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GRK 1482 Jahrbuch 2011-2014

Publications [1] Daniel H, Spanier B, Kottra G, Weitz D. From bacteria to man: Archaic proton-dependent peptide transporters at work. Physiology. 2006, 21: 93-102. [2] Daniel H, Kottra G. The proton oligopeptide cotranspor- ter family SLC15 in physiology and pharmacology. Pflügers Archiv. 2004, 447(5): 610-618. [3] Liang R, Fei YJ, Prasad PD, Ramamoorthy S, Han H, Yang-Feng TL, Hediger MA, Ganapathy V, Leibach FH. Human intestinal H+/peptide cotransporter. Cloning, functional expression, and chromosomal localization. J. Biol. Chem. 1995, 270 (12): 6456–6463. [4] Wollscheid B, Bausch-Fluck D, Henderson C, O‘Brien R, Bibel M, Schiess R, Aebersold R, Watts J.D. Mass-spec- trometric identification and relative quantification of N-linked cell surface glycoproteins. Nat. Biotechnol. 2009, 27: 378-386. PhD FELLOWS GRK Progress Report 2011-2014 | Page 41 Aim Based on previous findings, revealing differences of PEPT1 gly- cosylation depending on the intestinal segment, this study aims to i) verify predicted glycosylation sites in human and murine PEPT1 ii) identify function of the N-glycan structures of PEPT1 iii) clarify the composition of glycan structures attached to PEPT1 derived from different intestinal segments. Methods and Results To verify predicted glycosylation sites, site directed mutagene- sis will be used to delete single N-glycosylation sites. Therefore, asparagine of the glycosylation consensus motif is replaced by glutamine. Obtained gene-constructs will be analyzed in cell culture as well as in Xenopus laevis oocytes. Using western blot analysis of native as well as mutated PEPT1 expressed in both systems predicted glycosylation sites will be screened. Native and mutated gene constructs are furthermore transferred into a mammalian expression vector, carrying a fluorescent tag (GFP). Stable transfection of HT-29 cells, a human colon adenocarci- noma cell line, will enable live cell imaging of the intracellular localization and distribution of mutated PEPT1 by fluorescence microscopy. Co-transfection with SGLT1, an intestinal sodium- glucose transporter, fused with a fluorophore (DsRed), will be used as a control in all transfection experiments. Moreover, the protein transport activity of mutated PEPT1 for selective sub- strates, like the 14C marked dipeptide glycyl-sarcosine, will be determined. The two-electrode-voltage clamp technique (TEVC) will be used to analyze native as well as mutated PEPT1 expressed in Xenopus laevis oocytes. This electrophysiologi- cal measurement enables, via an electrical current flow, the determination of specific Km-values (transport capacity) and the affinities of PEPT1 for the transport of selected peptides (substrate specificity). The composition of the glycan structu- res attached to PEPT1 will be investigated, in vitro (HT-29) as well as in ex vivo models (mouse tissue). Therefore, isolated in- testinal brush border membrane vesicles, expressing the native transporters, will be generated. After immunoprecipitation and enzymatic deglycosylation the general types of oligosaccharide structures, present on the mature PEPT1 protein, will be identi- fied by successive Matrix assisted laser desorption ionization (MALDI). Outlook The role of glycans attached to the surface of PEPT1 is rather unknown. Neither detailed structural information about the oligosaccharide composition is available nor have functional properties been investigated so far. For this reason, this project wants to elucidate the composition of the glycans sticking to this intestinal peptide transporter and clarify their contribution and necessity for the intestinal peptide transport. Figure: Predicted topology model for human PEPT1, containing twel- ve transmembrane domains and seven putative N-glycosylation sites, marked with „Y“. Supervisors Prof. Dr. Hannelore Daniel | TUM | Physiology of Human Nutrition Dr. Kerstin Geillinger | TUM I Physiology of Human Nutrition Prof. Dr. Dirk Haller I TUM | Nutrition and Immunology Start of project: July 2011 Academic background: Studies of Nutritional Science at Technische Universität München