AL amyloidosis

Wikipedia has a good page on AL amyloidosis. “AL” stands for amyloid light chain. The light chains are part of the antibody molecule. AL is linked to diseases associated with increased antibody production: B cell myeloma and Waldenström’s macroglobulinemia. The Wikipedia authors did not mention of the therapeutic antibodies being injected into patients to treat what seems like every conceivable disease. We can only hope that these therapeutic antibodies are fully assembled and do not contain rogue light chains that can form amyloids and other toxic compounds with Cu(II)

What is an amyloid anyway? The featured image comes from a structure deposited on RCSB.org, a public access database of protein structures. The image of the IgG antibody molecule was obtained from this link. Note the presence of disulfide bonds (S-S) in the light chains. Also note that the light chain has a constant C_L and a variable V_L region. For readers that like to attribute human characteristics to molecules, it is as if the misfolded deep red light chain says to a red/orange light chain buddy in the process of folding and getting attached to a heavy chain, “Why do you want to conform when you can be deviant like me?” Deep Red and Red Orange spoon together in the same deviant pattern. Then the deviant pair say to the orange light chain in line to be made into an antibody, “Why are you conforming when you can be deviant?” The deviant trio recruit the Bright Yellow light chain into their pack of biological aberration. The pack grows and grows until they become large enough to get trapped in places where they can cause trouble. The heart, of course the heart! The reader is invited to go to the RCSB.org, website and rotate the structure around to get a feel for what an amyloid really is. The curious thing is that this structure is based on a fibril isolated from an actual patient. [1] How scary is that?

Basics of the amyloid structure

Radamaker and colleagues from several universities in Germany isolated AL amyloid fibrils from the heart of a woman suffering from advanced heart failure due to AL amyloidosis. [1] The patient was diagnosed with a monoclonal plasma cell disorder called “smoldering myeloma” one year before she was diagnosed at the same time as AL amyloidosis. The patient was treated for the bone marrow plasma cell cancer but required a heart transplantation anyway due to amyloid deposit build up in her heart. [1] The authors used an established protocol to isolate the amyoid fibrils from her diseased heart. [1]

The authors used cryoelectron microscopy to predict the structure of her light chain amyloid deposits. CryoEM involves taking a transmission electron microscopy image of a structure and fitting in fitting in known structures of the protein, or related proteins, based on X-ray crystallography data. In this case the scientist used a combination of the sequence of her light chain and the 1BJM entry of the rcsb.org database. To be specific, the X-ray crystal structure was based on Bence-Jones crystals of light chains found in the urine of multiple myeloma patients.

Okay, now that we are dealing with IgG light chains behaving badly, let us get on to our story of how Cu²⁺ makes a bad situation worse. This sequel comes from collaborators from Italy. [2]

Cu(II) / Cu²⁺ making a bad situation worse

Histidine is generally considered an amino acid that likes to bind to Cu with the imidizole nitrogen shown in blue below. Cysteine has a thiol that also likes to bind Cu, but in our case they can’t because they are bound to each other in a disulfide bond.

Only the side chains are shown just to make the point that a lot of Cu²⁺ binding groups are lost in going from histidine to alanine with a side chain consisting of one carbon with three hydrogens that chemists don’t generally draw unless they can come off.

Most lay readers will want to know about toxicity. Rosso and coworkers used a nematode called Caenorhabditis elegans or C elegans for short. They have an ancestral heart and circulatory system that can be observed to “beat.” These wormies were fed eh IgG light chain proteins with and without small amounts of Cu²⁺. Note that the wild type H7 light chain becomes more toxic with 50 µM Cu²⁺. [2] In panel 3B, M7 are non amyloid genic IgG light chains from multiple myeloma patients that just happen to be non amyloid genic.

This post is going to skip some very complicated biophysical techniques that tested thermo stability of the amyloidgenic light chains. [2] Data not shown demonstrate Cu²⁺ binding to the V_L of the cardiotropic H7 with a low micromolar affinity. [2] Alanine-substitution of two histidine residues in the constant domain of H7 (H7-H188A/H197A mutant) did not alter Cu²⁺ binding or impair H₂O₂ generation in vitro. [2] The His-to-Ala mutations destabilized the constant domain and reduced the toxicity of the H7 mutant. Rosso and coauthors could not explain the toxicity of Cu²⁺ only to the His188/His197 binding site alone. [2]

Two things to look into, in my opinion, would be

1. Cys221 at the end of the sequence and whether or not the disulfide bond was really there.
2. Free -SH groups, as opposed to -S-S- in disulfide bonds, could bind Cu²⁺.

This is an exciting paper in so many ways.

What if we were to take a casein digest mix of peptides, add a small amount of Cu²⁺ or Cu⁺ and feed them to C elegans? A next step could also be to use florescent dyes that bind to amyloid structures. Thioflavin T is just one example.

References

1. Radamaker L, Lin YH, Annamalai K, Huhn S, Hegenbart U, Schönland SO, Fritz G, Schmidt M, Fändrich M. (2019) Cryo-EM structure of a light chain-derived amyloid fibril from a patient with systemic AL amyloidosis. Nat Commun. 2019 Mar 20;10(1):1103. PMC free article
2. Russo R, Romeo M, Schulte T, Maritan M, Oberti L, Barzago MM, Barbiroli A, Pappone C, Anastasia L, Palladini G, Diomede L, Ricagno S. Cu(II) Binding Increases the Soluble Toxicity of Amyloidogenic Light Chains. Int J Mol Sci. 2022 Jan 16;23(2):950. PMC free article