A ROLE FOR DESTABILIZING AMINO-ACID REPLACEMENTS IN LIGHT-CHAIN AMYLOIDOSIS

Light-chain (L-chain) amyloidosis is characterized by deposition of fibrillar aggregates composed of the N-terminal L-chain variable region (V-L) domain of an immunoglobulin, generally in individuals overproducing a monoclonal L chain. In addition to proteolytic fragmentation and high protein concentration, particular amino acid substitutions may also contribute to the tendency of an L chain to aggregate in L-chain amyloidosis, although evidence in support of this has been limited and difficult to interpret. In this paper we identify particular amino acid replacements at specific positions in the V-L domain that are occupied at frequencies significantly higher in those L chains associated with amyloidosis. Analysis of the structural model for the V-L domain of the Bence-Jones protein REI suggests that these positions play important roles in maintaining domain structure and stability. Using an Escherichia coli expression system, we prepared single-point mutants of REI V-L incorporating amyloid-associated amino acid replacements that are both rare and located at structurally important positions. These mutants support ordered aggregate formation in an in vitro L-chain fibril formation model in which wild-type REI V-L remains soluble. Moreover, the ability of these sequences to aggregate in vitro correlates well with the extent to which domain stability is decreased in denaturant-induced unfolding. The results are consistent with a mechanism for the disease process in which the V-L domain, either before or after proteolytic cleavage from the L-chain constant region domain, unfolds by virtue of one or more destabilizing amino acid replacements to generate an aggregation-prone nonnative state.