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.
Center for Research on Reproduction and Women's Health, Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, USA.
H
H
Watari
E J
EJ
Blanchette-Mackie
N K
NK
Dwyer
M
M
Watari
E B
EB
Neufeld
S
S
Patel
P G
PG
Pentchev
J F
JF
Strauss
D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine; D001665:Binding Sites; D016466:CHO Cells; D002352:Carrier Proteins; D002784:Cholesterol / Q000378:metabolism; D006224:Cricetinae; D011992:Endosomes / Q000378:metabolism; D005973:Glutamine; D006031:Glycosylation; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D016350:Leucine Zippers; D008247:Lysosomes / Q000378:metabolism; D008562:Membrane Glycoproteins; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D000077296:Niemann-Pick C1 Protein; D009542:Niemann-Pick Diseases / Q000235:genetics*; D010641:Phenotype; D017354:Point Mutation; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013261:Sterols / Q000378:metabolism*; D014162:Transfection
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; D011506:Proteins; D011994:Recombinant Proteins; D013261:Sterols; D005973:Glutamine; D001216:Asparagine; D002784:Cholesterol