Abstract
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.
Highlights
Prion diseases, occurring in humans and a wide range of animals, are believed to be caused by misfolding of PrPC into a disease-associated form, PrPSc [1,2]
PrPC is a glycosylphosphatidylinositol (GPI)-anchored protein residing in detergent-resistant membranes (DRMs) and removed from DRMs in order to be internalized via clathrin-coated endocytosis
Because the role of glycosylation in sorting of PrPC is poorly understood, in this study we investigated the role of N-glycans and the GPI-anchor as potential polar sorting signals of PrPC expressed in Madin-Darby canine kidney (MDCK) cells
Summary
Prion diseases, occurring in humans and a wide range of animals, are believed to be caused by misfolding of PrPC into a disease-associated form, PrPSc [1,2]. PrPSc is enriched in b-sheets and forms partially protease-resistant aggregates which mainly accumulate in the central nervous system [3]. PrPC is a glycosylphosphatidylinositol (GPI)-anchored protein residing in detergent-resistant membranes (DRMs) and removed from DRMs in order to be internalized via clathrin-coated endocytosis. DRMs have been postulated as sites of conversion from PrPC to PrPSc either directly at the cell surface or in the early endocytic pathway [9]. Divergence or absence of GPIanchorage of PrPC influences development of prion disease [10,11]
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