Table 1.
Patient data and LC nomenclature.
Fig 1.
Analyses of AL or MM patient-derived LCs by SDS-PAGE and western blot.
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.
Fig 2.
Recruitment of VL domains and LC proteins by rVλ6Wil fibrils.
(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 (n = 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 (n = 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 (**, p < 0.001; ***, p < 0.0001).
Table 2.
Binding of 125I-labeled proteins to non-fibrillar substrates in a pulldown assay.
Fig 3.
Kinetic recruitment of LC by rVλ6Wil fibrils.
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 (*, p < 0.05). (E) Calculated recruitment rates of AL (red) and MM (blue) LC were not significantly different.
Fig 4.
Monomeric LC proteins are preferentially sequestered by rVλ6Wil fibrils.
(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.
Fig 5.
Thermodynamic folding stability of AL- and MM-associated LC does not correlate with fibril recruitment efficacy.
(A) Estimates of the thermodynamic folding stability expressed as the apparent melting midpoint at a scan rate of 0.5°C/min (Tmapp). Numbers indicate the Tmapp of individual LCs. Correlation analyses of (B), Tmapp and rVλ6Wil fibril recruitment efficacy at 24 h of incubation (% bound) and of (C), Tmapp 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 (p = 0.68).