Glycans in the intestinal peptide transporter PEPT1 contribute to function and protect from proteolysis

Abstract

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.

Post-translational modifications of transporters

“…Therefore, it is expected that deglycosylation at this residue, or alterations in the glycosylation pattern, will positively modulate PEPT1 function (56). However, further investigation suggested that the fundamental role of N -glycosylation at N50 is to protect the extracellular loops from protease degradation in oocytes, and further studies are needed to determine whether this mechanism is consistent in other expression models (57). This finding may mechanistically explain the variability in function between species and tissues (57).…”

A Novel Heterogalactan from Antrodia camphorata and Anti-Angiogenic Activity of Its Sulfated Derivative

“…Finally, the retentate was lyophilized to obtainthe final sulfated derivate ACW0-Sul. The degree of sulfation (DS), which is the average number of sulfate groups on each sugar residue, was calculated from the sulfur content based on the barium chloride gelatin method [30], using the following formula:DS = 162 × S% (32 − 102 × S%)…”

Influence of physiological and chemical factors on the absorption of bioactive peptides

“…The study showed that removal of glycans increased the maximal Gly‐Sar peptide transport rate. In addition, these experiments demonstrated that glycosylation of PepT1 stabilised this transporter against the proteolytic action of trypsine and proteinase K; however, in the case of trypsin, the transporter was stable independently of N‐linked glycans (Stelzl et al ., ).…”

“…Stelzl et al . () investigated the influence of glycosylation on the rate of Gly‐Sar transport. The study showed that removal of glycans increased the maximal Gly‐Sar peptide transport rate.…”