No abstract

The most common form of systemic amyloidosis originates from antibody light chains. The large number of amino acid variations that distinguish amyloidogenic from nonamyloidogenic light chain proteins has impeded our understanding of the structural basis of light-chain fibril formation. Moreover, even among the subset of human light chains that are amyloidogenic, many primary structure differences are found. We compared the thermodynamic stabilities of two recombinant k4 light-chain variable domains~V L s! derived from amyloidogenic light chains with a V L from a benign light chain. The amyloidogenic V L s were significantly less stable than the benign V L . Furthermore, only the amyloidogenic V L s formed fibrils under native conditions in an in vitro fibril formation assay. We used site-directed mutagenesis to examine the consequences of individual amino acid substitutions found in the amyloidogenic V L s on stability and fibril formation capability. Both stabilizing and destabilizing mutations were found; however, only destabilizing mutations induced fibril formation in vitro. We found that fibril formation by the benign V L could be induced by low concentrations of a denaturant. This indicates that there are no structural or sequence-specific features of the benign V L that are incompatible with fibril formation, other than its greater stability. These studies demonstrate that the V L b-domain structure is vulnerable to destabilizing mutations at a number of sites, including complementarity determining regions~CDRs!, and that loss of variable domain stability is a major driving force in fibril formation.

In primary (light chain-associated) amyloidosis, immunoglobulin light chains deposit as amyloid fibrils in vital organs, especially the kidney. Because the kidney contains high concentrations of urea that can destabilize light chains as well as solutes such as betaine and sorbitol that serve as protein stabilizers, we investigated the effects of these solutes on in vitro amyloid fibril formation and thermodynamic stability of light chains. Two recombinant light chain proteins, one amyloidogenic and the other nonamyloidogenic, were used as models. For both light chains, urea enhanced fibril formation by reducing the nucleation lag time and diminished protein thermodynamic stability. Conversely, betaine or sorbitol increased thermodynamic stability of the proteins and partially inhibited fibril formation. These solutes also counteracted urea-induced reduction in protein thermodynamic stability and accelerated fibril formation. Betaine was more effective than sorbitol. A model is presented to explain how the thermodynamic effects of the solutes on protein state equilibria can alter nucleation lag time and, hence, fibril formation kinetics. Our results provide evidence that renal solutes control thermodynamic and kinetic stability of light chains and thus may modulate amyloid fibril formation in the kidney.

The primary structural features that render human monoclonal light chains amyloidogenic are presently unknown. To gain further insight into the physical and biochemical factors that result in the pathologic deposition of these proteins as amyloid fibrils, we have selected for detailed study three closely homologous protein products of the light-chain variable-region single-gene family VKIV. Two of these proteins, REC and SMA, formed amyloid fibrils in vivo. The third protein, LEN, was excreted by the patient at levels of 50 g/day with no indication of amyloid deposits. Sequences of amyloidogenic proteins REC and SMA differed from the sequence of the nonpathogenic protein LEN at 14 and 8 amino acid positions, respectively, and these amino acid differences have been analyzed in terms of the three-dimensional structure of the LEN dimer. To provide a replenishable source of these human proteins, we constructed synthetic genes coding for the REC, SMA, and LEN variable domains and expressed these genes in Escherichia coli. Immunochemical and biophysical comparisons demonstrated that the recombinant VKIV products have tertiary structural features comparable to those of the patient-derived proteins. This welldefined set of three clinically characterized human KIV light chains, together with the capability to produce these KIV proteins recombinantly, provide a system for biophysical and structural comparisons of two different amyloidogenic light-chain proteins and a nonamyloidogenic protein of the same subgroup. This work lays the foundation for future investigations of the structural basis of light-chain amyloidogenicity.Keywords: amyloid proteins; immunoglobulin light chains; kappa IV domains; recombinant human VL; synthetic genes; variable-domain dimerization Over the past 30 years, detailed analyses of the structure and biomutagenesis to investigate effects of the changes on biological physical properties of immunoglobulin molecules have probed activities; synthetic Ig genes have been generated for the promany aspects of Ig interactions and effector functions (Padlan, duction of unique antibody reagents for medical and diagnos-1994). More recently, Ig genes have been cloned and altered by tic purposes (Borrebaeck, 1992 its include the assembly of a primarily p-structure protein intoThe first two authors contributed equally to this project.nonbranching, insoluble fibrils of diameter 7-10 nm and a char-IPTG, isopropyl P-D-thiogalactopyranoside; TES, Tris-EDTA-sucrose lead to death' Defining features Of amy1oid depos-42 1