Mutations in the Leucine Zipper Motif and Sterol-sensing Domain Inactivate the Niemann-Pick C1 Glycoprotein

doi:10.1074/jbc.274.31.21861

Mutations in the Leucine Zipper Motif and Sterol-sensing Domain Inactivate the Niemann-Pick C1 Glycoprotein*

From the ^‡Center for Research on Reproduction and Women’s Health, Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104, the ^§Section of Lipid Cell Biology, NIDDK, National Institutes of Health, the ^¶Neurology Research Laboratory, Department of Veterans Affairs Connecticut Healthcare System, Newington, Connecticut 06111, and the ^‖Cellular and Molecular Pathophysiology Section, Developmental and Metabolic Neurology Branch, NINDS, National Institutes of Health, Bethesda, Maryland 20892

Abstract

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 isN-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.

Footnotes

↵* This work was supported by NIH Grants HD06274 (to J. F. S.) and NS34339 (to S. P.) and a grant from the Ara Parseghian Medical Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵** To whom correspondence should be addressed: 1354 BRBII/III, 421 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-0147; Fax: 215-573-5408; E-mail: jfs3@mail.med.upenn.edu.
Abbreviations:

NPC

Niemann-Pick type C

HMG-CoA

3-hydroxy-3-methylglutaryl coenzyme A

SCAP

sterol response element-binding protein cleavage-activating protein

EGFP

enhanced green fluorescent protein
- Received March 17, 1999.
- Revision received May 6, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.