Interaction of alpha-crystallin with four major phospholipids of eye lens membranes

Abstract

It is well-studied that the significant factor in cataract formation is the association of α-crystallin, a major eye lens protein, with the fiber cell plasma membrane of the eye lens. The fiber cell plasma membrane of the eye lens consists of four major phospholipids (PLs), i.e., phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM). Despite several attempts to study the interaction of α-crystallin with PLs of the eye lens membrane, the role of individual PL for the binding with α-crystallin is still unclear. We recently developed the electron paramagnetic resonance (EPR) spin-labeling method to study the binding of α-crystallin to the PC membrane (Mainali et al., 2020a). Here, we use the recently developed EPR method to explicitly measure the binding affinity (K_a) of α-crystallin to the individual (PE*, PS, and SM) and two-component mixtures (SM/PE, SM/PS, and SM/PC in 70:30 and 50:50 mol%) of PL membranes as well as the physical properties (mobility parameter and maximum splitting) of these membranes upon binding with α-crystallin. One of the key findings of this study was that the K_a of α-crystallin binding to individual PL membranes followed the trends: K_a(PC) > K_a(SM) > K_a(PS) > K_a(PE*), indicating PE* inhibits binding the most whereas PC inhibits binding the least. Also, the K_a of α-crystallin binding to two-component mixtures of PL membranes followed the trends: K_a(SM/PE) > K_a(SM/PS) > K_a(SM/PC), indicating SM/PC inhibits binding the most whereas SM/PE inhibits binding the least. Except for the PE* membrane, for which there was no binding of α-crystallin, the mobility parameter for all other membranes decreased with an increase in α-crystallin concentration. It represents that the membranes become more immobilized near the headgroup regions of the PLs when more and more α-crystallin binds to them. The maximum splitting increased only for the SM and the SM/PE (70:30 mol%) membranes, with an increase in the binding of α-crystallin. It represents that the PL headgroup regions of these membranes become more ordered after binding of α-crystallin to these membranes. Our results showed that α-crystallin binds to PL membranes in a saturable manner. Also, our data suggest that the binding of α-crystallin to PL membranes likely occurs through hydrophobic interaction between α-crystallin and the hydrophobic fatty acid core of the membranes, and such interaction is modulated by the PL headgroup's size and charge, hydrogen bonding between headgroups, and PL curvature. Thus, this study provides an in-depth understanding of α-crystallin interaction with the PL membranes made of individual and two-component mixtures of the four major PLs of the eye lens membranes.

Keywords: Binding affinity; EPR; Maximum splitting; Mobility parameter; Phospholipid membranes; Physical properties; Spin-label; α-crystallin.

Fig. 1.

The chemical structure of cholesterol analog spin-label (CSL), and four major PLs of fiber cell plasma membranes of the eye lens, i.e., POPC, POPS, SM, and POPE. Approximate locations of these molecules across the lipid bilayer membrane are indicated.

Fig. 2.

Representative EPR spectra of CSL in individual PL membranes in the absence of α-crystallin (black) and 52.6 μM α-crystallin (red). Each spectrum was normalized with respect to peak to peak intensity of the central line. (A), (C), and (E) represent the EPR spectra for SM, POPS, and POPE* (asterisk indicates the presence of 30 mol% of POPC) membranes, respectively. (B), (D), and (F) represent the zoomed low field line of the spectra in (A), (C), and (E), respectively. The concentration of PL membranes was fixed at 9.4 mM, and the concentration of α-crystallin was varied (0 – 52.6 μM). All the samples were incubated at 37 °C for 16 hr, and EPR measurements were taken at 37 °C. The ratio of peak to peak intensity of low field line (h₊) and the central line (h₀) of EPR spectra was used to calculate the mobility parameter (h₊/h₀) of the PL membrane (see Fig. 2A). Peak to peak intensity of low field EPR line of unbound (U₀) and unbound plus bound (U₀ + B₀) contributions was used to calculate the binding affinity of α-crystallin to PL membrane (see Fig. 2B). The horizontal distance between the low field and high field lines in the EPR spectra was used to calculate the maximum splitting (see Fig. 2C)

Fig. 3.

(A) The percentage of membrane surface occupied plotted as a function of α-crystallin concentration for individual PL membranes, i.e., POPE*, POPS, SM, and POPC membranes. The data for the POPC membrane was taken from our recent study (Mainali et al., 2020a). The concentration of α-crystallin was varied (0 – 52.6 μM) and PL membranes was fixed at 9.4 mM. The mixture of α-crystallin and membrane samples were incubated at 37 °C for 16 hr. The percentage of membrane surface occupied by the α-crystallin was calculated by using equation (2). The data points were fitted with a one-site ligand binding model in GraphPad Prism (San Diego, CA) to estimate the binding affinity (K_a). The error bars were calculated from the average of three independent experiments. (B) Bar plot of K_a (obtained from (A)) for four major PL membranes. The error bars in (B) were calculated from the 95% confidence interval (profile likelihood) for the value of K_a.

Fig. 4.

The physical properties of individual PL membranes plotted as a function of α-crystallin concentration. (A) Profiles of the mobility parameter (h₊ ⁄h₀) plotted as a function of α-crystallin concentration. (B) Profiles of the maximum splitting plotted as a function of α-crystallin concentration. The data (mobility parameter) for the POPC membrane was taken from our previous study (Mainali et al., 2020a). Error bars were estimated from the average of three independent experiments. The concentration of α-crystallin was varied (0 – 52.6 μM), and the concentration of PL membranes was fixed at 9.4 mM. Each sample was incubated at 37 °C for 16 hr. The mobility parameter and maximum splitting were calculated by using the method explained in section 2.6.

Fig. 5.

Representative EPR spectra of CSL in two-component mixtures of PL membranes (70:30 mol%) in the absence (black) and the presence of 52.6 μM α-crystallin (red). Each spectrum was normalized with respect to peak to peak intensity of the central EPR line. (A), (C), and (E) represent the EPR spectra for SM/POPS, SM/POPE, and SM/POPS in 70:30 mol%, respectively. (B), (D), and (F) represent the zoomed low field line of the spectra in (A), (C), and (E), respectively. The concentration of two-component mixtures of PL membranes was fixed at 9.4 mM, and the concentration of α-crystallin was varied (0 – 52.6 μM). Each sample was incubated at 37 °C for 16 hr, and EPR spectra were recorded at 37 °C.

Fig. 6.

(A) The percentage of membrane surface occupied plotted as a function of α-crystallin concentration for two-component mixtures of PL membranes SM/POPC, SM/POPS, and SM/POPE in 70:30 mol%. (B) Bar plot of K_a of α-crystallin to two-component mixtures of PL membranes in 70:30 mol% (obtained from (A)). (C) The percentage of membrane surface occupied plotted as a function of α-crystallin concentration for two-component mixtures of PL membranes SM/POPC, SM/POPS, and SM/POPE in 50:50 mol%. (D) Bar plot of K_a of α-crystallin to two-component mixtures of PL membranes in 50:50 mol% (obtained from (C)). The concentration of α-crystallin was varied (0 – 52.6 μM), and the concentration of PL membranes was fixed at 9.4 mM. The mixture of α-crystallin and membrane samples were incubated at 37 °C for 16 hr. The data points in (A) and (C) were fitted with a one-site ligand binding model by using GraphPad Prism (San Diego, CA) to estimate the binding affinity K_a. The error bars in (A) and (C) were estimated from the average of three independent experiments. The error bars in (B) and (D) were calculated from the 95% confidence interval (profile likelihood) for the respective value of K_a.

Fig. 7.

The physical properties of two-component mixtures of PL membranes plotted as a function of α-crystallin concentration. (A) and (B) represent the profiles of mobility parameter (h₊/h₀) and maximum splitting, respectively, plotted as a function of α-crystallin concentration for two-component mixtures of PL membranes in 70:30 mol%. (C) and (D) represent the profiles of mobility parameter (h₊/h₀) and maximum splitting, respectively, plotted as a function of α-crystallin concentration for two-component mixtures of PL membranes in 50:50 mol%. The concentration of α-crystallin was varied (0 – 52.6 μM), and the concentration of PL membranes was fixed at 9.4 mM. Each sample was incubated at 37 °C for 16 hr. The mobility parameter and maximum splitting were calculated by using the method explained in section 2.6. The error bars were estimated from the average of three independent experiments.