Counteracting Effects of Renal Solutes on Amyloid Fibril Formation by Immunoglobulin Light Chains

Abstract

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.

Kinetics and Energetics of Assembly, Nucleation, and Growth of Aggregates and Fibrils for an Amyloidogenic Protein

“…Materials-Recombinant LC variable domain (V L ) SMA was expressed in Escherichia coli and purified (Ͼ99% by SDS-PAGE) as described previously (13). Recombinant SMA has the same sequence as the V L that originated from lymph node-derived amyloid fibrils of a patient with light chain-associated amyloidosis (14).…”

“…To prepare seeds from amyloid fibrils generated at atmospheric pressure, 1 mg/ml SMA in buffer A was agitated at 37°C for 96 h (13) and sonicated as described above. To study effects of seeding on aggregation, samples containing 1 mg/ml SMA and 0.1 mg/ml seeds were incubated either at 200 MPa and 32°C with 10 mM DTT or at atmospheric pressure and 37°C with agitation in buffer A alone (13).…”

Solute effects on the irreversible aggregation of serum albumin

“…in somewhat more concentrated solutions [10,12,15]. One plausible interpretation of these dual effects is that the solute modulates the two steps in eq.…”

“…[7,8]), is the two-step molecular picture originally introduced by Lumry and Eyring [9]. [7,[10][11][12][13][14][15]. However, this strict coupling between equilibrium and kinetic stability is not universal and may be valid only at conditions where K is below or close to unity (i.e.…”

Current and New Perspectives on the Molecular and Cellular Mechanisms of Amyloid Formation and Toxicity in Light Chain Amyloidosis

“…Urea, a known protein denaturant, decreased the thermodynamic stability and the fibril formation kinetics of both SMA and LEN while betaine and sorbitol (organic osmolytes) had the opposite effect. A concentration of 1.5 M urea was enough to increase fibril formation of both SMA and LEN (Kim et al, 2001). Conversely, the presence of 0.5 M betaine or sorbitol partially inhibited SMA fibril formation showing the interplay between stabilizing and denaturing forces that may occur in physiological environments.…”