MOESM1 of Evaluation of the effect of d-amino acid incorporation into amyloid-reactive peptides

Additional file 1: Figure S1. Purified peptides appeared, by RP-HPLC chromatography, as a single peak with a retention time of ~ 9 mi

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

<div><p>Background</p><p>Monoclonal 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.</p><p>Methods and findings</p><p>We 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.</p><p>Conclusion</p><p>The 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.</p></div

Binding of ¹²⁵I-labeled proteins to non-fibrillar substrates in a pulldown assay.

<p>Binding of ¹²⁵I-labeled proteins to non-fibrillar substrates in a pulldown assay.</p

Recruitment of VL domains and LC proteins by rVλ6Wil fibrils.

<p>(A) The recruitment (% bound) of ¹²⁵I-labeled rVλ6Wil, AL1κ and MM1κ assessed using a pulldown assay after 1 h (red) or 24 h (blue) incubation. Mean ± SD (<i>n</i> = 2 or 3). (B) Inhibition of ¹²⁵I-rVλ6Wil binding to rVλ6Wil fibrils by the presence of a 100- or 1000-fold molar excess non-radiolabeled rVλ6Wil (red), AL1κ (blue) or MM1κ (black). Mean ± SD (<i>n</i> = 3). (C) Recruitment of LC and VL by rVλ6Wil fibrils measured by surface plasmon resonance. Representative sensorgrams for the binding of rVλ6Wil (red), AL1κ (blue) and MM1κ (black) showing binding (arrow) and washout phases (bar). (D) Immunogold electron micrographs of AL1κ recruited by rVλ6Wil fibrils. Left panel, 10 nm-diameter gold particles (arrows) indicate the presence of the anti-κ mAb (binding AL1κ) on the fibrils (arrowheads). Right panel, shows control sample with no anti-κ LC mAb added. In A and B, data were analyzed by ANOVA with a Bonferroni post hoc test for multiple comparisons (**, <i>p</i> < 0.001; ***, <i>p</i> < 0.0001).</p

Analyses of AL or MM patient-derived LCs by SDS-PAGE and western blot.

<p>Electrophoresis was performed under non-reducing (NR) and reducing (R) conditions. Western blotting (W) was performed using reduced protein samples with a panel of anti-κ and or λ LC mAbs. Monomeric and dimeric LC appeared at ~ 24 KDa and 48 KDa, respectively.</p

Kinetic recruitment of LC by rVλ6Wil fibrils.

<p>Recruitment of ¹²⁵I-labeled AL and MM κ (A) or λ (B) LC proteins by rVλ6Wil fibrils (each point represents the mean of n = 2 observations, error bars are not visible). Rates were calculated using a single exponential binding equation, R² for each fit was > 0.95. (C) Binding of ¹²⁵I-labeled κ (circle) and λ (square) LC by rVλ6Wil fibrils after 1 h (red), 3 h (blue) or 24 h (green) of incubation. (D) Recruitment of AL- (circle) and MM-associated (square) LC was significantly different at 1 h (red), 3 h (blue) or 24 h (green) of incubation (*, <i>p</i> < 0.05). (E) Calculated recruitment rates of AL (red) and MM (blue) LC were not significantly different.</p

Monomeric LC proteins are preferentially sequestered by rVλ6Wil fibrils.

<p>(A) Quantitative densitometry of representative reduced SDS-PAGE gel electrophoretic profiles (arrowhead) of urea-treated ¹²⁵I-labeled LC. (B) Analysis of urea-treated rVλ6Wil fibrils following recruitment of ¹²⁵I-labeled LC. The monomer:dimer ratio was calculated for each LC. Where: DLU, digital light units; M, monomer; D, dimer; LMW, low molecular weight component.</p

Patient data and LC nomenclature.

<p>Patient data and LC nomenclature.</p

Thermodynamic folding stability of AL- and MM-associated LC does not correlate with fibril recruitment efficacy.

<p>(A) Estimates of the thermodynamic folding stability expressed as the apparent melting midpoint at a scan rate of 0.5°C/min (<i>Tm</i>_<i>app</i>). Numbers indicate the <i>Tm</i>_<i>app</i> of individual LCs. Correlation analyses of (B), <i>Tm</i>_<i>app</i> and rVλ6Wil fibril recruitment efficacy at 24 h of incubation (% bound) and of (C), <i>Tm</i>_<i>app</i> and the calculated recruitment rate. Black and gray symbols in B and C represent the MM- and AL-associated LC, respectively. Correlation coefficients in B and C were not significant (<i>p</i> = 0.68).</p

Peptide p5R exhibits enhanced α-helical secondary structure.

<p>CD spectra of peptides p5R (A – arrow shows isodichroic point) and p5 (B) in increasing concentrations of trifluoroethanol (TFE). The ratio of 222 nm and 205 nm was calculated (C) as a measure of α-helical content for p5 (○) and p5R (•). The 222 nm/205 nm ratio for human serum albumin in PBS was 1.0 (not shown).</p