Aprotinin binding to amyloid fibrils
2000
Cardoso, Isabel | Pereira, Pedro J Barbosa | Damas, Ana M. | Saraiva, Maria J. M.
Different low molecular mass ligands have been used to identify amyloid deposits. Among these markers, the dyes Thioflavin T and Congo Red interact specifically with the β‐sheet structure arranged in a cross‐β conformation, which is characteristic of amyloid. However, the molecular details of this interaction remain unknown. When labelled with technetium‐99m, the proteinase inhibitor aprotinin has been shown to represent a very important radiopharmaceutical agent for in vivo imaging of extra‐abdominal deposition of amyloid in amyloidosis of the immunoglobulin type. However, no information is available as to whether aprotinin binds other types of amyloid fibrils and on the nature and characteristics of the interaction. The present work shows aprotinin binding to insulin, transthyretin, β‐amyloid peptide and immunoglobulin synthetic amyloid fibrils by a specific dot‐blot ligand‐binding assay. Aprotinin did not bind amorphous precipitates and/or the soluble fibril precursors. A Ka of 2.9 µm−1 for the binding of aprotinin to insulin amyloid fibrils was determined by Scatchard analysis. In competition experiments, analogues such as an aprotinin variant, a spermadhesin and the soybean trypsin inhibitor were tested and results suggest that both aprotinin and the spermadhesin interact with amyloid fibrils through pairing of β‐sheets of the ligands with exposed structures of the same type at the surface of amyloid deposits. An electrostatic component may also be involved in the binding of aprotinin to amyloid fibrils because important differences in binding constants are observed when substitutions V15L17E52 are introduced in aprotinin; on the other hand β‐sheet containing acidic proteins, such as the soybean trypsin inhibitor, are unable to bind amyloid fibrils.
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