Stelzl Tamara, Geillinger-Kästle Kerstin E, Stolz Jürgen, Daniel Hannelore
Nutritional Physiology, Technische Universität München, Freising, Germany.
Nutritional Physiology, Technische Universität München, Freising, Germany
Am J Physiol Gastrointest Liver Physiol. 2017 Jun 1;312(6):G580-G591. doi: 10.1152/ajpgi.00343.2016. Epub 2017 Mar 23.
Despite the fact that many membrane proteins carry extracellular glycans, little is known about whether the glycan chains also affect protein function. We recently demonstrated that the proton-coupled oligopeptide transporter 1 (PEPT1) in the intestine is glycosylated at six asparagine residues (N50, N406, N439, N510, N515, and N532). Mutagenesis-induced disruption of the individual -glycosylation site N50, which is highly conserved among mammals, was detected to significantly enhance the PEPT1-mediated inward transport of peptides. Here, we show that for the murine protein the inhibition of glycosylation at sequon N50 by substituting N50 with glutamine, lysine, or cysteine or by replacing S52 with alanine equally altered PEPT1 transport kinetics in oocytes. Furthermore, we provide evidence that the uptake of [C]glycyl-sarcosine in immortalized murine small intestinal (MODE-K) or colonic epithelial (PTK-6) cells stably expressing the PEPT1 transporter N50Q is also significantly increased relative to the wild-type protein. By using electrophysiological recordings and tracer flux studies, we further demonstrate that the rise in transport velocity observed for PEPT1 N50Q is bidirectional. In line with these findings, we show that attachment of biotin derivatives, comparable in weight with two to four monosaccharides, to the PEPT1 N50C transporter slows down the transport velocity. In addition, our experiments provide strong evidence that glycosylation of PEPT1 confers resistance against proteolytic cleavage by proteinase K, whereas a remarkable intrinsic stability against trypsin, even in the absence of -linked glycans, was detected. This study highlights the role of N50-linked glycans in modulating the bidirectional transport activity of the murine peptide transporter PEPT1. Electrophysiological and tracer flux measurements in oocytes have shown that removal of the N50 glycans increases the maximal peptide transport rate in the inward and outward directions. This effect could be largely reversed by replacement of N50 glycans with structurally dissimilar biotin derivatives. In addition, -glycans were detected to stabilize PEPT1 against proteolytic cleavage.
尽管许多膜蛋白携带细胞外聚糖，但对于聚糖链是否也影响蛋白质功能却知之甚少。我们最近证明，肠道中的质子偶联寡肽转运体1（PEPT1）在六个天冬酰胺残基（N50、N406、N439、N510、N515和N532）处发生糖基化。诱变诱导破坏在哺乳动物中高度保守的单个N-糖基化位点N50，被检测到可显著增强PEPT1介导的肽向内转运。在此，我们表明，对于小鼠蛋白，通过用谷氨酰胺、赖氨酸或半胱氨酸替代N50或用丙氨酸替代S52来抑制N50位点的糖基化，同样会改变卵母细胞中PEPT1的转运动力学。此外，我们提供的证据表明，在稳定表达PEPT1转运体N50Q的永生化小鼠小肠（MODE-K）或结肠上皮（PTK-6）细胞中，相对于野生型蛋白，[C]甘氨酰肌氨酸的摄取也显著增加。通过使用电生理记录和示踪剂通量研究，我们进一步证明，观察到的PEPT1 N50Q转运速度的增加是双向的。与这些发现一致，我们表明，将重量与两到四个单糖相当的生物素衍生物连接到PEPT1 N50C转运体上会减慢转运速度。此外，我们的实验提供了有力证据，表明PEPT1的糖基化赋予了对蛋白酶K蛋白水解切割的抗性，而即使在没有N-连接聚糖的情况下，也检测到对胰蛋白酶具有显著的内在稳定性。这项研究突出了与N50相连的聚糖在调节小鼠肽转运体PEPT1双向转运活性中的作用。卵母细胞中的电生理和示踪剂通量测量表明，去除N50聚糖会增加向内和向外方向的最大肽转运速率。用结构不同的生物素衍生物替代N50聚糖可在很大程度上逆转这种效应。此外，检测到N-聚糖可稳定PEPT1以抵抗蛋白水解切割。
