The specific structural features of Ig LC primary sequence which lead to deposition as the pathological amyloid fibril are as yet undefined. The goal of this study is to investigate the biophysical basis of amyloid formation using the Immunoglobulin (1g) light chain (LC) proteins as the model system. A combined approach of molecular biology, biophysical and ultrastructural. studies, coupled with the significant resource of amyloid clinical data and purified proteins available in the Arthritis Center of BUSM, the vast literature on LC primary sequences, andrelationship of defined mutations in these sequences to amyloid disease provides a unique opportunity - to design, produce and investigate systematically - the structural and functional properties of specifically altered proteins - to define the precise molecular mechanism of LC amyloidosis (AL). These studies test the hypothesis that AL amyloid fibril deposition occurs as a result of amino acid substitutions in critical regions of the variable domain of the LC, destabilizing them, and resulting in their insolubility. The goals are: (1) To produce human 19 light chains as recombinant proteins in E. coli from clones that were genetically engineered based on the primary sequences of clinically derived amyloidogenic and non-amyloidogenic LO proteins. These recombinant LCs are being studied using biophysical methods and compared to clinically derived amyloidogenic and non-amyloidogenic LCs from the same sub-class. (2) To investigate the biophysical properties of recombinant and clinically derived LCs of the kappal sub-class for their stability and amyloid-forming tendencies under controlled conditions of temperature, pH, ionic strength, salt type, protein concentration, and interaction with components known to be associated with fibril formation, such as P-component, glycosaminoglycans (GAG's), and apoprotein E (apoF). These biophysical studies are supplemented with chromatographic and spectroscopic studies to observe theconversion of monomer to aggregate. (3) New proteins will be synthesized to incorporate mutations that are specifically engineered to examine the effect of modifying critical amino acid(s), domain(s), or domain type(s) on the biophysical properties of LC and their ability to form amyloid fibrils. This information may allow correlation of insolubility with disease severity, allow clinicians to determine prognosis and make treatment recommendations based on additional scientific data and design therapies to retard specific steps in the pathway or prevent amyloid deposition completely.
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