Mutations in the leucine zipper motif and sterol-sensing domain inactivate the Niemann-Pick C1 glycoprotein.

Niemann-Pick type C (NPC) disease, characterized by accumulation of low density lipoprotein-derived free cholesterol in lysosomes, is caused by mutations in the NPC1 gene. We examined the ability of wild-type NPC1 and NPC1 mutants to correct the NPC sterol trafficking defect and their subcellular localization in CT60 cells. Cells transfected with wild-type NPC1 expressed 170- and 190-kDa proteins. Tunicamycin treatment resulted in a 140-kDa protein, the deduced size of NPC1, suggesting that NPC1 is N-glycosylated. Mutation of all four asparagines in potential N-terminal N-glycosylation sites to glutamines resulted in a 20-kDa reduction of the expressed protein. Proteins with a single N-glycosylation site mutation localized to late endosome/lysosomal compartments, as did wild-type NPC1, and each corrected the cholesterol trafficking defect. However, mutation of all four potential N-glycosylation sites reduced ability to correct the NPC phenotype commensurate with reduced expression of the protein. Mutations in the putative sterol-sensing domain resulted in inactive proteins targeted to lysosomal membranes encircling cholesterol-laden cores. N-terminal leucine zipper motif mutants could not correct the NPC defect, although they accumulated in lysosomal membranes. We conclude that NPC1 is a glycoprotein that must have an intact sterol-sensing domain and leucine zipper motif for cholesterol-mobilizing activity.