Influence of the germline sequence on the thermodynamic stability and fibrillogenicity of human lambda 6 light chains

Abstract

Light chain-associated amyloidosis is a fatal disease characterized by the aggregation and pathologic deposition of monoclonal light chain-related fragments as amyloid fibrils in organs or tissues throughout the body. Notably, it has been observed that proteins encoded by the lambda variable light chain (V(L)) gene segment 6a are invariably associated with amyloid deposition; however, the contribution of the gene to this phenomenon has not been established. In this regard, we have determined the thermodynamic stability and kinetics of in vitro fibrillogenesis of a recombinant (r) V(L) protein, designated 6aJL2, which contains the predicted sequences encoded by the 6a and JL2 germline genes. Additionally, we studied a 6a mutant (6aJL2-Arg25Gly), that is present in approximately 25% of all amyloid-associated lambda6 light chains. Remarkably, the wild-type 6aJL2 protein was more stable than were all known amyloidogenic kappa and lambda light chains for which stability parameters are available; more importantly, it was even more so (and less fibrillogenic) than the only clinically proven nonamyloidogenic lambda6 protein, Jto. Conversely, the mutated 6aJL2-R25G molecule was considerably less stable and more fibrillogenic than was the native 6aJL2. Our data indicate that the propensity of lambda6 light chains to form amyloid can not be attributed to thermodynamic instability of the germline-encoded Vlambda6 domain, but rather, is dependent on sequence alterations that render such proteins amyloidogenic.

Site-directed Mutagenesis Reveals Regions Implicated in the Stability and Fiber Formation of Human λ3r Light Chains

“…The unfolding of 6aJL2 was evaluated to assess the thermodynamic stability difference between the 3 and 6 families. The results for 6aJL2 were similar to those previously reported (22). Far UV CD data showed that 6aJL2 germ line is completely reversible (Fig.…”

“…X-ray crystallography revealed that the three-dimensional topology of the single and double 3r mutants was not significantly different. The majority of 3 variants evaluated in this study showed higher stability compared with other germ lines associated with AL, such as 6a and IO18/O8 (22,27,28). This mutagenic approach helped to confirm or identify other key regions implicated in 3 AL.…”

“…The unfolding kinetics of 3rJL2/Y and 3mJL2 were very similar, as deduced from the comparison of the midpoints of chemical or thermal unfolding (C m and T m , respectively; Table 2) and the values for the cooperativity of the unfolding process (m and ⌬H VH , respectively; Table 2). The ⌬G values showed that 3rJL2/Y and 3mJL2 were more stable than 6aJL2 and IO18/08, the only amyloid germ lines previously evaluated (22,27,28). We found that 3rJL2/Y had refolding transitions that were not superimposable with the unfolding transitions (Fig.…”

“…The periplasmic proteins were extracted as described previously (22). The periplasmic extracts were precipitated twice with 30 -90% saturated ammonium sulfate and stored at 4°C.…”

“…Despite the predominance of the isotype in the disease, only one germ line has been characterized for its propensity to form amyloids. Our group previously reported the characterization of the recombinant germ line 6aJL2, a protein encoded by the 6a and the jl2 gene segments (22). 6aJL2 is thermodynamically more stable than other 6 light chains derived from patients with multiple myeloma, although it is capable of forming fibers in vitro after long periods of incubation (22).…”