Abstract
Mutations in the NPC1 gene cause Niemann-Pick type C disease, which appears to result from a defect in intracellular cholesterol trafficking. NPC1 is a member of the resistance-nodulation-cell division (RND) permease superfamily and contains a sterol-sensing domain, yet its cellular function and the identity of its substrates remain unknown. FLAG-tagged human NPC1 was purified from NPC1-expressing Chinese hamster ovary cells by solubilization in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), followed by affinity chromatography. Purified NPC1 in detergent solution appeared to be oligomeric as determined by gel filtration fast protein liquid chromatography and was photolabeled by an azido-cholesterol derivative. Fluorescent cholesterol analogs, including dehydroergosterol, cholestatrienol, and 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol), displayed enhanced fluorescence upon binding to NPC1 and also resulted in saturable, concentration-dependent quenching of NPC1 intrinsic Trp fluorescence. The apparent binding affinity for these three sterols was in the 0.5-6 microm range. Binding of NBD-cholesterol to NPC1 at low detergent concentration (2 mm CHAPS) was of high apparent affinity (0.5-0.6 microm) and occurred rapidly (<1 min). However, binding of a BODIPY-labeled cholesterol derivative was very slow, requiring approximately 3 h to reach equilibrium. The apparent NBD-cholesterol binding affinity was greatly reduced at higher detergent concentration. The stoichiometry of NBD-cholesterol binding to NPC1 was approximately 1. Various sterols, including native cholesterol and 25-hydroxycholesterol, inhibited NBD-cholesterol binding, suggesting that they compete for binding to the protein. Dynamic quenching studies showed that bound NBD-cholesterol was almost completely shielded from the aqueous medium, suggesting that it is buried in a deep hydrophobic pocket in NPC1. The use of fluorescent cholesterol analogs provides novel information on the molecular properties of the sterol-binding site in the full-length NPC1 protein.
Highlights
NPC1 is a member of the resistance-nodulation-cell division (RND) permease superfamily, a ubiquitous group of proteins found in all the major kingdoms [14]
A recent study reported that mutant mice lacking either NPC1 or NPC2 do not show any abnormalities in cholesterol absorption or uptake, indicating that the two proteins do not play a role in these processes in mammals [18]
NPC1 was purified from this extract using an antiFLAG antibody affinity column, eluting with FLAG peptide in buffer containing 2 mM CHAPS, which is below the detergent critical micelle concentration (CMC)
Summary
Materials—CHAPS was purchased from Biosynth (Naperville, IL). Tridecylphosphocholine (fos-choline 13; FC-13) was obtained from Anatrace (Maumee, OH). For measuring the binding of DHE, CTL, and NBD-cholesterol to purified NPC1, 1 l of sterol stock (in methanol) was added to 59 l of NPC1 (40 g/ml; 0.29 M; protein molecular mass 139 kDa/ mol) to a final concentration of 1 M. Kinetics of Binding of Fluorescent Sterols to NPC1—Cholesteryl-BODIPY (0.5 M) was added to purified NPC1 (0.29 M) in 2 mM CHAPS, 0.2 mM FC-13 in HEPES buffer. Quenching of NPC1 Intrinsic Trp Fluorescence—Binding of fluorescent sterols to NPC1 was measured in 2 mM CHAPS/ HEPES buffer by adding 1 l of increasing concentrations of sterol working solutions in methanol to a series of microcentrifuge tubes containing 59 l of NPC1 (0.29 M), followed by incubation at 4 °C for ϳ3 h (for DHE and CTL) and 30 min for NBD-cholesterol. A plot of F0/F versus [Q] gives a linear plot with a slope of KSV
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