Structure and Fibrillogenicity of 11-1F4 Epitope Peptide

Conformation-dependent antibodies serve as important tools for understanding the mechanism of aggregation on the pathway of amyloid fibril formation. Furthermore these antibodies may be used for diagnostic and therapeutic purposes because of their fibril-reactive nature. One of the important drawbacks of these antibodies is their potential for cross-reactivity with the soluble precursor proteins in addition to the amyloid fibrils.

11-1F4 is a murine monoclonal antibody generated against the amyloid fibrils of the immunoglobulin light chain Len. This antibody has been shown to recognize amyloid fibrils composed of other light chains regardless of their subgroup (λ or κ). In addition, this reagent binds amyloid fibrils composed of non-light chain precursor proteins. The 11-1F4 antibody is also selectively reactive with the fibrillar form of the precursors and does not recognize the soluble protein. It therefore binds a common conformational epitope within amyloid fibrils. More specifically, the epitope recognized by 11-1F4 on the Len peptide was recently localized to the first 22 N-terminal amino acids.

In the studies presented here, Eu⁺³-linked Immunoabsorbant Assays (EuLISA), in vitro fibril formation assays, and limited proteolysis experiments have been used to provide further insights into the conformation of the Len 1-22 peptide and its role in light chain fibrillogenesis. In vitro fibril formation assays demonstrated the fibrillogenic nature of this region. Single amino acid substitutions of Len 1-22, designated P8A and P8S, were also used in these studies and the results were compared with the native Len 1-22 molecule. The proline residue at the 8^th position, previously hypothesized to be critical in the conformation of Len 1-22, was shown to affect the binding of the 11-1F4 antibody and the kinetics of fibril formation of the Len 1-22 peptide. In addition, different fragmentation patterns were obtained when Len 1-22 and P8A Len 1-22, both in the soluble and fibrillar forms, were subjected to limited proteolysis experiments. This further supports the idea that a single site mutation at this position determines the conformation of this peptide, which in turn alters its serological reactivity and amyloidogenic propensity.

Understanding the conformation of the epitope on amyloid fibrils recognized by this antibody may be important in designing drugs in future which may diminish amyloid fibril formation or disaggregate already formed fibrils.