N-glycans and glycosylphosphatidylinositol-anchor act on polarized sorting of mouse PrP c in Madin-Darby canine kidney cells

Abstract

The cellular prion protein (PrP C) plays a fundamental role in prion disease. PrP C 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 PrP C 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 PrP C and also replaced the GPI-anchor of PrP C by the Thy-1 GPI-anchor in order to investigate the role of these signals in sorting of PrP C 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 PrP C. Exchange of the PrP C GPI-anchor for the one of Thy-1 redirects PrP C to the apical membrane. In conclusion, both N-glycosylation and GPI-anchor act on polarized sorting of PrP C, with the GPI-anchor being dominant over N-glycans. © 2011 Puig et al.