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

Abstract

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-α-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. 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-α-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. Cholesterol is important for a wide variety of physiological processes, including serving as a major component of lipid bilayers and droplets and as a precursor in steroid hormone biosynthesis. One of the major pathways by which cholesterol enters cells is through receptor-mediated endocytosis of LDL 2The abbreviations used are: LDL, low density lipoprotein; NPC, Niemann-Pick C; DHE, ergosta-5,7,9,(11),22-tetraen-3β-ol; PNGase F, peptide:N-glycosidase F; Endo H, endoglycosidase H; LBPA, lysobisphosphatidic acid; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; GC, gas chromatography; bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PBS, phosphate-buffered saline; ACTH, adrenocorticotropic hormone. particles. The LDL is delivered to lysosomes where cholesteryl esters are hydrolyzed by acid lipase and the liberated free cholesterol is then transported to other subcellular sites for further utilization and also for feedback regulation of cholesterol biosynthesis and LDL uptake. Although many of the steps involved in this pathway are well characterized, the mechanism by which free cholesterol exits the lysosome is poorly understood. Studies of two hereditary childhood neurodegenerative disorders with similar phenotypes, Niemann-Pick C (NPC) type 1 and type 2 diseases, have begun to provide insights into lysosomal cholesterol transport. On a cellular level, both diseases are characterized by lysosomal storage of cholesterol and other lipids, including sphingomyelin, glycolipids, and phospholipids. Fibroblasts from individuals with NPC disease accumulate LDL-derived cholesterol in lysosomes and are impaired in homeostatic responses that normally follow LDL uptake (1Liscum L. Ruggiero R.M. Faust J.R. J. Cell Biol. 1989; 108: 1625-1636Crossref PubMed Scopus (253) Google Scholar, 2Pentchev P.G. Kruth H.S. Comly M.E. Butler J.D. Vanier M.T. Wenger D.A. Patel S. J. Biol. Chem. 1986; 261: 16775-16780Abstract Full Text PDF PubMed Google Scholar). The gene defective in NPC type 1 disease, NPC1, encodes an ∼1278-amino acid protein containing 13 transmembrane domains and a putative sterol-sensing domain (3Carstea E.D. Morris J.A. Coleman K.G. Loftus S.K. Zhang D. Cummings C. Gu J. Rosenfeld M.A. Pavan W.J. Krizman D.B. Nagle J. Polymeropoulos M.H. Sturley S.L. Ioannou Y.A. Higgins M.E. Comly M. Cooney A. Brown A. Kaneski C.R. Blanchette-Mackie E.J. Dwyer N.K. Neufeld E.B. Chang T.Y. Liscum L. Strauss J.F. II I Ohno K. Ziegler M. Carmi R. Sokol J. Markie D. O'Neill R.R. van Diggelen O.P. Elleder M. Patterson M.C. Brady R.O. Vanier M.T. Pentchev P.G. Tagle D.A. Science. 1997; 277: 228-231Crossref PubMed Scopus (1251) Google Scholar). The NPC1 protein has primary to the Y.A. Cell Biol. PubMed Scopus Google and in endolysosomal Although lysosomal cholesterol accumulates in NPC type 1 disease, is the direct of NPC1 in cholesterol M. Chang Chang T.Y. S. A. PubMed Scopus Google Scholar, L. Sturley S.L. PubMed Scopus Google Scholar). The gene defective in NPC type 2 disease, NPC2, encodes a acid with a acid that is to the lysosome S. A. Vanier M.T. R. M. Science. PubMed Scopus Google Scholar). protein, which is also present in C. C. L. Biol. PubMed Scopus Google and A. L. J. 1997; PubMed Scopus Google binds cholesterol S. M. S. PubMed Scopus Google and as a cholesterol protein in Biol. Scholar, R. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of NPC2 important for sterol binding have S. A. PubMed Scopus Google and J. A. S. A. PubMed Scopus Google NPC1 and NPC2 that both function in a pathway in lysosomal lipid J.A. M. L. Vanier M.T. S. A. PubMed Scopus Google Scholar). In this as a the of NPC2 in and disease processes, we have characterized biochemical and binding properties of functional recombinant human NPC2 a The preparation consists of NPC2 glycoforms that are of binding cholesterol, and we find that binding of some a in the properties of Based on this observation, we have developed a chromatography-based binding and have this to to potential and to NPC2 as the of in cholesterol transport. results that NPC2 a in the cellular of a variety of in to cholesterol, an that important in the that in the of this protein. was from lysobisphosphatidic acid also and from and from sterols, lipids, and from protein protein (Endo to as Endo and PNGase from from protein was from was by with by NPC2 cells with the human NPC2 into the D. J. Biol. Chem. Full Text PDF PubMed Google which contains a by the mouse a and a mouse with and with of One that in the of was used for of recombinant protein. was as L. J. PubMed Scopus Google that was in the some cells in containing of the Cell In a from C. C. L. Biol. PubMed Scopus Google for and to a All The was three with of PBS, and the and into by a with a The was to a and an in to the mixture of was The was and into NPC2 was and glycoforms by on a that was a of to a 2 To the to from for was an to the major in with Endo H in in for by of and then with PNGase in by by and and of from the in was with to the used by the M. for and then with a acid in or acid in acid containing a mass in the to a of 1 protein in acetate, a of protein was to a and a of to a and purified NPC2 and to to In some the was with 2 of 1 to and to protein for further In other by the with an of a of and the was either by gas with as J.A. M. L. Vanier M.T. S. A. PubMed Scopus Google or into a of for further analysis by or cholesterol, and and the J. J. Full Text PDF PubMed Google Scholar). Cholesterol in a on in with and with containing NPC2 of protein from the in the and of cells to three with for with in for three with PBS, and with complex and 1 in for the cells three with PBS, and the with in The an with and and with a of this was used for the to of and Binding NPC2 in acetate, was with of the of for and by cation-exchange as of NPC2 the poorly soluble cholesterol cholesterol and cholesterol acetate, where the NPC2 in a of cholesterol that the and not the to binding the in an and the of in the mixture was of binding we determined the of from its and its its of and and 1986; PubMed Scopus Google Scholar, R. J. Biol. Chem. 277: Full Text Full Text PDF PubMed Scopus Google Scholar). to or to NPC2 in that free in and with NPC2 similar The of NPC2 was determined the of the on the for the of The of was in the of for Binding as for the direct binding that NPC2 was with for by with 1 of a in to the The was for cation-exchange chromatography. 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PubMed Scopus Google Scholar). mass revealed that the from the NPC2 the of that the mass for the mature NPC2 protein is and from this the of from to on the NPC2 PNGase to a mass of that the observed by was of in glycosylation was further a of and mass the glycosylation of the three potential N-linked glycosylation on NPC2 are in and the for is utilization of in a to the sites and of the was on the NPC2 contains three and on the of the mature in the of a potential glycosylation These are by the and that have of and are PNGase or Endo H have on and of the a potential glycosylation is this the mass in the of the from the observed mass with PNGase N-linked and the to a PNGase have a mass that of the unmodified an N-linked is to Endo H, which and some the two of a glycosylated a which has a mass the unmodified by the in the of the NPC2 PNGase and Endo H and then further the with and the mass is to a is glycosylated and also the of the analysis revealed that NPC2 present in 1 Endo H and PNGase analysis of of from PNGase and Endo 1 revealed that the Asn-19 was present with the of that this potential glycosylation is not In the unmodified Asn-39 was not PNGase and Endo H the 1 and of and that Asn-39 was with an Endo H-sensitive an to the unmodified was not in the 1 of in the PNGase PNGase 1 also a that to the 1 of an that Asn-116 The unmodified was not detected in the Endo 1 the to or not detected and not that Asn-116 on the NPC2 present in 1 is linked to an Endo H-resistant analysis of of human NPC2 containing the potential Asn-39 was as in of the are for and PNGase and Endo All from analysis of glycosylated with or PNGase of the for and or and containing the putative Asn-116 as in All from and or and analysis of glycosylated with or Endo H of human NPC2 containing the putative Asn-116 was by as are for and the for Endo or for the of Endo The in 2 that have similar to The protein present in as a by Endo H two with a and the other with a The was for 2 and and the was for PNGase a to the by PNGase of mass analysis of the and PNGase results to for that Asn-39 and Asn-116 not Asn-19 glycosylated and mass of the by Endo H of results to the 1 In analysis of the by Endo H of revealed that in to the for the we also a that to an that was with we not the for that of Asn-116 by results that NPC2 with either Endo H-sensitive Asn-39 and Asn-116 form in 2 and and form in or with an Endo H-sensitive Asn-39 and an Endo H-resistant Asn-116 form in and forms in 2 and and results a with both Endo H and PNGase mass as in Asn-19 is not Asn-39 contains an Endo H-sensitive the of and for and detected for the and that Asn-116 is not and a Endo H-sensitive NPC2 of and Although the NPC2 to in further revealed an In the of a for NPC2 on the in of upon binding to protein, we that of protein, a significantly S. 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The that accumulates that this potential NPC2 transported by NPC2 in by and A. J.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that of and LDL uptake was in primary from individuals with NPC type 1 and 2 diseases, to a from impaired lysosomal of cholesterol. that some accumulate in mouse Pentchev S. A. J. PubMed Scopus Google and that lipid storage is similar in both NPC1 and NPC2 J.A. M. L. Vanier M.T. S. A. PubMed Scopus Google Scholar, M.T. S. C. Pentchev E.D. J. Google Scholar). Although is the of Biol. PubMed Scopus Google is that some of molecules normally the lysosome and are the NPC as and of and other and accumulation of molecules in the lysosome to the of NPC disease, either by direct on the lysosome or by the and of molecules or for function in some other for with and for for preparation of and and for and with protein and with