STRUCTURAL STUDIES ON FAP FIBRILS - REMOVAL OF CONTAMINANTS IS ESSENTIAL FOR THE INTERPRETATION OF X-RAY DATA

Amyloid fibrils were isolated from heterozygous Met 30 familial amyloi dotic polyneuropathy (FAP) patients. A chelating agent, collagenase di gestion and defatting agents were used to remove the serum amyloid P-c omponent, collagen and other adherent tissue components from the fibri ls. 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 r ings, indicate the presence of a lipid structure in all the samples th at had not been previously delipidated. In contrast, defatted fibrils exhibited only two reflections in the 4-30 Angstrom region: a sharp re flection corresponding to a 4.76 Angstrom spacing and a broad and diff use one centered at 10.5 Angstrom spacing, both characteristic of a be ta-pleated sheet structure. The low angle pattern, from the purified m aterial, shows one unique reflection at 63 Angstrom spacing. Analysis of the supernatant after the removal of the adherent material using th in-layer chromatography, confirmed the presence of different types of lipids, namely sphingolipids, phospholipids, acylglycerides, fatty aci ds, cholesterol and cholesterol esters. Since the purified fibrils exh ibit the green birefringence when stained with Congo red and at the sa me time maintain the characteristic beta-structure, as revealed by the X-ray pattern, we conclude that the removal of the lipids does not in terfere with the integrity of the fibrils and constitutes a prerequisi te for a careful analysis of X-ray diffraction patterns of FAP (transt hyretin) fibrils.