Kinetic evidence for multiple aggregation pathways in antibody light chain variable domains

Abstract

Aggregation of antibody light chain proteins is associated with the progressive disease light chain amyloidosis. Patient-derived amyloid fibrils are formed from light chain variable domain residues in non-native conformations, highlighting a requirement that light chains unfold from their native structures in order to aggregate. However, mechanistic studies of amyloid formation have primarily focused on the self-assembly of natively unstructured peptides, and the role of native state unfolding is less well understood. Using a well-studied light chain variable domain protein known as WIL, which readily aggregates in vitro under conditions where the native state predominates, we asked how the protein concentration and addition of pre-formed fibril "seeds" alter the kinetics of aggregation. Monitoring aggregation with thioflavin T fluorescence revealed a distinctly non-linear dependence on concentration, with a maximum aggregation rate observed at 8 μM protein. This behavior is consistent with formation of alternate aggregate structures in the early phases of amyloid formation. Addition of N- or C-terminal peptide tags, which did not greatly affect the folding or stability of the protein, altered the concentration dependence of aggregation. Aggregation rates increased in the presence of pre-formed seeds, but this effect did not eliminate the delay before aggregation and became saturated when the proportion of seeds added was greater than 1 in 1600. The complexity of aggregation observed in vitro highlights how multiple species may contribute to amyloid pathology in patients.

Keywords: AL amyloidosis; aggregation kinetics; amyloid fibrils; antibody variable domain; protein misfolding; systemic light chain amyloidosis.

FIGURE 1

Aggregation kinetics of WIL‐V have a nonlinear dependence on concentration. WIL‐V solutions were incubated in microplates in PBS, pH 7.4, containing 1 μM ThT at 37°C and shaken at 500 rpm. Data shown from two independent microwell plates, each with two wells per condition. (a) ThT fluorescence as a function of time. Colors indicate initial WIL‐V concentration. (b) Calculated aggregation midpoint (T ₅₀) as a function of concentration from the data shown in (a). (c) Maximum fluorescence intensity from the data shown in (a). (d) Electron micrographs showing examples of aggregate morphology observed after 72 h of incubation. Initial WIL‐V concentrations and appropriate scale bars are shown below each image. Multiple aggregate species were observed in each reaction.

FIGURE 2

WIL‐variants have similar folded states and stability. (a) Intrinsic fluorescence spectra (λ _ex = 280 nm) of WIL variants showing increased fluorescence intensity and red‐shifted fluorescence maxima upon unfolding in 6 M urea, which is characteristic of antibody variable domains. Intensities are normalized relative to the maximum intensity of the sample in 6 M urea at 25°C for each variant. Data are shown at 25 and 37°C, consistent with the LCs remaining folded at 37°C. (b) Urea titration of WIL‐V variants in PBS, pH 7.4, 37°C, followed by intrinsic fluorescence. Colors and shapes indicate the variant: green circles, WIL‐V; blue triangles, WIL‐V‐Chis; orange squares, WIL‐V‐Nhis.

FIGURE 3

WIL‐V variants aggregate with distinct kinetics. WIL‐V solutions were incubated in microplates in PBS, pH 7.4, containing 1 μM ThT at 37°C and shaken at 500 rpm. Data shown from two independent microwell plates, each with two wells per condition. The data for untagged WIL‐V are reproduced from Figure 1. Colors are the same as in Figure 2. (a) ThT fluorescence as a function of time and concentration. (b) Calculated midpoint times (T ₅₀) for the data shown in (a). Lines show the average T ₅₀ at each timepoint.

FIGURE 4

Aggregation of WIL‐V variants can be accelerated by addition of homologous pre‐formed seeds. WIL‐V solutions were incubated in microplates, with (solid lines) or without (dashed lines) addition of 1% (v/v) pre‐formed seeds (PBS, pH 7.4, 1 μM ThT, 37°C, 500 rpm). Colors indicate the identity of the seed protein. (a) ThT fluorescence as a function of time. (b) Calculated midpoint times (T ₅₀) for the data shown in (a).

FIGURE 5

Concentration dependence of seeds on the aggregation kinetics of WIL‐V‐Nhis. Aggregation of WIL‐V‐Nhis in microplates (PBS, pH 7.4, 1 μM ThT, 37°C, 500 rpm) was monitored by ThT fluorescence after addition of varying concentrations of pre‐formed fibril seeds. Colors indicate the proportion of seeds added. Unseeded reactions are shown with dashed lines. (a) ThT fluorescence as a function of time and seed concentration. (b) Calculated midpoint times (T ₅₀) for the data shown in (a). Circles indicate T ₅₀ values observed at 8 μM, triangles indicate T ₅₀ values observed at 16 μM.

FIGURE 6

Heterologous seeds accelerate WIL‐V aggregation with varying efficiency. Aggregation reactions in microplates were initiated in the presence or absence of 1% (v/v) seeds (PBS, pH 7.4, 1 μM ThT, 37°C, 500 rpm). Colors indicate the identity of the seed protein. (a) ThT fluorescence as a function of time. Unseeded reactions are shown with dashed lines. (b) Calculated midpoint times (T ₅₀) for the data shown in (a).