Effects of mutations in Aedes aegypti sterol carrier protein-2 on the biological function of the protein

Abstract

Sterol carrier protein-2 (SCP-2) is a nonspecific intracellular lipid carrier protein. However, the molecular mechanism of ligand selectivity and the in vivo function of SCP-2 remain unclear. In this study, we used site-directed mutagenesis to investigate the ligand selectivity and in vivo function of the yellow fever mosquito sterol carrier protein-2 protein (AeSCP-2). Mutations to amino acids in AeSCP-2 known to interact with bound ligand also weakened NBD-cholesterol binding. Substitution of amino acids in the ligand cavity changed the ligand specificity of mutant AeSCP-2. Overexpressing wild-type AeSCP-2 in the Aedes aegypti cultured Aag-2 cells resulted in an increase in the level of incorporation of [(3)H]cholesterol. However, overexpressing mutants that were deleterious to the binding of NBD-cholesterol in AeSCP-2 showed a loss of ability to enhance uptake of [(3)H]cholesterol in cultured cells. Interestingly, when [(3)H]palmitic acid was used as the substrate for incorporation in vivo, there was no change in the levels of incorporation with overexpression of wild-type protein or mutated AeSCP-2s. The in vivo data suggest that AeSCP-2 is involved in sterol uptake, but not fatty acid uptake. This is the first report that the cholesterol binding ability may directly correlate with AeSCP-2's in vivo function in aiding the uptake of cholesterol.

Figure 1

Binding of NBD-cholesterol (1.25 µM) to AeSCP-2 under the conditions of a competitive ligand (0.005 to 5 µM). (A). Cholesterol. (B). β-sitosterol. (C). Palmatic acid. (D). 7-dehydrocholesterol. The background NBD-cholesterol fluorescence (NBD-cholesterol alone in the reaction buffer) was deducted from each assay. The trend line (the slope was shown by the trend line) was draw from the mean of three replicates and the bars represent standard deviation. R² values of each non-linear regression curve were shown.

Figure 2

Ribbon diagrams (Sybyl) of the AeSCP-2 (pdb entry 1PZ4) at 1.35 Å resolution and selected amino acid residues for point mutations that directly interact with bound ligand (blue colored sticks, F9, F32, M90, and F105) and that are not in direct contact with the ligand (orange colored stick, Y30 and W44). The C16 fatty acid is highlighted as a ball and stick model with carbons (white) and oxygen (red).

Figure 3

Binding of NBD-cholesterol to recombinant wild type and mutant AeSCP-2s. The background NBD-cholesterol fluorescence (NBD-cholesterol alone in the reaction buffer) was deducted from each assay. Shown are net changes in NBD-cholesterol fluorescence in intensity (RFU = Relative fluorescence unit) at 0.5 µM concentration in the presence of increasing concentrations of each protein (0.01 to 10 µM). The data represent mean values from three replicates and processed using GraphPad PRISM4.0 (single binding site and non-linear regression model). R² values of each non-linear regression curve were shown.

Figure 4

The effect of point mutation in AeSCP-2 on [³H]cholesterol uptake in Aedes aegypti Aag-2 cells. Aag-2 cells were transfected with over-expression vectors (see Materials and Methods) and the empty vector was used as negative controls. Over-expression of each protein was verified via western blotting analysis (Suppl. Fig. 2). The mean and standard deviation are shown (N=3). Stars mark significant differences (p<0.05) between wild type AeSCP-2 and mutant AeSCP-2s and the negative control.

Figure 5

The effect of point mutation in AeSCP-2 on [³H]palmitic acid uptake in Aedes aegypti Aag-2 cells. Aag-2 cells were transfected with over-expression vectors (see Materials and Methods) and the empty vector was used as negative controls. Over-expression of each protein was verified via western blotting analysis (Suppl. Fig. 2). The mean and standard deviation are shown (N=3).

Figure 6

Intrinsic tryptophan fluorescent emission in wild type and mutants. A. Wild type AeSCP-2 recombinant proteins in the presence or absence of ligand or inhibitors. B. Wild type AeSCP-2 recombinant protein denatured in 50 mM Tris-HCl, 6.5 M GuHCl, pH 7.4 in the presence and absence of ligand or inhibitors. C. F9W/W44F mutant AeSCP-2 in the presence and absence of inhibitors. D. M90L mutant AeSCP-2 in the presence and absence of inhibitors.