Differential recruitment efficacy of patient-derived amyloidogenic and myeloma light chain proteins by synthetic fibrils—A metric for predicting amyloid propensity

Abstract

BackgroundMonoclonal free light chain (LC) proteins are present in the circulation of patients with immunoproliferative disorders such as light chain (AL) amyloidosis and multiple myeloma (MM). Light chain-associated amyloid is a complex pathology composed of proteinaceous fibrils and extracellular matrix proteins found in all patients with AL and in ~10–30% of patients who presented with MM. Amyloid deposits systemically in multiple organs and tissues leading to dysfunction and ultimately death. The overall survival of patients with amyloidosis is worse than for those with early stage MM.Methods and findingsWe have developed a sensitive binding assay quantifying the recruitment of full length, patient-derived LC proteins by synthetic amyloid fibrils, as a method for studying their amyloidogenic potential. In a survey of eight urinary LC, both AL and MM-associated proteins were recruited by synthetic amyloid fibrils; however, AL-associated LC bound significantly more efficiently (p < 0.05) than did MM LCs. The LC proteins used in this study were isolated from urine and presumed to represent a surrogate of serum free light chains.ConclusionThe binding of LC to synthetic fibrils in this assay accurately differentiated LC with amyloidogenic propensity from MM LC that were not associated with clinical amyloid disease. Notably, the LC from a MM patient who subsequently developed amyloid behaved as an AL-associated protein in the assay, indicating the possibility for identifying MM patients at risk for developing amyloidosis based on the light chain recruitment efficacy. With this information, at risk patients can be monitored more closely for the development of amyloidosis, allowing timely administration of novel, amyloid-directed immunotherapies—this approach may improve the prognosis for these patients.

Kinetic stability and sequence/structure studies of urine-derived Bence-Jones proteins from multiple myeloma and light chain amyloidosis patients

“…AL1κ and MM2λ are the proteins that presented similar rate constants of recruitment into amyloid fibrils in our previous study on recruitment of light chains [19]. However, their %LC bound to fibrils is dramatically different.…”

“…In addition, the modulating effect of the constant domain, and host factors may play important role in amyloid aggregation. Previously, we have reported that both MM and AL BJPs were recruited by amyloid-like V L fibrils; however the extent of recruitment of AL-derived BJPs was significantly greater, allowing discrimination of the two disease populations [19]. …”

“…AL1λ showed the lowest rate constant among the AL BJPs and the lowest %LC bound to fibrils. In our study, AL1λ was one of the two proteins that we could not characterize due to its poor solubility at the concentrations required for the current study (~50 μg) compared to the ones required for the recruitment report (5–10 ng) [19]. Our data confirms AL1λ and MM2λ have very different stability and kinetic properties for folding and that that the %LC bound is the right parameter to discriminate between amyloidogenic and non-amyloidogenic proteins (The location of non-conservative mutations for AL1λ and MM2λ is shown in supplementary figure 2).…”

“…These components were isolated from urine specimens as previously described [20]. The V L -subgroup classification was determined serologically using polyclonal antihuman V L -subgroup-specific antisera: antihuman total κ light chain (14-6E4) and total λ light chain (21-3F4) monoclonal antibodies [20, 21]; or by amino acid sequence analysis [19, 22]. Isolated BJPs were lyophilized and stored at room temperature until used.…”

“…In this study, we have characterized the thermodynamics of 9 BJPs from 5 patients with AL amyloidosis and 4 MM patients previously described for their differential ability to be recruited in amyloid fibril elongation experiments [19]. From the five AL BJPs included in this study, partial unfolding and solubility issues precluded the analysis of two of them, so we conducted scan rate dependency experiments on the remaining seven proteins.…”