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GRK 1482 Jahrbuch 2011-2014

Abstract The intestinal peptide transporter PEPT1 plays an important role in the absorption of dietary di- and tripeptides. Recently, a mass shift bet- ween the molecular weight of PEPT1 isolated from the small intestine compared to preparations of the large intestine was observed. First experiments provide evidence that differences in glycosylation result in the observed mass shift. Therefore, we aim to verify predicted gly- cosylation sites of human and murine PEPT1 and elucidate the struc- ture and function of these. In addition, tissue-specific glycosylation of PEPT1 will be investigated. Introduction Ingested proteins that enter the intestinal tract are absorbed either as free amino acids or oligopepti- des. The uptake of di- and tripeptides is mediated via a proton-dependent transport system, the oligo- peptide transporter 1 (PEPT1) PEPT1, also known as solute carrier family 15 member 1 (SLC15A1), a high capacity and low affinity transporter, located in the apical brush border membrane of enterocy- tes [1]. PEPT1 uses a proton-motive force for up- hill transport of short chain peptides and se-lective peptidomimetics from the intestinal lumen into the epithelial cells [2]. Expression cloning of the human PEPT1 trans- porter predicted a molecular weight of 78kDa, con- sisting of 708 amino acid residues and possessing 12 putative membrane-spanning segments [3]. Moreover, seven putative N-glycosylation sites at the consensus sequence motif (Asn-X-Thr/Ser) are predicted (www.uniprot.org, Acc. No.: P46059). While one N-glycosylation sites is located in the ex- tracellular loop between transmembrane domain (TMD) 1 and 2, the other six N-sequons can be found in a 202 amino acid large extracellular loop between TMD 9 and 10. In the mouse PEPT1 trans- porter (Acc. No.: Q9JIP7), five putative N-glycosyla- tion sites are suspected, while two of them have already been confirmed by mass-spectrometry [4]. In procaryotes like E. coli or Lc. lactis, expressing structural similar peptide transporters (TppB/DtpT) to the mammalian PEPT1, the extracellular loop between the TMD 9 and 10, comprising most of the putative N-glycosylation sites, is missing [1]. This raises the question about the function of the glycans,attachedtotheproteinsurfaceofthePEPT1 transporter. Glycans are highly complex sugar moi- eties, with importance for proper protein function. Beneath correct protein folding, they are essen- tial for protein trafficking and targeting. Moreover, glycans ensure protein stability, attenuate proteo- lytic degradation and increase apical surface ex- pression. However, until now little is known about the function of the glycan structures of PEPT1 re- garding possible influence on localiza-tion, trans- port activity or specificity. PhD FELLOWS Page 40 | GRK Progress Report 2011-2014 Tamara Stelzl (M.Sc.) Physiology of Human Nutrition PhD 5/2 Glycosylation of membrane proteins in the intestine- structure and functions

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