Role of the mechanisms for antibody repertoire diversification in monoclonal light chain deposition disorders: when a friend becomes foe

Abstract

The adaptive immune system of jawed vertebrates generates a highly diverse repertoire of antibodies to meet the antigenic challenges of a constantly evolving biological ecosystem. Most of the diversity is generated by two mechanisms: V(D)J gene recombination and somatic hypermutation (SHM). SHM introduces changes in the variable domain of antibodies, mostly in the regions that form the paratope, yielding antibodies with higher antigen binding affinity. However, antigen recognition is only possible if the antibody folds into a stable functional conformation. Therefore, a key force determining the survival of B cell clones undergoing somatic hypermutation is the ability of the mutated heavy and light chains to efficiently fold and assemble into a functional antibody. The antibody is the structural context where the selection of the somatic mutations occurs, and where both the heavy and light chains benefit from protective mechanisms that counteract the potentially deleterious impact of the changes. However, in patients with monoclonal gammopathies, the proliferating plasma cell clone may overproduce the light chain, which is then secreted into the bloodstream. This places the light chain out of the protective context provided by the quaternary structure of the antibody, increasing the risk of misfolding and aggregation due to destabilizing somatic mutations. Light chain-derived (AL) amyloidosis, light chain deposition disease (LCDD), Fanconi syndrome, and myeloma (cast) nephropathy are a diverse group of diseases derived from the pathologic aggregation of light chains, in which somatic mutations are recognized to play a role. In this review, we address the mechanisms by which somatic mutations promote the misfolding and pathological aggregation of the light chains, with an emphasis on AL amyloidosis. We also analyze the contribution of the variable domain (V_L) gene segments and somatic mutations on light chain cytotoxicity, organ tropism, and structure of the AL fibrils. Finally, we analyze the most recent advances in the development of computational algorithms to predict the role of somatic mutations in the cardiotoxicity of amyloidogenic light chains and discuss the challenges and perspectives that this approach faces.

Keywords: V(D)J rearrangement; amyloid; antibodies; immune system; light chain (AL) amyloidosis; protein aggregation; somatic hypermutation.

Figure 1

Structural characteristics of human immunoglobulins. (A) Crystal structure of the intact human IgG B12 with broad and potent activity against primary HIV-1 isolates (Method: X-ray diffraction - PDB 1HZH). (B) Solution structure of human Immunoglobulin M (Method: Solution X-ray scattering – PDB 2RCJ) (C) Solution structure of human secretory IgA1 (Method: Solution X-ray scattering – PDB 3CHN). (D) Semi-extended solution structure of human myeloma immunoglobulin D (Method: solution X-ray scattering – PDB 1ZVO). (E) Crystal structure of amyloidogenic and cardiotoxic Bence-Jones Λ2 LC dimer H9 (Method: X-ray crystallography – PDB 5M6A). The insert in A shows the spatial conformation of the light chain (L1 to L3, colored in deep blue) and heavy chain (H1 to H3, colored in brown) CDRs. CM stands for carbohydrate moiety attached to the C_H2 domain. The Fab (fragment antigen-binding) and Fc (fragment crystallizing) regions of each antibody are indicated. In C, the secretory component of the dimeric IgA is shown in red. The long hinge connecting the Fab and Fc regions in IgA1 antibodies is also indicated. Due to limitations of the structural method applied, the spatial location of the J chain was not determined. In C and D, the light and heavy chains are shown in blue and green, respectively. In E, the variable domains of the light chain dimer are shown in blue and light blue, while the constant domains are shown in red and salmon. Note that the Bence Jones dimer is stabilized by a LC-LC interface that mimics the LC-HC interface of a functional antibody. All structures were prepared with PyMOL (PyMOL Molecular Graphics System, Version 2.5.2, Schrödinger, LLC).

Figure 2

B cell differentiation pathway and mechanisms for generation of antibody diversity. (A) The human polyclonal antibody repertoire is generated by two molecular mechanisms: 1) V(D)J gene segments recombination, and 2) somatic hypermutation, which occurs at specific stages of B-cell differentiation. Plasma cells, the final stage of B cell differentiation, secrete large amounts of the specific antibody (IgG, IgA, or IgE). Under certain circumstances, a clone of plasma cells can overproduce the antibody LC and secret it in a free state, which entails the risk of aggregation and disease. MFLC stands for monoclonal free light chain. (B) Schematic representation of the IGLV-IGLJ-IGLC gene segment recombination in the Λ LC locus. Note that the V_L domain of the LC is encoded by the IGLV and IGLJ gene segments, while the C_L domain is encoded by the IGLC gene. (C) Structural characteristics of the immunoglobulin LCs and AL amyloid fibrils. The LC structures correspond to the full-length (FL) Λ6 LC of the anti-Hepcidin Fab (PDB 3H0T) and the Λ1 monoclonal free LC (Bence Jones protein) LOCW, both determined by x-ray diffraction. The variable (V_L) and constant (C_L) domains are indicated. The regions in β-strand conformation are colored red. The AL fibril structures correspond to the ex-vivo Λ6 (PDB 6HUD) and Λ1 (PDB 6IC3) AL fibrils obtained from the cardiac deposits in patients with AL amyloidosis determined by cryo-EM. The β core of the AL fibrils is composed only of segments of the V_L domain. The FL LCs and AL fibrils shown in the figure are not related to each other. They were included in the same figure only for comparative purposes. Structures shown in C were prepared with PyMOL (PyMOL Molecular Graphics System, Version 2.5.2, Schrödinger, LLC).

Figure 3

Anatomopathological features of light chain deposition disease (LCDD). (A) Hematoxylin & eosin (H&E) and (B) PAS staining of kidney biopsies from two patients in the early and late stages of the disease, respectively. Note in B the nodular mesangium with a markedly increased tenascin-rich extracellular matrix. (C) Electron microscopy (EM) analysis that shows expanded nodular mesangium with an increased extracellular matrix and punctate, powdery, ground-pepper-like extracellular deposits.

Figure 4

Anatomopathological characteristics of renal AL amyloidosis (A–C) H&E, Congo red, and immunofluorescence analysis for human κ LC of a renal biopsy of a κ AL amyloidosis patient in advance stage of the disease. (D, E) show images taken in EM analysis of the same sample shown in (A–C). (F) The H&E staining of the renal biopsy of a Λ AL amyloidosis in early stage of the disease is presented for comparative purpose. In A, arrows indicate AL amyloid deposits stained with H&E that appear as a waxy, homogenous, eosinophilic material that has almost completely replaced the mesangium. In B and C, arrows indicate the same material stained with Congo red, and detected by the anti-human κ LC antibody, respectively. In C, the primary, and secondary antibodies were goat anti-human κ LC antibody and fluorescein-conjugated rabbit anti-goat IgG, respectively. Note in D and E the abundant randomly disposed fibrils with diameter in the range of 7.3 nm to 12.3 nm, characteristic of the AL amyloid fibrils.

Figure 5

Impact of somatic mutations Ile21Phe and Leu104Val on the structure and biophysical properties of the amyloidogenic Λ6 LC AR. (A) to (C) show the crystallographic structure of the rV_L proteins AR, 6aJL2(G25), and 6aJ2(R25). Mutant residues (m) Phe21 and mVal104, and wild type Ile21 and Leu104 are represented in stick format and indicated. For reference purposes, the highly conserved Trp35, the disulfide bond Cys23-Cys88, Phe2, and the residue at position 25 are also represented in stick format and indicated. The inset in A and B show a more detailed representation of the spatial relationship of mPhe21, mVa104, Ile21, and Leu104 with non-polar residues that compose the core of the V_L domain fold. The mutant and wild-type residues, Trp35, and the disulfide bond Cys23-Cys88 are represented in a combination of semi-transparent spheres/sticks representation, to highlight the side chain-to-side chain interactions. Proteins 6aJL2(R25) and 6aJL2(G25) have the germline sequence of the IGLV6-57 (Λ6) segments. They have the same sequence, except in position 25, which is an allotypic variation of the IGLV6-57. Variant G25 suppresses a cation-π interaction between Phe2 and Arg25, determining a less thermodynamically stable and more fibrillogenic protein (169). (D-F) show the in vitro fibrillogenesis of the rV_L Λ6 proteins AR, 6aJL2(G25), and 6aJL2(R2) performed at 200 µg/ml in presence of 20 µM thioflavin T. The protein solutions were incubated at 37°C with constant stirring of 350 r.p.m. with a 5x2 mm Teflon-coated magnetic micro-stir bar. Amyloid-like fibril formation was detected by serially measuring ThT fluorescence, exciting the sample at 450 nm, and recording the ThT fluorescence at 482 nm. The lag time (t _lag) for nucleation was calculated by extrapolating the linear region of the hyperbolic phase back to the abscissa (213). The table in the bottom shows the thermodynamic parameters of the rV_L protein AR, its single and double mutants AR(F21I), AR(V104L), and AR(F21I-V104L), and the germline proteins 6aJL2(G25) and 6aJL2(R25) determined by reversible unfolding with increasing concentration of guanidinium hydrochloride (GdnHCl). (A) Data were taken from reference (205). (B, C) Data were taken from reference (211). ΔΔG values were calculated considering AR as the wild type; positive values indicate a higher stability than that of AR. The structures shown in (A) to (C) were prepared with PyMOL (PyMOL Molecular Graphics System, Version 2.5.2, Schrödinger, LLC) using PDB 5IR3 for rVL AR, PDB 5C9K for rVL 6aJL2 (G25), and PDB 2W0K for rVL 6aJL2 (R25).

Figure 6

Structure of ex-vivo AL fibrils determined by cryo-EM. (A) AL fibril formed by a Λ1 LC encoded by the V_L gene segment IGLV1-44. PDB ID 6IC3 (233). (B) FOR001 AL fibril formed by a Λ1 LC encoded by the V_L gene segment IGLV1-51*02. PDB ID 7NSL (234). (C) FOR005 AL fibril formed by a Λ3 LC encoded by the V_L gene segment IGLV3-19*01. PDB OD 6Z1O (235). (D) AL fibril formed by the Λ6 LC AL55 encoded by the V_L gene segment IGLV6-57*02. PDB ID 6HUD (232). The four AL fibrils were extracted from cardiac deposits of patients with cardiac AL amyloidosis. Column 1 shows cartoon representations of the V_L domain polypeptide chain conformation in the amyloid fibrils. Regions in β-strand are shown as arrow and colored red. Disordered regions are represented as dashed lines in an arbitrary arrangement. Column 2 shows the same structures of column 1, but with the projection of the amino acid side chains in a combination of semi-transparent spheres/sticks representation to highlight the side chain-to-side chain interactions that contribute to fibril stability. The amino acid residues spanning the CDR1, and the loop connecting the β-strands E and F (loop E-F) are colored red and blue, respectively. Column 3 shows the assembly of the AL fibrils by stacking the monomers one on top of the other, with parallel in-register arrangement of β strands. In all structures, the conserved intradomain disulfide bond Cys23-Cys88 is represented in sticks or spheres format and colored yellow. N and C stand for N- and C-terminal. (E) and (F) show a cartoon representation of the same AL Λ6 fibril shown in D, with a detailed representation of the different types of interactions that stabilize the fibril structure. In (E), the amino acid side chains are shown in a combined semi-transparent spheres/stick format, using the following color code: blue for negatively charged (acid) amino acids, red for positively charged (basic) amino acids, salmon for polar non-charged amino acids, light blue for non-polar amino acids, black for proline, and yellow for cysteines. Some residues are indicated for structural reference. Note that non-polar side chains tend to cluster buried in the fibril core, while charged and polar non-charged amino acids tend to locate in the solvent exposed surface. The image in (F) shows the stacking of the AL55 LC monomers, one on top of the other, with parallel in-register arrangement of β strands. The H bonds between the peptide backbones of adjacent monomers are shown as black dotted lines. Aromatic amino acids interacting by intermolecular stacking are shown. In both images, the conserved intradomain disulfide bond Cys23-Cys88 (colored yellow) and the fibril-specific salt bridge formed by Lys17 and Asp92 are shown. Note that this salt bridge is intermolecular, that is, it is established between residues located in monomers that are contiguous in the fibrillar structure. Regions in β-strands are represented as arrows. Amino acids are identified using a one-letter code. All structures were prepared with PyMOL (PyMOL Molecular Graphics System, Version 2.5.2, Schrödinger, LLC).

Figure 7

Intralisosomal processing and fibrillar aggregation of an amyloidogenic LC in human mesangial cells. EM micrographs of lysosomes obtained from cultured human mesangial cells that were incubated with an amyloidogenic Λ LC purified from the urine of a patient with AL amyloidosis. After (A) 30 min and (B) 2 h, the cells were harvested, and the lysosomal fraction was obtained by density gradient ultracentrifugation. Note in (A) the intralysosomal amyloid fibrils arranged in perfect circles (black arrows). In (B), the amyloid fibrils show a less orderly arrangement (black arrow inside the lysosome). Note some amyloid fibrils that appear to have escaped from the lysosome (black arrows outside the lysosome).