Functional analysis of N‐glycosylation in the intestinal peptide transporter PEPT1

The intestinal peptide transporter PEPT1 (SLC15A1) mediates electrogenic proton‐coupled influx of dietary di‐ and tripeptides as well as of peptidomimetics into epithelial cells. Western blot analyses performed with protein isolates of mouse intestine revealed the existence of diverse PEPT1 glycoforms (Wuensch, T., et al. Am J Physiol Gastrointest Liver Physiol 305: G66‐73, 2013). In small intestine PEPT1 exhibits a mass of ~95 kDa, while in colon the transporter appears with a mass ~105 kDa. Enzymatic deglycosylation with PNGaseF uniformly reduced the PEPT1 mass in both gut segments to ~65 kDa. These mass differences addressed the question of whether glycosylation has an effect on function. We thus assessed whether different glycosylation states alter PEPT1 transport activity. By targeted mutagenesis, putative N‐glycosylation sites in murine PEPT1 were disrupted and mutant transporters heterologously expressed in Xenopus laevis oocytes. Replacement of six asparagine residues (N) at positions N50, N406, N439, N510, N515 and N532 by glutamine (Q) resulted in a decrease of the PEPT1 mass by around 35 kDa. Electrophysiology revealed all glycosylation‐deficient transporters to be functional with comparable expression levels in oocyte membranes. Remarkably, in the mutant transporter N50Q affinity for glycyl‐sarcosine (Gly‐Sar) decreased from 0.66 ±0.12 mM to 1.33 ± 0.23 mM while maximal inward currents increased 2.5‐fold from 0.85 ± 0.21 nA to 2.11 ± 0.21 nA. Tracer flux studies with [ 14 C]‐Gly‐Sar confirmed the reduction in substrate affinity of the N50Q mutant while maximal influx rate increased from 2.32 ± 0.30 to 4.47 ± 0.32 pmol/min/oocyte. In the human protein (hPEPT1) the N50Q mutant as well presented a lower Gly‐Sar affinity (0.8 ± 0.1 mM in WT versus 1.2 ± 0.2 mM in mutant) and a maximal inward current increased from 1.6 ± 0.2 to 4.1 ± 1.4 nA. Since neither the surface density of the mutant transporter N50Q was changed, nor its conductance, we assume that N‐glcyosylation at N50 constraints the movement of the N‐terminal domain during the transport cycle. Attachment of N‐linked oligosaccharides at sequon N50, with an identified overall mass of 3–5 kDa, seems to attenuate PEPT1 mobility while the absence of the glycans could accelerate the Support or Funding Information This work was funded by GRK 1482 by Deutsche Forschungsgemeinschaft