College of Animal Science, Zhejiang University, Hangzhou 310058, China.
College of Animal Science, Zhejiang University, Hangzhou 310058, China; Department of Animal and Veterinary Sciences, University of Vermont, Burlington 05405.
J Dairy Sci. 2020 Jul;103(7):6679-6683. doi: 10.3168/jds.2019-16858. Epub 2020 Apr 22.
Bovine peptide transporter 2 (bPepT2), which mediates the absorption of di- and tripeptides in the bovine mammary gland, was predicted to contain multiple putative N-glycosylation sites of asparagine residues. N-Linked glycosylation is proven to be essential for the folding, stability, localization, and substrate binding of nutrient transporters and could therefore potentially have an essential role in the function of bPepT2. This study investigated the effect of mutagenesis of N-glycosylation sites on the transport function of bPepT2 in Chinese hamster ovary (CHO) cells. The bPepT2 cDNA was cloned and sequenced. BioXM (http://202.195.246.60/BioXM/) and TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0/) software were used to predict the AA composition and transmembrane domain of bPepT2, respectively. The AA sequence of bPepT2 was predicted to have 12 transmembrane domains, with a large extracellular loop between the ninth and tenth transmembrane domains. All 5 putative N-glycosylation sites in this loop were altered by site-directed mutagenesis, and the mutant construct was transfected into CHO cells for transport activity assay. Compared with the wild type, the bPepT2 mutant had significantly lower uptake activity of β-alanyl-l-lysyl-Nε-7-amino-4-methyl-coumarin-3-acetic acid (β-Ala-Lys-AMCA), a model dipeptide. Treatment with tunicamycin, an inhibitor of N-linked glycosylation, reduced the uptake of β-Ala-Lys-AMCA in CHO cells relative to the control group. Kinetic studies indicated that the Michaelis constant of bPepT2 was not affected by the mutation (98.03 ± 8.30 and 88.33 ± 4.23 µM for the wild type and the mutant, respectively), but the maximum transport activity was significantly reduced (40.29 ± 8.30 and 13.02 ± 2.95 pmol/min per milligram of protein for the wild type and the mutant, respectively). In summary, this study demonstrated that N-glycosylation is critical for the function of bPepT2.
牛肽转运蛋白 2（bPepT2）介导牛乳腺中二肽和三肽的吸收，据预测其包含多个天冬酰胺残基的潜在的 N-糖基化位点。已经证明 N-连接糖基化对于营养转运蛋白的折叠、稳定性、定位和底物结合至关重要，因此可能对 bPepT2 的功能具有重要作用。本研究探讨了突变 N-糖基化位点对中国仓鼠卵巢（CHO）细胞中 bPepT2 转运功能的影响。克隆并测序了 bPepT2 cDNA。使用 BioXM（http://202.195.246.60/BioXM/）和 TMHMM（http://www.cbs.dtu.dk/services/TMHMM-2.0/）软件分别预测 bPepT2 的 AA 组成和跨膜结构域。bPepT2 的 AA 序列预测有 12 个跨膜结构域，在第九和第十个跨膜结构域之间有一个大的细胞外环。该环中的所有 5 个潜在的 N-糖基化位点都通过定点突变进行了改变，并将突变构建体转染到 CHO 细胞中进行转运活性测定。与野生型相比，bPepT2 突变体对β-丙氨酰-L-赖氨酰-Nε-7-氨基-4-甲基香豆素-3-乙酸（β-Ala-Lys-AMCA）的摄取活性显著降低，β-Ala-Lys-AMCA 是一种模型二肽。用 N-连接糖基化抑制剂衣霉素处理，与对照组相比，CHO 细胞中 β-Ala-Lys-AMCA 的摄取减少。动力学研究表明，bPepT2 的米氏常数不受突变的影响（野生型和突变型分别为 98.03±8.30 和 88.33±4.23µM），但最大转运活性显著降低（野生型和突变型分别为 40.29±8.30 和 13.02±2.95 pmol/min/mg 蛋白）。总之，本研究表明 N-糖基化对 bPepT2 的功能至关重要。
