Biochemical Characterization of Niemann-Pick C: a Disease of Cholesterol Transport

Despite intense scientific interest, the mechanism by which cholesterol is transported between membrane compartments in animal cells remains obscure. One transport pathway begins in lysosomes where cholesterol is liberated from plasma lipoproteins that have entered the cell through receptor-mediated endocytosis. This cholesterol is transported from the lysosome to other cellular membranes to perform structural and regulatory roles. A clue to the mechanism of this cholesterol movement comes from observations in cells from patients with Niemann-Pick Type C (NPC) disease. These individuals accumulate large amounts of cholesterol throughout the body caused by mutations in either one of two genes encoding the lysosomal proteins NPC1 and NPC2. Unlike the membrane protein NPC1, evidence suggests that the soluble protein NPC2 is a cholesterol binding protein.

In the course of isolating a cholesterol-homeostasis membrane protein that binds sterols, we encountered NPC1. Using rabbit membranes, an integral membrane protein that bound sterols was isolated with a 14,000-fold purification while maintaining 8% final yield. Mass spectrometry identified the protein to be NPC1. Recombinant human NPC1 was expressed, purified, and confirmed to be a high affinity sterol receptor. NPC1Õs sterol binding domain was localized to itÕs N-terminal luminal soluble domain, which can be prepared as a soluble protein of 240 amino acids, NPC1(NTD), that is secreted by cells. The binding properties of NPC1(NTD) binds cholesterol similar to NPC2 with a Kd of ~130nM. Cross-competition studies between purified NPC1(NTD) and the soluble NPC2 protein revealed differences in sterol specificity depicting the different parts of the cholesterol moiety the NPC proteins bind. [Keywords: Niemann-Pick C Disease; cholesterol trafficking; cholesterol binding; oxysterols; lysosomes; Niemann-Pick C1 protein; Niemann-Pick C2 protein]