Early events in light chain aggregation at physiological pH reveal new insights on assembly, stability, and aggregate dissociation

Abstract

Early events in immunoglobulin light chain (AL) amyloid formation are especially important as some early intermediates formed during the aggregation reaction are cytotoxic and play a critical role in the initiation of amyloid assembly. We investigated the early events in in vitro aggregation of cardiac amyloidosis AL proteins at pH 7.4. In this study we make distinctions between general aggregation and amyloid formation. Aggregation is defined by the disappearance of monomers and the detection of sedimentable intermediates we call non-fibrillar macromolecular (NFM) intermediates by transmission electron microscopy (TEM). Amyloid formation is defined by the disappearance of monomers, Thioflavin T fluorescence enhancement, and the presence of fibrils by TEM. All proteins aggregated at very similar rates via the formation of NFM intermediates. The condensed NFM intermediates were composed of non-native monomers. Amyloid formation and amyloid yield was variable among the different proteins. During the stationary phase, all proteins demonstrated different degrees of dissociation. These dissociated species could play a key role in the already complex pathophysiology of AL amyloidosis. The degree of dissociation is inversely proportional to the amyloid yield. Our results highlight the importance and physiological consequences of intermediates/fibril dissociation in AL amyloidosis.

Keywords: Immunoglobulin light chain; Thioflavin T; aggregate dissociation; circular dichroism; intrinsic fluorescence; light chain amyloidosis; non-fibrillar macromolecular intermediates.

Figure 1.

Composite kinetic data analysis of monomer disappearance, Thioflavin T fluorescence, and structural variations determined by intrinsic fluorescence and CD of κIO18/O8 (A, C and E) and κIO18/O8 Y87H (B, D and F) during aggregation. Shown are (A and B) the representative kinetic traces obtained from sedimentation assay to calculate the % monomer remaining in the reaction (■) vs ThT fluorescence assay (●) (For information regarding number of replicates [technical and biological], see Methods section). The highlighted area represents the different phases (based on concentration of monomers): [█ – nucleation, █ – elongation, █ – stationary, and █ – dissociation phase]. The colour coded dotted lines represents the exact time points when aliquots from the ongoing aggregation reaction were analysed by EM. The brown box highlights the aggregate dissociation in these proteins. (C and D) Intrinsic tryptophan fluorescence assay representing changes in the ${\lambda }_{max}^{em}$ and emission intensity during aggregation. (E and F) Secondary structural information obtained from far UV-CD spectra during aggregation.

Figure 2.

Composite kinetic data analysis of monomer disappearance, Thioflavin T fluorescence, and structural variations determined by intrinsic fluorescence and CD of AL-09 (A, C and E) and AL-09 H87Y (B, D and F) during aggregation. Shown are (A and B) the representative kinetic traces obtained from sedimentation assay to calculate the % monomer remaining in the reaction (■) vs ThT fluorescence assay (●) (For information regarding number of replicates [technical and biological], see Methods section). The highlighted area represents the different phases (based on concentration of monomers): [█ – nucleation, █ – elongation, █ – stationary, and █ – dissociation phase]. The colour coded dotted lines represents the exact time points when aliquots from the ongoing aggregation reaction were analysed by EM. The brown box highlights the aggregate dissociation in these proteins. (C and D) Intrinsic tryptophan fluorescence assay representing changes in the ${\lambda }_{max}^{em}$ and emission intensity during aggregation. (E and F) Secondary structural information obtained from far UV-CD spectra during aggregation.

Figure 3.

Composite kinetic data analysis of monomer disappearance, Thioflavin T fluorescence, and structural variations determined by intrinsic fluorescence and CD of AL-12 (A, C and E) and AL-12 R65S (B, D and F) during aggregation. Shown are (A and B) the representative kinetic traces obtained from sedimentation assay to calculate the % monomer remaining in the reaction (■) vs ThT fluorescence assay (●) (For information regarding number of replicates [technical and biological], see Methods section). The highlighted area represents the different phases (based on concentration of monomers): [█ – nucleation, █ – elongation, █ – stationary, and █ – dissociation phase]. The colour coded dotted lines represents the exact time points when aliquots from the ongoing aggregation reaction were analysed by EM. The brown box highlights the aggregate dissociation in these proteins. (C and D) Intrinsic tryptophan fluorescence assay representing changes in the ${\lambda }_{max}^{em}$ and emission intensity during aggregation. (E and F) Secondary structural information obtained from far UV-CD spectra during aggregation.

Figure 4.

Schematic representation of the aggregation mechanism of κIO18/O8, AL-09 and AL-12 at pH 7.4. During the early phases we observe the formation of NFM intermediates. κIO18/O8 was devoid of any fibrillar species throughout aggregation. AL-09 demonstrated a significant increase in amyloid yield concomitant with the decrease in the population of NFM intermediates. AL-12 presented very low amyloid yield during the course of aggregation.