Glycans in the intestinal peptide transporter PEPT1 contribute to function and protect from proteolysis.
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 individualN-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 [14C]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 ofN-linked glycans, was detected.NEW & NOTEWORTHYThis 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 inXenopusoocytes 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,N-glycans were detected to stabilize PEPT1 against proteolytic cleavage.
Nutritional Physiology, Technische Universität München, Freising, Germany.
Tamara
T
Stelzl
Nutritional Physiology, Technische Universität München, Freising, Germany.
Kerstin E
KE
Geillinger-Kästle
Nutritional Physiology, Technische Universität München, Freising, Germany.
Jürgen
J
Stolz
Nutritional Physiology, Technische Universität München, Freising, Germany hannelore.daniel@tum.de.
Hannelore
H
Daniel
D000818:Animals; D001692:Biological Transport; D019921:Biotinylation; D002460:Cell Line; D004151:Dipeptides / Q000378:metabolism*; D019286:Endopeptidase K / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism*; D006031:Glycosylation; D007413:Intestinal Mucosa / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials; D051379:Mice; D009154:Mutation; D000074011:Peptide Transporter 1; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D059748:Proteolysis; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014357:Trypsin / Q000378:metabolism; D014982:Xenopus laevis
D004151:Dipeptides; D000074011:Peptide Transporter 1; C489941:Slc15a1 protein, mouse; D027981:Symporters; C004194:glycylsarcosine; D014357:Trypsin; D019286:Endopeptidase K
N-linked glycosylation; Xenopus laevis; glycoprotein; peptide transport; peptide transporter 1