No abstract

The deposition of certain Bence Jones proteins as tubular casts, basement membrane precipitates, or amyloid fibrils results in the human light-chain-associated renal and systemic dis -myeloma (cast) nephropathy, light-chain deposition disease, and immunocyte-derived (primary or AL) amyloidosis. To determine if light-chain nephrotoxicity or amyloidogenicity is related to the propensity of these components to form high molecular weight aggregates under physiological conditions, we used a size-exclusion chromatographic system to study 40 different Bence Jones proteins. Each sample was tested over a wide range of protein concentration in three different buffers varying in pH, osmolality, and the presence or absence oflow concentrations ofurea. Thirty-three of the 35 proteins found clinically and/or experimentally to form in vivo pathologic light-chain deposits were shown to undergo high-order self-association and form hh molecular weight aggregates. In contrast, of five nonpathologic proteins, one showed polymerization under the chromatographic conditions used. The correlation between the in vitro results achieved by size-exclusion chromatography and that found in vivo provides (i) a rapid dlagnostic method to identify potential nephrotoxic or amyloidogenic Bence Jones proteins and (ii) an experimental means to gain new insight into the physicochemical basis of light-chain aggregtion and the treatment of those invariably fatal disorders associated with pathologic lightchain deposition.The human light-chain-related renal and systemic diseasesmyeloma (cast) nephropathy, light-chain deposition disease, and immunocyte-derived (primary or AL) amyloidosisresult from the pathologic deposition of monoclonal light chains (i.e., Bence Jones proteins) in the form of casts, basement membrane precipitates, or fibrils, respectively (1). These light-chain deposits ultimately result in the impairment ofrenal and other organ function and account for much ofthe morbidity and mortality found in patients with these disorders. The fact that pathologic light-chain deposits are not an invariant accompaniment of clinical or experimental (1) Bence Jones proteinuria and are not necessarily directly related to the amount of monoclonal light chain synthesized or excreted implies that certain light chains are inherently nephrotoxic or amyloidogenic.Several in vitro and in vivo models have been devised that provide an experimental means to assess the pathologic potential of Bence Jones proteins (2-5). For example, in one model we demonstrated that the injection into mice ofcertain Bence Jones proteins resulted in the deposition in the mouse kidney of the human proteins in the form of tubular casts, basement membrane precipitates, crystals, or amyloid fibrils (6). Through studies involving >40 different Bence Jones proteins, we found that the renal lesions induced experimentally were comparable to those of patients from whom the proteins were derived and that the experimental mouse model was capable of differentiating "nephrotoxic" from "...

Amyloidosis is a pathological process in which normally soluble proteins polymerize to form insoluble fibrils (amyloid). Amyloid formation is found in a number of diseases, including Alzheimer's disease, adult-onset diabetes, and light-chain-associated amyloidosis. No pharmaceutical methods currently exist to prevent this process or to remove the fibrils from tissue. The search for treatment and prevention methods is hampered by a limited understanding of the biophysical basis of amyloid formation. Glycosaminoglycans (GAGs) are long, unbranched heteropolysaccharides composed of repeating disaccharide subunits and are known to associate with amyloid fibrils. The interaction of amyloid-associated free light chains with GAGs was tested by both size-exclusion high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments. The results indicated that heparin 16 000 and chondroitin sulfate B and C precipitated both human intact light chains and recombinant light chain variable domains. Although all light chains interacted with heparin, the strongest interactions were obtained with proteins that had formed amyloid. Molecular modeling indicated the possibility of interaction between heparin and the conserved saddlelike surface of the light chain dimer opposite the complementarity-determining segments that form part of the antigen-binding site of a functional antibody. This suggestion might offer a new path to block the aggregation of amyloid-associated light chain proteins, by design of antagonists based on properties of GAG binding. A hexasaccharide was modeled as the basis for a possible antagonist.

Small-zone gel filtration chromatography, combined with analytical-scale columns and fast run times, provides a useful system for the study of protein-protein interactions. A computer simulation (SCIMMS, or Simulated Chromatography of Interactive MacroMolecular Systems) that replicates the small-zone behavior of interacting proteins has been developed. The simulation involves an iterative sequence of transport, equilibration, and diffusion steps. This chapter illustrates the use of the simulation to study the homodimerization of rapidly equilibrating immunoglobulin light chain proteins and for determination of association constants. The simulation can also be used to study heterogeneous interactions, kinetically controlled interactions, and higher-order oligomerization, and it can replicate large-zone and Hummel-Dreyer conditions.