Immunoglobulin amyloidosis (AL) which is characterized by extracellular deposition of fibril complexes composed of monoclonal immunoglobulin light chains (LC) is the most common form of systemic amyloidosis and is generally associated with the worst prognosis. Little is known about the pathogenic mechanisms involved in fibril formation, but two major factors may be involved. One is the synthesis of abnormal protein structure and a second involves defective protein degradation. This proposal seeks to test the hypothesis that the amino acid sequence of Ig LC variable regions plays a determining role in the pathogenesis of AL amyloid. This hypothesis is based on preliminary data of amino acid sequences of amyloid proteins which show unique variations. To test this hypothesis it will be necessary to isolate and determine the primary structure of a number of amyloid proteins and evaluate the effect of these substitutions on secondary and tertiary structure. Amyloid fibrils or their precursors will be isolated by standard methods and subjected to complete amino acid sequence analysis. An important part of these studies will be the selection of appropriate LC proteins for structural analyses. LC proteins, which have already been assigned to subgroups by sequence analysis, will be used to make monoclonal antibodies that are specific for the subgroups that are of interest (e.g. kappa I, lambda I and VI). All LC (amyloid and myeloma) proteins can then be typed so that only those belonging to the desired subgroups will be subjected to complete sequence analysis. In this way immunologic techniques will be used to restrict the chemical studies to a level that can be more easily accomplished. In addition, attempts will be made to identify monoclonal antibodies which recognize amyloid LC but not myeloma LC. The effects of amino acid substitutions on the structure of amyloid LC will be investigated in several ways. 1) Secondary structure analysis using tested prediction models will be used to search for alterations in beta sheet and turn forming potential. 2) Association constants for LC dimers will be determined to identify any increase or decrease in intradimer binding. 3) Computer graphics will be used to identify new intra and intermolecular interactions generated by the amino acid substitutions found in the amyloid subunit proteins. Using these methods we hope to define the mechanisms of amyloid fibril formation.
