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 CL and a variable VL 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.

Supplemental figure 1A from Radamker2019 showng a transmission electron microscope image of an AL fibril. also shown is are some Bence-Jones crystals of IgG light chains from Wikipedia.

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.

The Cu2+ sites in relation to structure. A. sequence and structural information of the amyloid from RCSB.org, The golden bars are beta sheets, the structures that sort of look like folded bed sheets in the featured image. The disulfide bond cysteines are shown for reference. B. The sequence of amyloid forming light chain from Russo 2022 [2] These authors mutated the Cu2+ binding sites His188 and His197 to alanines. The magenta paint brush sweeps represent other amino acids of the IgG light chain in the vicinity of the histidines being mutated to alanines.

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 is the link to the image on the left. H7 mutant with destabilized CL domain is less toxic to C. elegans. (A) Dose–response curves reveal the diminished toxicity of H7-H188A/H197A (H7 mut) compared to H7 (WT), as higher concentrations are required to inhibit the pumping rate of worms. Worms were fed for 2 h with different concentrations of WT or mutated H7 suspended in 10 mM PBS, pH 7.4, and the pharyngeal pumping was scored 24 h after the administration. Control worms received vehicle alone (dotted line). Each value is the mean ± SE, n = 30. IC50 was 28.9 and 46.8 µg/mL for WT and mutated H7, respectively, p < 0.01, Student’s t-test. (B) Worms were fed for 2 h with 10 or 100 µg/mL H7 or H7-H188A/H197A, or 100 µg/mL M7 dissolved in 10 mM PBS, pH 7.4, with or without 50 µM Cu (II). Control worms received 10 mM PBS, pH 7.4 with or without 50 µM Cu (II) (vehicle). Pharyngeal pumping was determined 24 h after the administration. Each value is the mean ± SE, n = 20. * p < 0.05, ** p < 0.01 and **** p < 0.001 vs. the corresponding vehicle, °°°° p < 0.001, one-way ANOVA and Bonferroni’s post hoc test.
A close up to show just how toxin Cu(II) is in the presence of IgG light chains!

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 VL 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

Published by BL

I like to write educational websites

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