterocytes and provided the first evidence for the participation of PepT2 in nutrient absorption. This first description of segment-specific expression and localization points to a complementary role of the different peptide transporters, corresponding to the changes in nutrient availability along the intestine. Both gene expression and absorption activity assays showed that an increase in water salinity shifted the localization of the PepT genes transcription and activity down along the intestinal tract. Additionally, an unexpected pH effect was found on the absorption of small peptides, with increased activity at higher pH levels. This work emphasizes the relationships between different functions of the fish intestine and how they are affected by environmental conditions.

肽转运蛋白（peptide transporter, PepT）系统因在所有脊椎动物物种的蛋白质吸收过程中发挥关键作用而广为人知。这类同向转运体利用肠上皮细胞顶膜的氢离子（H+）梯度，介导小分子肽的吸收过程。对于鱼类而言，肠道是兼具多重功能的器官，参与渗透压调节、酸碱平衡调控以及营养物质吸收等生理过程。因此我们推测，环境刺激会对肽类吸收过程产生影响。本研究以莫桑比克罗非鱼（Oreochromis mossambicus）为研究对象，探究了盐度、pH值以及摄食三种环境因素对其肠道内三种肽转运蛋白（PepT1a、PepT1b、PepT2）的表达、活性与定位的影响。实时荧光定量PCR（quantitative real time PCR, qPCR）分析结果表明，两种PepT1亚型的表达主要集中于肠道近端区段，而PepT2则特异性分布于肠道远端区段。采用定制抗体进行的免疫荧光分析验证了qPCR的结果：两种转运蛋白均定位于肠上皮细胞的顶膜，且该实验首次证实PepT2参与了营养物质吸收过程。这种针对区段特异性表达与定位的首次描述，揭示了不同肽转运蛋白间的互补功能，这与肠道内营养物质可获得性的梯度变化相契合。基因表达与吸收活性实验均显示，水体盐度升高会使PepT基因的转录及其活性沿肠道向下（远端）转移。此外，本研究还发现了一个此前未被预期的pH值对小分子肽吸收的影响：在较高pH环境下，肽吸收活性会增强。本研究阐明了鱼类肠道各项功能之间的内在关联，以及这些功能如何受环境条件调控。