Structural studies on FAP fibrils: removal of contaminants is essential for the interpretation of X-ray data

Abstract

Amyloid fibrils were isolated from heterozygous Met 30 familial amyloidotic polyneuropathy (FAP) patients. A chelating agent, collagenase digestion and defatting agents were used to remove the serum amyloid P-component, collagen and other adherent tissue components from the fibrils. The characteristic birefringence of amyloid fibrils remained after purification. X-ray diffraction experiments were performed before and after the removal of adherent material. The predominant features from the X-ray diffraction pattern, consisting of a series of concentric rings, indicate the presence of a lipid structure in all the samples that had not been previously delipidated. In contrast, defatted fibrils exhibited only two reflections in the 4-30 Å region: a sharp reflection corresponding to a 4.76 Å spacing and a broad and diffuse one centered at 10.5 Å spacing, both characteristic of a β-pleated sheet structure. The low angle pattern, from the purified material, shows one unique reflection at 63 Å spacing.

Analysis of the supernatant after the removal of the adherent material, using thin-layer chromatography, confirmed the presence of different types of lipids, namelysphingolipids, phospholipids, acylglycerides, fatty acids, cholesterol and cholesterol esters.

Since the purified fibrils exhibit the green birefringence when stained with Congo red and at the same time maintain the characteristic β-structure, as revealed by the X-ray pattern, we conclude that the removal of the lipids does not interfere with the integrity of the fibrils and constitutes a prerequisite for a careful analysis of X-ray diffraction patterns of FAP (transthyretin) fibrils.

Key Words:

• TRANSTHYRETIN (TTR)
• AMYLOID
• FAMILIAL AMYLOID POLYNEUROPATHY (FAP)
• X-RAY DIFFRACTION