Mutations in specific structural regions of immunoglobulin light chains are associated with free light chain levels in patients with AL amyloidosis

Abstract

Background: The amyloidoses are protein misfolding diseases characterized by the deposition of amyloid that leads to cell death and tissue degeneration. In immunoglobulin light chain amyloidosis (AL), each patient has a unique monoclonal immunoglobulin light chain (LC) that forms amyloid deposits. Somatic mutations in AL LCs make these proteins less thermodynamically stable than their non-amyloidogenic counterparts, leading to misfolding and ultimately the formation of amyloid fibrils. We hypothesize that location rather than number of non-conservative mutations determines the amyloidogenicity of light chains.

Methodology/principal findings: We performed sequence alignments on the variable domain of 50 kappa and 91 lambda AL light chains and calculated the number of non-conservative mutations over total number of patients for each secondary structure element in order to identify regions that accumulate non-conservative mutations. Among patients with AL, the levels of circulating immunoglobulin free light chain varies greatly, but even patients with very low levels can have very advanced amyloid deposition.

Conclusions: Our results show that in specific secondary structure elements, there are significant differences in the number of non-conservative mutations between normal and AL sequences. AL sequences from patients with different levels of secreted light chain have distinct differences in the location of non-conservative mutations, suggesting that for patients with very low levels of light chains and advanced amyloid deposition, the location of non-conservative mutations rather than the amount of free light chain in circulation may determine the amyloidogenic propensity of light chains.

Figure 1. VL structure.

A) Topological Diagram of the protein structure for AF490909 adapted from Schiffer, et al . The two β-sheets of the domains have been separated. Residues that point towards the core are circled. CDR segments are highlighted in yellow. The β-strands have been encased within the arrows and are connected to their respective loops. B) Structural model of a VL (1BRE.pdb) showing CDR regions in pink, β-hairpin between strands D and E in blue and dimer interface in green ribbons.

Figure 2. Non-conservative mutations over total number of patients for each secondary structure element for AL Vλ and Vκ proteins.

The x-axis shows the different elements of secondary structure in the VL, while the y-axis gives the ratio of non-conservative mutations in each secondary structure element per total number of patients. The secondary structure boundaries used were based on the germline donor for each protein. Numbering is based on Kabat (http://vbase.mrc-cpe.cam.ac.uk/). A) Vκ sequences. B) Vλ sequences. C) Comparison of Vλ VI and other lambdas (Vλ I, II and III). The percentage of patients with mutations (% PWM) in each secondary structure element is listed per group.

Figure 3. Non-conservative mutations over total number of patients for each secondary structure element for normal control Vλ, Vκ, and multiple myeloma control proteins.

The x-axis shows the different elements of secondary structure in the VL, while the y-axis gives the ratio of non-conservative mutations in each secondary structure element per total number of sequences. The secondary structure boundaries used were based on the germline donor for each protein. Numbering is based on Kabat (http://vbase.mrc-cpe.cam.ac.uk/). A) Vκ normal control sequences. B) Vλ normal control sequences. C) Multiple Myeloma control sequences (combined Vλ and Vκ). The percentage of sequences with mutations (% PWM) in each secondary structure element is listed per group.

Figure 4. Structural models showing the common locations of non-conservative mutations in ALVλ proteins in our study.

Protein models were based on the crystal structure for Vλ VI germline (2CDO.pdb). The β-strands in the structure are shown as red ribbons; mutation regions discussed in the captions are shown in green.

Figure 5. Comparison of non conservative mutations per total number of patients for low (I), medium (II) and high (III) iFLC levels in selected Mayo proteins per secondary structure.

iFLC levels have been shown to be a good clinical parameter to follow disease progression , . The sequences were gathered into three groups based on their iFLC levels at the time of diagnosis. Whenever there is no data shown for a particular group/secondary structure element, the value is zero. The secondary structure boundaries used were based on the germline donor for each protein. Numbering is based on Kabat (http://vbase.mrc-cpe.cam.ac.uk/). The %PWM in each secondary structure element is listed per group.

Figure 6. Structural models showing regions of high and low non-conservative mutation accumulation in iFLC level groups.

Protein models were based on the crystal structure for Vλ VI germline (2CDO.pdb). The β-strands in the structure are shown as red ribbons; mutation regions discussed in the captions are shown in green.