Protein Misfolding - Marina Ramirez-Alvarado

Role of the Location of Mutations in Light Chain Amyloidosis

Light Chain Amyloidosis (AL) is characterized by the abnormal proliferation of monoclonal plasma B cells, which secrete monoclonal immunoglobulin light chains. B-cell development involves the rearrangement of immunoglobulin (Ig) gene segments, conferring on each mature B cell a unique combination of one heavy and one light chain variable region gene (VH and VL) that subsequently may undergo somatic hypermutation in the germinal centers of lymph nodes. It has been reported that VH and VL genes associated with AL display evidence of hypermutation adding further support that AL VL clones derive from post-germinal center B cells (Perfetti et al., 1998).

The secreted monoclonal AL light chains misfold and deposit as amyloid fibrils in vital organs leading to organ failure and death. There are two VL gene subtypes, λ and κ. There is a preponderance of L versus κ in light chain genes associated with AL (λ:κ= 3:1) compared to normal individuals (λ: κ =1:2). The most commonly used germline donor sequences in AL patients are λ6a, λ3r, λ2 and λ1 (Abraham et al., 2003). It has also been reported that patients whose clones belong to VL λ6a are predominantly associated with renal involvement whereas AL VL clones derived from λ1c, λ2a2 λ3r are more likely to present with cardiac and multisystem disease (Comenzo et al., 2001). Our analyses have shown that mutations are located throughout the protein and do not always concentrate on the hypervariable regions of the VL domain.

Protein stability, fibril formation and structure

We are interested in studying the effect the different mutations in the AL proteins have on the secondary structure, protein folding and stability and in amyloid formation. We use the kappa germline protein as our control. We have followed protein unfolding by thermal and chemical denaturation using Circular Dichroism and Fluorescence spectroscopy. Amyloid fibril formation has been carried out at the melting temperature of the proteins and verified by Thioflavine T binding and electron microscopy. We have used X-ray crystallography and Nuclear Magnetic Resonance (NMR) spectroscopy to study the structural effects of mutations across several AL proteins.

Our laboratory is conducting the first systematic studies of restorative mutations in AL proteins, allowing us to assess the contributions of individual residues or combinations of residues to amyloidogenicity. Although the mutations in AL proteins are unique to each patient, an underlying structural mechanism may be involved in fibril formation that is common to all pathogenic LC proteins. We feel that these features are best explored through the relationship of protein stability, fibril formation kinetics and protein structure. We have shown that the dimer interface of AL-09 protein is twisted 90° relative to the protein from its germline sequence, κI O18/O8 (wild type). Three of the seven mutations in AL-09 are non-conservative, and all are located within the dimer interface. In order to understand the specific role of each of the non-conservative mutations, we have generated restorative single and double mutants. The restoration of one particular mutant (AL-09 H87Y) completely restores thermodynamic stability and delays fibril formation to κI O18/O8 (wild type) levels, and restores the canonical dimer interface, thereby emphasizing the potential importance of the structural integrity of these proteins to protect against amyloidogenicity. To understand the cooperativity and interplay of the mutations within different AL proteins, we are performing similar systematic mutational studies. These studies allow us to see how combinations of the somatic mutations in AL proteins drive amyloidogenicity. Despite the proteins we study being approximately 95% similar they have widely different properties. It is important to understand the impact of not only specific mutations and their locations within the protein but also how they act together, in concert within the VL domain system.