Differential effects on light chain amyloid formation depend on mutations and type of glycosaminoglycans

Abstract

Amyloid light chain (AL) amyloidosis is a protein misfolding disease where immunoglobulin light chains sample partially folded states that lead to misfolding and amyloid formation, resulting in organ dysfunction and death. In vivo, amyloid deposits are found in the extracellular space and involve a variety of accessory molecules, such as glycosaminoglycans, one of the main components of the extracellular matrix. Glycosaminoglycans are a group of negatively charged heteropolysaccharides composed of repeating disaccharide units. In this study, we investigated the effect of glycosaminoglycans on the kinetics of amyloid fibril formation of three AL cardiac amyloidosis light chains. These proteins have similar thermodynamic stability but exhibit different kinetics of fibril formation. We also studied single restorative and reciprocal mutants and wild type germ line control protein. We found that the type of glycosaminoglycan has a different effect on the kinetics of fibril formation, and this effect seems to be associated with the natural propensity of each AL protein to form fibrils. Heparan sulfate accelerated AL-12, AL-09, κI Y87H, and AL-103 H92D fibril formation; delayed fibril formation for AL-103; and did not promote any fibril formation for AL-12 R65S, AL-103 delP95aIns, or κI O18/O8. Chondroitin sulfate A, on the other hand, showed a strong fibril formation inhibition for all proteins. We propose that heparan sulfate facilitates the formation of transient amyloidogenic conformations of AL light chains, thereby promoting amyloid formation, whereas chondroitin sulfate A kinetically traps partially unfolded intermediates, and further fibril elongation into fibrils is inhibited, resulting in formation/accumulation of oligomeric/protofibrillar aggregates.

Keywords: AL Amyloidosis; Amyloid; Amyloidosis; Fibril; Glycosaminoglycan; Immunoglobulin Fold; Immunoglobulin Light Chain; Light Chain Amyloidosis; Protein Misfolding.

FIGURE 1.

Chemical structure of sulfated glycosaminoglycans consensus sequences (A–D) and the control branched glycan dextran sulfate (E) and dextran sulfate (F). Negatively charged groups (sulfate and carboxylic acid groups) on the saccharide sequences are colored in red. The average number of negative charges per saccharide unit is displayed by the numbers in parentheses in light blue.

FIGURE 2.

Structural and stability properties of the AL proteins prior to amyloid formation reactions. Shown are far-UV CD spectra (A) and thermal unfolding (B) of AL-103 H92D (olive), AL-103 (blue), AL-09 (red), AL-12 (green), κI Y87H (violet), AL-12 R65S (orange), AL-09 H87Y (yellow), κI O18/O8 (brown), AL-09 I34N/H87Y (cyan), and AL-103 delP95aIns (black). All proteins display β-sheet structure with the characteristic two minima (235 and ∼217 nm) for these proteins. Experimental conditions were as follows: 20 μm protein in 10 mm Tris-HCl, pH 7.4. Far-UV CD spectra were acquired at 4 °C. Thermal denaturation experiments were performed from 4–90 °C at a rate of 0.5 °C min⁻¹. All experiments were followed by a refolding experiment (not shown).

FIGURE 3.

Differential effect of GAGs on the t₅₀ value of fibril formation kinetics of AL proteins at physiological pH. All reactions were performed with 20 mm protein and 1.0 mg ml⁻¹ GAG. Data are from fibril formation reactions conducted in triplicate. Reaction was considered positive when ThT fluorescence increased 4-fold (>200,000 arbitrary units). Arrowheads, fibril formation reaction was negative for the given protein and GAG. AL-09 H87Y, κI O18/O8, AL-09 I34N H87Y, and AL-103 delP95aIns did not form fibrils at pH 7.4 alone or in the presence of GAGs. None of the proteins tested were able to form fibrils in the presence of chondroitin sulfate A under physiological solution conditions, and therefore, differential effects on the t₅₀ values were not included for this GAG.

FIGURE 4.

TEM images of AL-12 at the end point of the reaction. A, pH 7.4; B, heparin; C, chondroitin sulfate A; D, heparan sulfate; E, dermatan sulfate; F, dextran sulfate; G, dextran. Scale bar, 200 nm. Shown are fibril formation kinetics at pH 7.4 (control) (H) in the absence (black) or presence (red) of 1.0 mg/ml heparin (I), chondroitin sulfate A (J), heparan sulfate (K), dermatan sulfate (L), dextran sulfate (M), or dextran (N). Kinetic traces are average of triplicates. Solid lines, fits to the Boltzmann equation. Data were collected for 800 h, but the last data points were discarded due to inner filter effects, as reported previously (38). A.U., arbitrary units. Error bars, S.E.

FIGURE 5.

TEM images of AL-12 R65S at the end point of the reaction. A, pH 7.4; B, heparin; C, chondroitin sulfate A; D, heparan sulfate; E, dermatan sulfate; F, dextran sulfate; G, dextran. Scale bar, 200 nm (except where indicated). Shown are fibril formation kinetics at pH 7.4 (control) (H) in the absence (black) or presence (red) of 1.0 mg/ml heparin (I), chondroitin sulfate A (J), heparan sulfate (K), dermatan sulfate (L), dextran sulfate (M), or dextran (N). Kinetic traces are the average of triplicates. Solid lines, fits to the Boltzmann equation. Data were collected for 800 h, but the last data points were discarded due to inner filter effects, as reported previously (38). A.U., arbitrary units. Error bars, S.E.

FIGURE 6.

TEM images of AL-103 at the end point of the reaction. A, pH 7.4; B, heparin; C, chondroitin sulfate A; D, heparan sulfate; E, dermatan sulfate; F, dextran sulfate; G, dextran. Scale bar, 200 nm. Shown are fibril formation kinetics at pH 7.4 (control) (H) in the absence (black) or presence (red) of 1.0 mg/ml heparin (I), chondroitin sulfate A (J), heparan sulfate (K), dermatan sulfate (L), dextran sulfate (M), and dextran (N). Kinetic traces are the average of triplicates. Solid lines, fits to the Boltzmann equation. Data were collected for 800 h, but the last data points were discarded due to inner filter effects, as reported previously (38). A.U., arbitrary units. Error bars, S.E.

FIGURE 7.

TEM images of AL-103 H92D at the end point of the reaction. A, pH 7.4; B, heparin; C, chondroitin sulfate A; D, heparan sulfate; E, dermatan sulfate; F, dextran sulfate; G, dextran. Scale bar, 200 nm. Shown are fibril formation kinetics at pH 7.4 (control) (H) in the absence (black) or presence (red) of 1.0 mg/ml heparin (I), chondroitin sulfate A (J), heparan sulfate (K), dermatan sulfate (L), dextran sulfate (M), or dextran (N). Kinetic traces are the average of triplicates. Solid lines, fits to the Boltzmann equation. Data were collected for 800 h, but the last data points were discarded due to inner filter effects, as reported previously (38). A.U., arbitrary units. Error bars, S.E.

FIGURE 8.

TEM images of AL-09 at the end point of the reaction. A, pH 7.4; B, heparin; C, chondroitin sulfate A; D, heparan sulfate; E, dermatan sulfate; F, dextran sulfate; G, dextran. Scale bar, 200 nm. Shown are fibril formation kinetics at pH 7.4 (control) (H) in the absence (black) or presence (red) of 1.0 mg/ml heparin (I), chondroitin sulfate A (J), heparan sulfate (K), dermatan sulfate (L), dextran sulfate (M), or dextran (N). Kinetic traces are the average of triplicates. Solid lines, fits to the Boltzmann equation. Data were collected for 800 h, but the last data points were discarded due to inner filter effects, as reported previously (38). A.U., arbitrary units. Error bars, S.E.

FIGURE 9.

TEM images of κI Y87H at the end point of the reaction. A, pH 7.4; B, heparin; C, chondroitin sulfate A; D, heparan sulfate; E, dermatan sulfate; F, dextran sulfate; G, dextran. Scale bar, 200 nm. Shown are fibril formation kinetics at pH 7.4 (control) (H) in the absence (black) or presence (red) of 1.0 mg/ml heparin (I), chondroitin sulfate A (J), heparan sulfate (K), dermatan sulfate (L), dextran sulfate (M), and dextran (N). Kinetic traces are the average of triplicates. Solid lines, fits to the Boltzmann equation. Data were collected for 800 h, but the last data points were discarded due to inner filter effects, as reported previously (38). A.U., arbitrary units. Error bars, S.E.

FIGURE 10.

TEM images of aggregates formed in the presence of chondroitin sulfate A at the end point of the reaction. A, AL-103 H92D; B, AL-103; C, AL-09; D, AL-12; E, κI Y87H; F, AL-12 R65S; G, AL-09 H87Y; H, κI O18/O8; I, AL-09 I34N/H87Y; J, AL-103 delP95aIns. For κI Y87H, a mixture of fibrillar and spherical species was observed. For AL-09, a mixture of fibrillar, amorphous, and spherical chain aggregates were observed. Scale bar, 200 nm (except where indicated). Proteins are shown in order of increasing stability as measured by T_m values, as shown in Table 1.

FIGURE 11.

Irreversible changes caused by partial unfolding in the presence of heparan sulfate in AL-103 H92D. AL-103 H92D and AL-09 secondary structure in the absence (A and B) or presence of 1.0 mg ml⁻¹ heparan sulfate (C and D) or chondroitin sulfate A (E and F), followed by far-UV CD spectra. 20 μm protein was incubated at 4 °C (black open circles) for 10 min and then incubated at 37 °C (black solid circles) for 10 min and finally incubated at 4 °C (red solid circles).

FIGURE 12.

A–C, suggested model of GAG mode of action on AL amyloid fibril formation. D, representative morphology of aggregated species observed by TEM. The morphological designations/definitions were based on empirical/visual comparison and contrasted with the description of amyloid aggregates and intermediates in the fibril formation pathway, reported for AL proteins as well as other proteins (47).