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.
Highlights
Primary systemic antibody light chain (AL)1 amyloidosis is a plasma cell disorder in which immunoglobulin light chains deposit pathologically as amyloid fibrils in the body, leading to progressive organ failure and eventual death [1,2,3]
Fibril Formation in PBS—Fig. 1 shows time courses for soluble native protein concentrations and Thioflavin T (ThT) fluorescence, which is indicative of fibril formation [6, 7, 36]
There was a concomitant increase in ThT fluorescence, indicative of fibril formation (Fig. 1B)
Summary
Vol 276, No 2, Issue of January 12, pp. 1626 –1633, 2001 Printed in U.S.A. Counteracting Effects of Renal Solutes on Amyloid Fibril Formation by Immunoglobulin Light Chains*. 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. Osmolytes (e.g. glycerol and sucrose), in the absence of urea, have been found to inhibit amyloid fibril formation from scrapie prion protein [34] and immunoglobulin light chains [8] by stabilizing the native states and reducing the levels of the aggregation-competent species. To examine the possible roles of renal solutes in AL amyloidosis and to gain further insight into the relationships between the thermodynamic stability of light chains and their propensity to form amyloid fibrils, we modulated protein stability by varying concentrations of renal solutes, i.e. urea, betaine, and sorbitol. It has previously been established that recombinant SMA forms fibrils in vitro that are indistinguishable from in vivo light chain fibrils, and recombinant LEN does not form fibrils in vitro in buffer alone [6, 10]
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