Polymorphic fibrillation of the destabilized fourth fasciclin-1 domain mutant A546T of the Transforming growth factor-β-induced protein (TGFBIp) occurs through multiple pathways with different oligomeric intermediates

Abstract

Mutations in the transforming growth factor β-induced protein (TGFBIp) are linked to the development of corneal dystrophies in which abnormal protein deposition in the cornea leads to a loss of corneal transparency and ultimately blindness. Different mutations give rise to phenotypically distinct corneal dystrophies. Most mutations are located in the fourth fasciclin-1 domain (FAS1-4). The amino acid substitution A546T in the FAS1-4 domain is linked to the development of lattice corneal dystrophy with amyloid deposits in the superficial and deep stroma, classifying it as an amyloid disease. Here we provide a detailed description of the fibrillation of the isolated FAS1-4 domain carrying the A546T substitution. The A546T substitution leads to a significant destabilization of FAS1-4 and induces a partially folded structure with increased surface exposure of hydrophobic patches. The mutation also leads to two distinct fibril morphologies. Long straight fibrils composed of pure β-sheet structure are formed at lower concentrations, whereas short and curly fibrils containing a mixture of α-helical and β-sheet structures are formed at higher concentrations. The formation of short and curly fibrils is preceded by the formation of a small number of oligomeric species with high membrane permeabilization potential and rapid fibril formation. The long straight fibrils are formed more slowly and through progressively bigger oligomers that lose their membrane permeabilization potential as fibrillation proceeds beyond the lag phase. These different fibril classes and associated biochemical differences may lead to different clinical symptoms associated with the mutation.

FIGURE 1.

Chemical and thermal denaturation of FAS1–4 A546T (■) and WT (○). A, shown is urea unfolding of FAS1–4 A546T monitored by the change in the CD signal at 222 nm versus urea concentration. Inset, CD spectra of FAS1–4 A546T and WT at 0 m urea are shown. Error bars are based on duplicates. B, thermal denaturation of FAS1–4 A546T and WT monitored by the change in the CD signal at 222 nm as a function of temperature is shown. C, shown is ANS fluorescence of FAS1–4 A546T and FAS1–4 WT with the λ_max indicated in the graph. a.u., arbitrary units.

FIGURE 2.

Trypsin digestion of FAS1–4 WT and FAS1–4 A546T. A, shown is a SDS-PAGE gel of FAS1–4 WT and FAS1–4 A546T incubated with 1, 0.1, 0.01, 0.001, and 0% of trypsin (w/w). B, shown are the relative intensities of the band on SDS-PAGE after trypsin digestion of FAS1–4 A546T (■) and FAS1–4 WT (○). The intensities of the band on the SDS-PAGE gel were determined using the software ImageJ (National Institutes of Health).

FIGURE 3.

Fibrillation of FAS1–4 A546T at various conditions. A, fibrillation of FAS1–4 A546T was monitored by ThT fluorescence over time in a plate reader for 0.4 (triangles), 0.8 (squares), and 1.2 mg/ml (circles) FAS1–4A546T with 20 mm NaCl (open symbols) or 0.001% heparin (closed symbols). B, shown is ThT fluorescence of 0.4 (squares) and 0.8 mg/ml (circles) FAS1–4 with (closed symbols) and without (open symbols) 10% fibril seeds of preformed fibrils of 1.2 mg/ml FAS1–4 0.001% heparin. C, ThT fluorescence of uniformly ¹³C,¹⁵N-labeled FAS1–4 A546T (●) compared with non-labeled FAS1–4 A546T (○) during fibrillation. The variation in lag times between these two samples is within the level of variation for identical samples. a.u., arbitrary units.

FIGURE 4.

Electrostatic screening of fibrillation. A, ThT fluorescence of 0.8 mg/ml FAS1–4 A546T with 20 mm (●), 100 mm (○), 137 mm (■), 200 mm (□), 300 mm (▴), and 400 mm (△) NaCl. The arrow indicates increasing NaCl concentration. B, left y axis, the logarithm of the slope of the elongation phase of the ThT curve seen during fibrillation is plotted against the square root of the NaCl concentration in mm for 0.8 mg/ml FAS1–4 A546T and gives a straight line (●). Right y axis, shown is the lag time of the fibrillation curves in A plotted against the square root of the NaCl concentration (○). The lag time is determined as the intersection between the two straight lines formed during the lag phase and the elongation phase respectively. a.u., arbitrary units.

FIGURE 5.

FTIR analysis of the secondary structure of FAS1–4 A546T. A, shown is a comparison of FTIR spectra and the second derivative of the spectra of uniformly ¹³C,¹⁵N-labeled FAS1–4 A546T (○) and non-labeled FAS1–4 A546T (●). B, comparison of FTIR spectra of fibrils of FAS1–4 A546T obtained at 0.4 (circles), 0.8 (squares), and 1.2 mg/ml (triangles) with 20 mm NaCl (open symbols) or 0.001% heparin (closed symbols).

FIGURE 6.

Structural analysis of fibrils formed at different concentrations with 20 mm NaCl, 400 mm NaCl, or 0.001% heparin. A, shown is TEM analysis of the morphology of fibrils of FAS1–4 A546T at 0.4, 0.8, and 1.2 mg/ml with 20 mm NaCl, 400 mm NaCl, or 0.001% heparin. B, shown are CD spectra of the secondary structure of fibrils of FAS1–4 A546T at 0.4 (circles), 0.8 (squares), and 1.2 mg/ml (triangles) with 20 mm NaCl (open symbols) or 0.001% heparin (closed symbols). C, shown are ThT fluorescence spectra of the two types of fibrils normalized to protein concentration, long and straight fibrils at 0.4 mg/ml 0.001% heparin (●) and short and curly fibrils at 1.2 m/ml 20 mm NaCl (○), and ThT in buffer (■). D, TEM analysis of the morphology of fibrils of FAS1–4 A546T at 0.1 and 0.2 mg/ml with 0.001% heparin is shown. a.u., arbitrary units.

FIGURE 7.

Structural analysis of first and second generation fibrils formed at different concentrations with 20 mm NaCl or 0.001% heparin. A, shown is limited trypsin digestion of the two types of fibrils of FAS1–4 A546T, long and straight fibrils at 0.4 mg/ml 0.001% heparin (○) and short and curly fibrils at 1.2 mg/ml 20 mm NaCl (■). B, TEM analysis of the morphology is shown. C, shown are CD spectra of the secondary structure of fibrils of FAS1–4 A546T at 0.4 mg/ml seeded with 1.2 mg/ml 0.001% heparin fibrils with 20 mm NaCl (○) and 0.001% heparin (●). D, shown are CD spectra of the secondary structure of fibrils of FAS1–4 A546T at 1.2 mg/ml seeded with 0.4 mg/ml 0.001% heparin fibrils with 20 mm NaCl (○) and 0.001% heparin (●).

FIGURE 8.

AF4 analysis of samples removed at different time point from a fibrillating sample with 1.2 mg/ml FAS1–4 A546T. A, shown is an elution profile of time point-samples taken during fibrillation of 1.2 mg/ml FAS1–4 A546T, with zoomed areas showing the dimer peak and the oligomer peak. Samples were removed at time point 0 h (○), 1 h (□), 2 h (♢), 3 h (×), 4 h (|), 5 h (△), 6 h (●), 7 h (■), 13 h (♦), and 24h (▴). B, peak integral of the monomer (●), dimer (○), and oligomer species (×) made using Gaussian curve fitting using the OriginPro 8 software.

FIGURE 9.

AF4 analysis of samples removed at different time points from a fibrillating sample with 0.4 mg/ml FAS1–4 A546T, 0.001% heparin. Shown is an elution profile of time-point samples taken during fibrillation of 0.4 mg/ml FAS1–4 A546T, 0.001% heparin with zoomed areas showing the monomer peak and the higher order oligomer peaks. Samples are removed at time point 0 h (○), 1 h (□), 3 h (♢), 5 h (×), 7.5 h (|), 11 h (△), and 24 h (●). Different oligomeric species can be seen.

FIGURE 10.

Calcein release from DOPG vesicles by samples removed at different time points during fibrillation of 0.4 mg/ml FAS1–4 A546T, 0.001% heparin (●) and 1.2 mg/ml FAS1–4 A546T 20 mm NaCl (■). Calcein release using 0.6 μm protein at different time points during fibrillation and ThT fluorescence for the same time points of 0.4 mg/ml FAS1–4 0.001% heparin (○) and 1.2 mg/ml FAS1–4 20 mm NaCl (□) is shown. Error bars based on duplicates. The calcein release is plotted against the primary y axis, and the ThT fluorescence is plotted against the secondary y axis.

FIGURE 11.

Proposed model for the formation of the two different types of fibrils. Initially the monomer with a structure of mixed α-helical and β-sheet structure is present. At high concentrations the monomer forms dimers and bigger oligomers and curly fibrils. The formation of fibrils occurs so rapidly that the monomer retains some of its native structure in the fibrils, leading to curly fibrils with a mixed α-helical and β-sheet structure. At 0.4 mg/ml the formation of fibrils occurs more slowly and through the formation of many different types of oligomers. This leads to the formation of long straight fibrils with β-sheet structure as the monomer has sufficient time to rearrange. At the final stage, the size of monomers and dimers is not scaled to match the size of the fibrils (these will be much bigger than the ones shown in the figure). MPP, membrane permeabilization potential.