The integrity of a cholesterol-binding pocket in Niemann-Pick C2 protein is necessary to control lysosome cholesterol levels

Abstract

The neurodegenerative disease Niemann-Pick Type C2 (NPC2) results from mutations in the NPC2 (HE1) gene that cause abnormally high cholesterol accumulation in cells. We find that purified NPC2, a secreted soluble protein, binds cholesterol specifically with a much higher affinity (K(d) = 30-50 nM) than previously reported. Genetic and biochemical studies identified single amino acid changes that prevent both cholesterol binding and the restoration of normal cholesterol levels in mutant cells. The amino acids that affect cholesterol binding surround a hydrophobic pocket in the NPC2 protein structure, identifying a candidate sterol-binding location. On the basis of evolutionary analysis and mutagenesis, three other regions of the NPC2 protein emerged as important, including one required for efficient secretion.

Figure 1

Purified tagged NPC2 protein complements cholesterol accumulation in npc2 cells with ≈4.5 × 10⁵ molecules of protein/cell at steady-state being sufficient for rescue. (A) NPC2 with C-terminal myc and 6xHis tags is as effective as wild-type NPC2 in rescuing npc2 fibroblasts. After treatment for 3 days with 10% conditioned media from CHO-KI cells transiently transfected with NPC2 or NPC2-myc-his, npc2 fibroblasts were stained with filipin. (Bar = 20 μm.) Dose–response curves quantifying the average filipin fluorescence/area show that NPC2 and NPC2-myc-his are similarly effective in clearing cholesterol accumulation in npc2 fibroblasts (EC₅₀ ≈ 0.5%). Much less activity is found in the enhanced GFP conditioned media (EC₅₀ ≈ 10%) due to the endogenous NPC2 produced by CHO cells. Error bars are standard deviations of fluorescence/area from seven imaged fields. (Inset) Protein blot of conditioned media probed with anti-NPC2. NPC2 and NPC2-myc-his are expressed at similar levels. (B) Assessment of NPC2-myc-his purification. NPC2-myc-his conditioned media and purified fractions after nickel-affinity purification and gel filtration were resolved by SDS/PAGE in a 12% gel and stained for total protein with SYPRO Ruby. NPC2-myc-his purified from conditioned media from stably expressing cells migrates as a doublet with an apparent molecular mass of 27–29 kDa. (C) The EC₅₀ of purified NPC2 in complementing npc2 cells is 1.5 nM. Addition of NPC2-myc-his purified by the two-step protocol to npc2 fibroblasts induces a dose-dependent decrease in lysosomal cholesterol, as assessed by filipin staining. Error bars are standard deviations of fluorescence/area from seven imaged fields. (D) Purification table of NPC2-myc-his two-step purification. Total protein at each step was determined by Bradford assay, and activity was determined by filipin staining with the amount necessary for 50% rescue defined as 1 unit. (E) The number of NPC2 molecules required in a single npc2 cell for rescue is ≈4.5 × 10⁵. npc2 cells were incubated overnight with the indicated amount of purified NPC2-myc-his. Longer incubation (3 days) did not alter the amount of NPC2-myc-his detected. Incubation of conditioned media along with 5 mM mannose-6-phosphate to block uptake of the NPC2 protein was done concurrently to ensure that the protein detected was specific. Cell extracts along with known amounts of purified NPC2-myc-his protein were immunoblotted with anti-NPC2. A single band of ≈22 kDa was detected. The amount of NPC2 protein within each cell was estimated based on standard curves of purified NPC2-myc-his and dividing by the total number of cells loaded into the lane (2 × 10⁵). The graph shows the mean and standard deviations from three experiments. Cells incubated with the indicated concentration of protein for 3 days were filipin stained concurrently to determine the degree of rescue.

Figure 2

Purified NPC2 protein binds cholesterol with a K_d of ≈30–50 nM. (A) Association and dissociation of cholesterol to NPC2-myc-his. Purified NPC2-myc-his (0.8 μg/ml) was incubated with 10 nM [³H]cholesterol in PBS (pH = 7.4) at 30°C. For the association curve, aliquots were removed at the times indicated and immediately centrifuged through a centrisep column to separate free and bound cholesterol, and bound counts were measured. For the dissociation curve, 500 nM cold cholesterol was added at time 0 to block free sites. At the times indicated, aliquots were taken and analyzed as above. Nonlinear regression analysis of the curves reveals k_off = 0.18 min⁻¹, k_on = 5.8 × 10⁶ M⁻¹⋅min⁻¹, and K_d = k_off/k_on = 30 nM. Error bars show the standard deviations of triplicate measurements. (B) Saturation binding of cholesterol to NPC2-myc-his. Purified NPC2-myc-his (0.5 μg/ml) was incubated with the indicated amount of [³H]cholesterol in PBS at pH 7.4 or 5.0 for 30 min at 30°C. Bound counts were collected by gel filtration and measured. Analysis of the saturation curves shown gives K_d = 20 nM for pH = 7.4 and K_d = 17 nM for pH = 5.0. From at least four similar measurements, we obtained K_d values of 27 ± 15 nM at pH = 7.4 and 32 ± 24 nM at pH = 5.0. (C) Cholesteryl oleate and oleic acid are unable to compete for the NPC2-myc-his cholesterol binding site. Purified NPC2-myc-his (0.5 μg/ml) was incubated with 10 nM [³H]cholesterol and the indicated amount of cold competitor for 30 min at 30°C. Bound counts were collected by gel filtration and measured. Cholesterol is able to compete for binding with a calculated K_d of 50 nM, whereas oleic acid and cholesteryl oleate show no competition even at the highest concentration tested (8 μM). Error bars show the standard deviations of measurements taken from three independent experiments.

Figure 3

Evolutionary analysis of the NPC2 protein reveals amino acids predicted to be important for function. A plot of local evolutionary rates across the NPC2 protein is shown, along with the multiple sequence alignment of vertebrate NPC2 homologs used to estimate those rates and the mutations constructed within the NPC2 protein. The values plotted along the y axis are derived from the average number of amino acid substitutions per site (Materials and Methods) for residues at the corresponding x axis position. The circles on the plot mark the four evolutionarily constrained regions (ECRs A, B, C, and D from left to right). The sequence is numbered beginning with the first amino acid after the signal sequence cleavage site (marked by an arrow). Asterisks, colons, or dots below the sequence alignment indicate identical, highly similar, and similar residues, respectively. Mutations made in the mouse sequence are shown as colored amino acid residues; the substituting residue is shown above the mouse sequence. Red indicates conserved charged residues; blue indicates conserved hydrophobic residues predicted to be at least partially surface-exposed; green indicates conserved aromatic residues; and yellow indicates other conserved residues (see text). The positions of the mutations are highlighted on the rate plot with diamonds of the same colors.

Figure 4

Mutations in NPC2 that specifically decrease cholesterol binding interfere with mobilization of lysosomal cholesterol. (A) Rescue of npc2 fibroblasts with NPC2-myc-his conditioned media. A single concentration of wild-type NPC2-myc-his conditioned media was chosen that results in an intermediate level of rescue (≈75%) when added to npc2 cells. Equivalent amounts of each of the mutants were added to npc2 cells and analyzed by filipin staining after incubation for 3 days. The level of rescue normalized to 100% for wild type is given as a mean percentage with the standard deviation from at least three independent experiments. Of the 17 mutants assayed, only six showed a significantly reduced level of rescue, and these are labeled in orange (<70% rescue) and red (<50% rescue). (B) Protein blot of the six loss-of-function mutants purified from conditioned media. Conditioned media from transiently transfected CHO-KI cells were concentrated and purified by nickel affinity. Protein blotting with anti-myc shows that NPC2-myc-his from transient transfection is secreted as three major bands between 27 and 32 kDa. All three forms are present in each of the mutants. Variation in the amount of protein was corrected for subsequent experiments by comparison with a wild-type standard curve (5–15 μl). (C) Dose–response curves for rescue of npc2 fibroblasts with purified NPC2 proteins. Each of the purified NPC2-myc-his proteins was added at the indicated concentration to npc2 cells for 3 days, fixed, and filipin stained, and the fluorescence from seven fields was quantified. The EC₅₀ value for each is given, along with the activity with wild type normalized to 100%. All six mutants identified by the conditioned media assay have severely reduced activity, with the three most severe mutations (F66A, V96F, and Y100A) exhibiting <10% of wild-type activity. (D) Assessment of the cholesterol-binding ability of mutant NPC2 proteins. Each of the purified NPC2-myc-his proteins was incubated with increasing amounts of [³H]cholesterol to measure saturation binding. Calculation of the K_d values from these curves shows that three of the mutants (K32A, D72A, and K75A) bind similarly to wild type, whereas the other three mutants (F66A, V96F, and Y100A) show no detectable binding.

Figure 5

NPC2 does not function as an extracellular sensor of cholesterol. (A) Modulation of extracellular levels of cholesterol does not affect NPC2 function. Purified NPC2 was delipidated through acetone precipitation. NPC2 was added at the indicated concentrations to npc2 fibroblasts in lipid-free media with or without 1 μM cholesterol. After 3 days, cells were filipin stained and the fluorescence was quantified as described in Materials and Methods. The EC₅₀ of the curve does not change significantly with or without cholesterol. (B) NPC2 can rescue individual transiently transfected npc2 cells. npc2 fibroblasts transiently transfected with NPC2-myc-his and GFP (10:1) to mark transfected cells were filipin-stained 3 days posttransfection. (Left) GFP fluorescence. (Center) Filipin fluorescence. (Bar = 20 μM.) Over 40% of GFP-positive cells were fully rescued (fluorescence in all pixels <750 units), and 23% were partially rescued (fluorescence between 750 and 1,000 units) when transfected with wild-type NPC2-myc-his. An anti-myc protein blot demonstrates that whereas D7A and I15F are expressed similarly to wild type, the amount secreted into the media is drastically reduced. Transfection of the mutants still resulted in a substantial fraction of fully (17 and 13%) and partially (27 and 31%) rescued cells, indicating that secretion is likely not required for rescue.

Figure 6

Four functional regions of NPC2. (A) A proposed cholesterol-binding site. Two views of the NPC2 crystal structure (Friedland et al., ref. 11) are shown in ribbons, with four key residues shown in stick representation. Three cavities detected within the crystal structure are shown in green. F66 (magenta) and Y100 (yellow) are proposed to be involved in ring-stacking interactions with cholesterol. The phenylalanine substitution at V96 (red) is proposed to project into the cavity (see especially Right) to sterically hinder cholesterol binding. (B) Assignment of functional regions on the NPC2 crystal structure. Three views of space-filling models of the NPC2 structure. Amino acids implicated in cholesterol binding (F66, V96, and Y100) are in green. D7 and I15 are required for efficient secretion of the protein and are in cyan. Two other regions of the protein marked in magenta (K32) and orange (D72 and K75) do not have a known function at this time. All of the residues are colored in the same hue as their corresponding ECR in Fig. 3, except for F66, which does not fall within an ECR.