Site-directed mutagenesis reveals regions implicated in the stability and fiber formation of human λ3r light chains

Abstract

Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this work, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, the second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40-60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. This mutagenic approach helped to identify key regions implicated in λ3 AL.

Keywords: Amyloid; Antibody; Fibril; Mutagenesis in Vitro; Thermodynamics.

FIGURE 1.

Sequence alignment of λ3 germ lines, λ3 proteins obtained from patients and healthy individuals, and 6aJL2 germ line. The amino acid residues are colored by relative conservation using the ALIGN program (54). The dots indicate gaps inserted to maximize the alignment. The top group shows the λ3germ line sequences, including the 6aJL2 germ line sequence. Green, slate, and purple triangles above the alignment mark the sites that were mutated in 3rJL2, 3rJL2/Y, and 3rJL2/YA, respectively. Black rectangles above the alignment mark the potential aggregation regions predicted by TANGO, PASTA, and Aggrescan servers. Framework (FR), complementarity determining regions (CDR), and joining (JL2) segments are indicated below the alignment with pink, indigo, and gray stripes, respectively. The secondary structure elements of 3rJL2/Y are marked below the alignment. The middle and lower groups comprise the λ3 sequences derived from patients with AL amyloidosis and healthy individuals, respectively. The germ line sequences were obtained from the VBASE database, and the λ3 sequences derived from patients and healthy individuals were obtained from the National Center for Biotechnology Information server.

FIGURE 2.

Denaturation curves of λ3 proteins. A and C, fraction of unfolded protein as a function of guanidine HCl concentration. The solid lines represent a two-state fit of the data from 0 to 5 m of the denaturant. B and D, temperature-induced unfolding. The inset in B shows the fluorescence spectra of 3rJL2/Y and 3rJL2/YA. The filled symbols indicate denaturation, and the open symbols indicate renaturation. The 6aJL2 protein is shown for comparison purposes. The calculated thermodynamic parameters are shown in Table 2.

FIGURE 3.

Far UV CD spectra at different temperatures of λ light chains. Unfolding and refolding traces correspond are shown as dashed lines and open circles, respectively. CD spectra were recorded during heating and cooling of the same sample. The continuous lines represent the native protein. The refolding of 3rJL2/Y, 3rJL2/YA, 3rJL2/YA/P7D, 3rJL2/YA/P40S, and 3rJL2/YA/I48M shows some changes in secondary structure as evidenced by the displacement of the refolding trace beyond the minimum (215 nm) or approximately at the maximum at 225 nm. The far UV CD recordings were carried out using a 0.2-cm-path length cuvette (200 μg/ml protein in 20 mm Na₂HPO₄, pH 7.5).

FIGURE 4.

Structural differences and dimer interfaces of germ line 3mJL2 and the 3rJL2 mutants. A, superposition of 3mJL2 (green), 3rJL2/Y (yellow), and 3rJL2/YA (cyan) monomers showing the location of residues Pro-7, Pro-8, Pro-40, Ile-48, and Ala-91 in 3rJL2/YA. The β-strands that form the variable domain are marked with capital letters. B, the conformation of the N-terminal sheet switch region is similar in 3mJL2 and 3rJL2, including the β-bulge in strand A. The two hydrogen bonds that formed between Pro-7 and Pro-8 in β-strand A and Thr-103 and Lys-104 in β-strand G are represented by dotted lines. In 3mJL2, the additional residue at CDR3(Y95b) forms a hydrogen bond with E3, tightening the N terminus to the core of the variable domain. The 6aJL2 monomer is shown in orange for comparison. C, superposition of the region between the Pro-40 and Ile-48 of 3mJL2, 3rJL2/Y, 3rJL2/YA, and 6aJL2. D, the unique hydrophobic interactions that formed between Trp-91 and Tyr-34 in 3rJL2/Y are marked. Notice the additional stacking with residue Val-96. Residues Tyr-32, Leu-46, and Tyr-49, which complement the hydrophobic patch, are shown. E, 3mJL2 (magenta and pink) presents the native dimer formed by most of the VL-VL crystal structures. F, the dimers of 3rJL2/Y (green and pale green) and 3rJL2/YA (cyan and slate) present a different arrangement at their asymmetric units. 3rJL2/Y and 3rJL2/YA only form the native dimer interface with their corresponding symmetry partners. Residue Arg-20, located on the strand edge of 3mJL2, prevents the formation of the arrangement observed in B).

FIGURE 5.

Analysis of the λ light chain variable domain oligomeric state. Purity of the samples was assessed by 15% SDS-PAGE. Fifty micrograms of protein was loaded into each well. The graph show the elution profile, and the inset shows the calibration plot. A calibration curve for λ light chains and standard proteins was performed in a Superdex 75 column. The calibration curve was used to estimate the molecular mass of the variable domains based on the elution position during analytical size exclusion chromatography. All of the purified variable domains eluted with a molecular mass corresponding to a monomer.

FIGURE 6.

Effect of protein concentration on λ light chain stability. A, denaturation curves in the presence of guanidine HCl. B, thermal denaturation curves: 20, 50, and 200 μg. The filled symbols and the open symbols indicate denaturation and renaturation, respectively. As can be seen, there is not a concentration dependence relationship with ΔG. The calculated thermodynamic parameters are shown in Table 5.

FIGURE 7.

Aggregation properties of 3rJL2 and 3mJL2 predicted from their amino acid sequences. Four regions with intrinsic aggregation propensity were identified on the λ3 sequences using the TANGO, PASTA, and Aggrescan servers. Only the representative TANGO prediction profiles are shown. The regions are identified with letters in the 3rJL2 profile as a (residues 32–37), b (residues 44–50), c (residues 70–76), and d (residues 93–100). Region b, which includes strand C′, has the highest aggregation score. The replacement of Ile-48 by Pro or Met diminished or eliminated the aggregation score, respectively. The fibril extension rate of mutant 3rJL2/YA/I48M was slower than that of 3rJL2/YA, confirming the importance of position 48 in β-sheet aggregation. Surprisingly, there were no predicted aggregation regions in the 6aJL2 sequence.

FIGURE 8.

Fibrillogenesis in vitro. A, kinetics of the in vitro fibrillogenesis of λ3 proteins. The 3rJL2/YA, 3rJL2/YA/I48M, and 3rJL2/YA/P7D mutants present different lag phase (t_lag) and fiber extension times. In contrast, 3rJL2/Y, 3rJL2/YA/P8S, 3rJL2/YA/P40S, and 3mJL2 did not form fibrils. The 6aJL2 protein formed fibrils under the conditions assayed at a similar rate as 3rJL2/YA/P7D. Samples containing 100 μg/ml protein were incubated in PBS at 37 °C under continuous stirring. Fibril formation was monitored by changes in the thioflavin T fluorescence. The error bars represent the standard deviations from three independent experiments. B, transmission electron microscopy analysis of fibrils. TEM images of 3rJL2/YA, 3JL2/YA/I48M, 3rJL2/YA/P7D, and 6aJL2 display the typical morphology. The samples were collected from in vitro fibrillogenesis experiments (see “Materials and Methods”). The black bars indicate 100 nm.