NPC2, the protein deficient in Niemann-Pick C2 disease, consists of multiple glycoforms that bind a variety of sterols
NPC2, the protein deficient in Niemann-Pick C2 disease, consists of multiple glycoforms that bind a variety of sterols
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DOI：
10.1074/jbc.m608743200
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发表时间：
2006-12-01
影响因子：
4.8
通讯作者：
Lobel, Peter
中科院分区：
文献类型：
--
作者：
Liou, Heng-Ling;Dixit, Sayali S.;Lobel, Peter
尼曼-皮克C病是一种致命的神经退行性疾病，其特征是体内胆固醇和其他脂质的积聚。一种形式的疾病是由NPC2缺乏引起的，NPC2是一种结合胆固醇的可溶性溶酶体糖蛋白。为了更好地了解NPC2的生物学功能以及它的缺失如何导致疾病，我们对重组人蛋白的结构和功能特性进行了表征。高度纯化的NPC2由糖基化的异构体组成的复杂混合物，类似于在人脑尸检标本中观察到的。质谱分析表明，在成熟蛋白质中存在的三个潜在的N-连接糖基化位点中，ASN-19没有被利用；ASN-39连接到内切糖苷酶H(Endo H)敏感的寡糖，而ASN-116被可变地利用，要么是未经修饰的，要么是连接到Endo H敏感或Endo H抗性的寡糖。所有的糖形式都是内吞的，并改善了NPC2缺陷的成纤维细胞的胆固醇储存表型。此外，纯化后的制剂含有游离态和脂结态蛋白质的混合物。所有的糖形式都与胆固醇结合，而固醇与NPC2的结合显著改变了它在阳离子交换层析中的行为。基于这一观察，我们发展了基于层析的结合分析，并确定NPC2与荧光胆固醇类似物脱氢麦角甾醇形成等摩尔复合体。此外，我们发现NPC2结合了一系列与胆固醇相关的分子(胆固醇前体、植物甾醇、一些氧合甾醇、胆固醇硫酸盐、胆固醇醋酸酯和5-α-胆甾烷-3-酮)，并且27-羟基甾醇在NPC2缺陷小鼠的肝脏中积累。没有检测到各种糖脂、磷脂或脂肪酸的结合。这些生化特性支持NPC2在溶酶体类固醇运输中的直接和专门功能。
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-alpha-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.