Somatic mutations of the L12a gene in V-kappa(1) light chain deposition disease: potential effects on aberrant protein conformation and deposition

Abstract

Light chain deposition disease (LCDD) and light chain amyloidosis (AL) are disorders of monoclonal immunoglobulin deposition in which normally soluble serum precursors form insoluble deposits in tissues. A common feature in both is the clonal proliferation of B-cells that produce pathogenic light chains. However, the deposits in LCDD differ from those in AL in that they are ultrastructurally granular rather than fibrillar and do not bind Congo red or colocalize with amyloid P component or apolipoprotein E. The reason(s) for their differences are unknown but are likely multifactorial and related to their protein conformation and their interaction with other molecules and tissue factors in the microenvironment. Knowledge of the primary structure of the light chains in LCDD is very limited. In the present study two new kappa(1) light chains from patients with LCDD are described and compared to seven other reported kappa-LCDD proteins. The N-terminal amino acid sequences of light chain GLA extracted from the renal biopsy and light chain CHO from myocardial tissue were each identical to the respective light chains isolated from the urines and to the V-region amino acid sequences translated from the cloned cDNAs obtained from bone marrow cells. The germline V-region sequences, determined from the genomic DNA in both and in MCM, a previously reported kappa(1) LCDD light chain, were identical and related to the L12a germline gene. The expressed light chains in all three exhibit amino acid substitutions that arise from somatic mutation and result in increased hydrophobicity with the potential for protein destabilization and disordered conformation.

Figure 1.

A and B: GLA renal biopsy tissue. A: Immunofluorescence micrograph demonstrates immunostaining for κ light chain in glomerular and tubular basement membranes. B: Electron micrograph of a tubule shows clustered granular electron-dense deposits (arrows) in basement membrane. C and D: CHO myocardial tissue. C: Immunofluorescence micrograph reveals perimyocytic deposits staining for κ. D: No staining for λ light chain. Magnification: A, ×200; B, ×10,000; C and D, ×300.

Figure 2.

Fifteen percent SDS-PAGE and Western blot analysis of GLA and CHO κ light chains. Extracted light chains from GLA kidney biopsy tissue (lane 1) and CHO cardiac tissue (lane 5) were stained with Coomassie blue. Immunoreactivity was demonstrated for the extracted proteins GLA (lane 2) and CHO (lane 6) with anti-κ. Urine eluates are for GLA (lane 3) and CHO (lane 7) after KappaLock purification, stained with Coomassie blue. Western blot analysis was made of GLA (lane 4) and CHO (lane 8) urine eluates immunostained with anti-κ.

Figure 3.

cDNA sequence of the V-region of the monoclonal κ₁ light chains GLA (A) and CHO (B) cloned from bone marrow plasma cells. Numbering is according to Kabat et al.

Figure 4.

Comparison of the amino acid sequence encoded by the V-κ₁ L12a subgroup germline gene with GLA, CHO and MCM sequences.

Figure 5.

Variable-region light chain dimers of GLA (A) and CHO (B), showing the spatial location of amino acid substitutions. The backbone structure of κ₁ light chain REI (Brookhaven Protein Databank entry REI) was computationally mutated to introduce the residues at the positions shown. Side-chain atoms represented in solid spheres are carbon (green), oxygen (red), and nitrogen (blue).

Figure 6.

The hydrophobic residue content of the CDR-presenting surfaces of V-region light chain dimers of proteins MCM, GLA, and CHO are compared to that of REI. Atoms of hydrophobic amino acids are represented by solid green spheres. The models were constructed by using the program Insight II (Molecular Simulations) to replace amino acids in the crystallographic structure of REI (pdb 1 rei) with the variations found in the amino acid sequences of the three V-region light chains associated with LCDD. All show an increased proportion of hydrophobic side chains accessible to solution. Arrows indicate Trp³², a genetically encoded feature of κ1a (gene: L12a) proteins not found in the κ1b (gene: O18–O8) protein REI. The fully exposed nature of the indole side chain of Trp³², as modeled, has been confirmed by the crystallographically determined structure of the human anti-gp41 Fab, 3D6 (pdb1dfb), which incorporates a κ1a light chain. The view is down the twofold axis of the V-domain dimer.