Macromolecular crowding induces holo α-lactalbumin aggregation by converting to its apo form

Abstract

Macromolecular crowding has been shown to have an exacerbating effect on the aggregation propensity of amyloidogenic proteins; while having an inhibitory effect on the non-amyloidogenic proteins. However, the results concerning aggregation propensity of non-amyloidogenic proteins have not been convincing due to the contrasting effect on holo-LA, which despite being a non-amyloidogenic protein was observed to aggregate under crowded conditions. In the present study, we have extensively characterized the crowding-induced holo-LA aggregates and investigated the possible mechanism responsible for the aggregation process. We discovered that macromolecular crowding reduces the calcium binding affinity of holo-LA resulting in the formation of apo-LA (the calcium-depleted form of holo-LA) leading to aggregate formation. Another finding is that calcium acts as a chaperone capable of inhibiting and dissociating crowding-induced holo-LA aggregates. The study has a direct implication to Alzheimer Disease as the results invoke a new mechanism to prevent Aβ fibrillation.

Figure 1. Temperature-dependent aggregation profiles of holo-LA.

(A) The temperature dependence of the light scattering intensity at 400 nm of holo-LA in the absence and presence of varying [Ficoll 70], the molar concentration of Ficoll 70. The lines represent the best-fits (using Equation 1) of data obtained in the presence of 0 g/l (solid circle), 100 g/l (solid triangle), 200 g/l (solid inverted triangle), 300 g/l (solid square) and 400 g/l (solid diamond) Ficoll 70. Inset shows the plot of T _agg (open triangle) and T _i (open circle) versus [Ficoll 70]. (B) Plot of T _i versus [holo-LA].

Figure 2. Characterization of the Ficoll 70-induced holo-LA aggregates.

(A) Plot of ANS binding profile (λ _max versus temperature) of holo-LA aggregates formed in the presence of Ficoll 70 at different temperatures. Inset shows the representative ANS binding profiles of holo-LA in the absence (20°C: solid line; 80°C: open circle) and presence of Ficoll 70 (20°C: dashed line; 80°C: dotted line). (B) Plot of ThT binding profile (ThT fluorescence intensity at 485 nm versus temperature) of holo-LA aggregates formed in the presence of Ficoll 70 at different temperatures. Inset shows the representative ThT binding spectra of holo-LA aggregates formed in the absence (20°C: solid line; 80°C: open circle) and presence of Ficoll 70 (20°C: dashed line; 80°C: dotted line). (C) Electron micrograph of holo-LA aggregates following heat-induced denaturation in the presence of 400 g/l Ficoll 70.

Figure 3. Reduction in the calcium-binding affinity of holo-LA in the presence of Ficoll 70.

(A) Far-UV CD spectra of holo-LA at 45°C under different solvent conditions: dilute (solid line); 1.2 mM EDTA (dashed line) and 300 g/l Ficoll 70 (dotted line). (B) Near-UV CD spectra of holo-LA at 45°C under different solvent conditions: dilute (solid line); 1.2 mM EDTA (dashed line) and 300 g/l Ficoll 70 (dotted line). (C) Representative thermal denaturation profiles of holo-LA in the presence of varying calcium chloride concentrations under dilute (solid symbols) and crowded (open symbol) conditions. In order to maintain clarity, some transition curves have not been shown.

Figure 4. Calcium inhibits crowding-induced holo-LA aggregation.

(A) Plot of ANS fluorescence intensity (at 473 nm) versus [CaCl₂] (solid circle) and percent soluble protein versus [CaCl₂] (solid triangle) under Ficoll 70 treated conditions. (B) Electron micrograph of holo-LA following heat-induced denaturation in the presence of 400 g/l Ficoll 70 and 100 mM calcium chloride. (C) Far-UV CD spectra of holo-LA at 45°C under different solvent conditions: dilute (solid line); 300 g/l Ficoll 70 (dashed line) and 300 g/l Ficoll 70 plus 100 mM CaCl₂ (dotted line). (D) Near-UV CD spectra of holo-LA at 45°C under solvent conditions: dilute (solid line); 300 g/l Ficoll 70 (dashed line) and 300 g/l Ficoll 70 plus 100 mM CaCl₂ (dotted line).

Figure 5. Calcium reverses crowding-induced holo-LA aggregates.

Aggregation kinetics of holo-LA monitored by observing change in light scattering intensity at 400 nm in the presence of 400 g/l Ficoll 70. After 4 hours, calcium chloride was added to the protein-crowder system.