N-Glycans and Glycosylphosphatidylinositol-Anchor Act on Polarized Sorting of Mouse PrPC in Madin-Darby Canine Kidney Cells

The cellular prion protein (PrPC) plays a fundamental role in prion disease. PrPC is a glycosylphosphatidylinositol (GPI)-anchored protein with two variably occupied N-glycosylation sites. In general, GPI-anchor and N-glycosylation direct proteins to apical membranes in polarized cells whereas the majority of mouse PrPC is found in basolateral membranes in polarized Madin-Darby canine kidney (MDCK) cells. In this study we have mutated the first, the second, and both N-glycosylation sites of PrPC and also replaced the GPI-anchor of PrPC by the Thy-1 GPI-anchor in order to investigate the role of these signals in sorting of PrPC in MDCK cells. Cell surface biotinylation experiments and confocal microscopy showed that lack of one N-linked oligosaccharide leads to loss of polarized sorting of PrPC. Exchange of the PrPC GPI-anchor for the one of Thy-1 redirects PrPC to the apical membrane. In conclusion, both N-glycosylation and GPI-anchor act on polarized sorting of PrPC, with the GPI-anchor being dominant over N-glycans

N-Glycosylation instead of cholesterol mediates oligomerization and apical sorting of GPI-APs in FRT cells

In contrast to MDCK cells, in FRT cells oligomerization and apical sorting of GPI-APs are mediated by N-glycosylation independent of cholesterol and raft association

Sorting GPI-anchored proteins

The study of glycosylphosphatidylinositol-anchored-protein sorting has led to some surprising new findings and concepts. Evidence is accumulating that, during their delivery to the surface, different types of plasma membrane protein might be sorted from each other early in this pathway, in the endoplasmic reticulum. Furthermore, membrane-lipid composition and microdomains might have a role in the process of protein sorting in both the secretory and endocytic pathways