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
• light chain amyloidosis
• Thioflavin T
• non-fibrillar macromolecular intermediates
• intrinsic fluorescence
• circular dichroism
• aggregate dissociation

Acknowledgements

We thank our former laboratory members Luis M. Blancas-Mejia for his creative inputs during the optimization of the intrinsic fluorescence assay and Marta Marin-Arganyfor providing AL-12 R65S protein for the present study. We also thank all other members of Dr. Marina Ramirez-Alvarado's laboratory for their critical and constructive comments.

Disclosure statement

The authors declare that they have no conflicts of interest with the contents of this article.

Additional information

Funding

This work was supported by National Institutes of Health (NIH R01) grant [GM128253], the Mayo Foundation, and the generosity of amyloidosis patients and their families.