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J. Biol. Chem., Vol. 281, Issue 48, 36710-36723, December 1, 2006

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NPC2, the Protein Deficient in Niemann-Pick C2 Disease, Consists of Multiple Glycoforms That Bind a Variety of Sterols^*>

Heng-Ling Liou, Sayali S. Dixit^¶, Sujuan Xu^¶, G. Stephen Tint^||, Ann M. Stock^¶**, and Peter Lobel¹

From the Center for Advanced Biotechnology and Medicine, ^**Howard Hughes Medical Institute, the Departments of Pharmacology and ^¶Biochemistry, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 and ^||Research Service, Veterans Affairs Medical Center, East Orange, New Jersey 07018

Niemann-Pick C disease is a fatal neurodegenerative disorder characterized by an endolysosomal accumulation of cholesterol and other lipids. One form of the disease is caused by a deficiency in NPC2, a soluble lysosomal glycoprotein that binds cholesterol. To better understand the biological function of NPC2 and how its deficiency results in disease, we have characterized the structural and functional properties of recombinant human protein. Highly purified NPC2 consists of a complex mixture of glycosylated isoforms, similar to that observed in human brain autopsy specimens. Mass spectrometric analysis revealed that of the three potential N-linked glycosylation sites present in the mature protein, Asn-19 is not utilized; Asn-39 is linked to an endoglycosidase H (Endo H)-sensitive oligosaccharide, and Asn-116 is variably utilized, either being unmodified or linked to Endo H-sensitive or Endo H-resistant oligosaccharides. All glycoforms are endocytosed and ameliorate the cholesterol storage phenotype of NPC2-deficient fibroblasts. In addition, the purified preparation contains a mixture of both free and lipid-bound protein. All glycoforms bind cholesterol, and sterol binding to NPC2 significantly alters its behavior upon cation-exchange chromatography. Based on this observation, we developed chromatography-based binding assays and determined that NPC2 forms an equimolar complex with the fluorescent cholesterol analog dehydroergosterol. In addition, we find that NPC2 binds a range of cholesterol-related molecules (cholesterol precursors, plant sterols, some oxysterols, cholesterol sulfate, cholesterol acetate, and 5--cholestan-3-one) and that 27-hydroxysterol accumulates in NPC2-deficient mouse liver. Binding was not detected for various glycolipids, phospholipids, or fatty acids. These biochemical properties support a direct and specialized function of NPC2 in lysosomal sterol transport.

Received for publication, September 11, 2006 , and in revised form, October 2, 2006.

^* This work was supported by a postdoctoral fellowship award from the National Niemann-Pick Disease Foundation (to H.-L. L.), a grant from the Office of Research and Development, Department of Veterans Affairs (to G. S. T.), and grants from the Ara Parseghian Medical Research Foundation (to A. M. S. and P. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ To whom correspondence should be addressed: Center for Advanced Biotechnology and Medicine, 679 Hoes Ln., Piscataway, NJ 08854. Tel.: 732-235-5032; Fax: 732-235-5289; E-mail: lobel{at}cabm.rutgers.edu.

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