Zinc and Copper Ions Induce Aggregation of Human β-Crystallins

Abstract

Cataracts are defined as the clouding of the lens due to the formation of insoluble protein aggregates. Metal ions exposure has been recognized as a risk factor in the cataract formation process. The γ and β crystallins are members of a larger family and share several structural features. Several studies have shown that copper and zinc ions induce the formation of γ-crystallins aggregates. However, the interaction of metal ions with β-crystallins, some of the most abundant crystallins in the lens, has not been explored until now. Here, we evaluate the effect of Cu(II) and Zn(II) ions on the aggregation of HβA1, as a representative of the acidic form, and HβB2, as a representative of the basic β-crystallins. We used several biophysical techniques and computational methods to show that Cu(II) and Zn(II) induce aggregation following different pathways. Both metal ions destabilize the proteins and impact protein folding. Copper induced a small conformational change in HβA1, leading to high-molecular-weight light-scattering aggregates, while zinc is more aggressive towards HβB2 and induces a larger conformational change. Our work provides information on the mechanisms of metal-induced aggregation of β-crystallins.

Keywords: cataracts; copper; crystallins; human beta crystallins; zinc.

Figure 1

β-crystallin structures and sequences. (a) Sequence alignment of HβA1 and HβB2 crystallins. The elements of the secondary structure of the N-terminal and C-terminal domains and the linker region are shown. Histidine residues are shown in blue, cysteines in yellow, and tryptophan in magenta. Three-dimensional structure models of (b) HβA1 and (c) HβB2. Each strand is named A through H for each domain.

Figure 2

Effect of Zn(II) and Cu(II) as reported by turbidity assays at 37 °C. Absorbance at 405 nm as function of time for (a) HβA1 in the absence (black) and presence of 0.5, 1, and 1.5 equivalents of Zn(II) (red gradient); (b) HβA1 in the absence (black) and presence of 0.5, 1, and 1.5 equivalents of Cu(II) (blue gradient); (c) HβB2 in the absence (black) and presence of 0.5, 1, and 1.5 equivalents of Zn(II) (red gradient); (d) HβB2 in the absence (black) and presence of 0.5, 1, and 1.5 equivalents of Cu(II) (blue gradient). The change in absorbance is due to the formation of aggregates that scatter the light.

Figure 3

Protein oligomerization induced by metal-binding. (a) HβA1 correlation coefficient in the absence (gray) and presence of 1.5 equivalents of Zn(II) (red). (b) HβA1 correlation coefficient in the absence (gray) and presence of 1.5 equivalents of Cu(II) (blue). (c) HβB2 correlation coefficient in the absence (gray) and presence of 1.5 equivalents of Zn(II) (red). (d) HβB2 correlation coefficient in the absence (gray) and presence of 1.5 equivalents of Cu(II) (blue). Measurements with metal ions yielded a shift to the right indicating an increase in size due to oligomerization. Calculated R_H are shown. (Replica spectra are shown in Figure S5).

Figure 4

Normalized Intrinsic fluorescence. (a) HβA1 in the absence of metal ions (black) and in the presence of Zn(II) (red). (b) HβA1 in the absence of metal ions (black) and in the presence of Cu(II) (blue). (c) HβB2 in the absence of metal ions (black) and in the presence of Zn(II) (red). (d) HβB2 in the absence of metal ions (black) and in the presence of Cu(II) (blue). The emission spectrum was recorded in the range of 300 and 500 nm using an excitation wavelength of 295 nm at 37 °C. (Replica spectra are shown in Figure S7).

Figure 5

Normalized ANS fluorescence intensity. HβA1 (red) without the metals and with 1.5 eq of Cu(II) and Zn(II). HβB2 (blue) without the metals and with 1.5 eq of Cu(II) and Zn(II).