PURIFICATION AND CHARACTERIZATION OF AMYLOID-RELATED TRANSTHYRETIN ASSOCIATED WITH FAMILIAL AMYLOIDOTIC CARDIOMYOPATHY

Analysis of amyloid fibril material associated with familial amyloidotic cardiomyopathy revealed that it contains a mixture of transthyretin-related polypeptides. The major protein band in SDS/polyacrylamide gel corresponding to a molecular mass of 14.5 kDa, consists of transthyretin fragments starting at positions 46, 49 and 59, the latter not previously identified, and one blocked fragment derived from the N-terminal part of transthyretin. in reverse-phase HPLC, the major fragment recovered was that starting at Thr49, indicating a trypsin-like cleavage (Lys at position 48). Two minor bands, corresponding to 17 kDa and 35 kDa, contained proteins with blocked N-termini, and migrated as monomeric and dimeric transthyretin, respectively. A 13-kDa protein band was found to contain transthyretin with a ragged N-terminus, mainly starting at positions 2 and 5. Three more bands, corresponding to 10, 25 and 29 kDa, consist of transthyretin molecules with blocked N-termini and most likely of aggregates of truncated molecules. A point mutation of amyloid transthyretin was identified at position 111 (Met instead of Leu in normal serum transthyretin) which confirms the mutation found for Danish siblings with familial amyloidotic cardiomyopathy. However, the presence of a non-variant amyloid transthyretin was also observed, indicating that the Danish kindred is heterozygous with respect to this point mutation. Isoelectric focusing of the amyloid fibril material resolved multiple protein bands ranging over pH 4.5-6.5, confirming heterogeneities. Methanol extraction of the cardiac amyloid fibril material prior to the purification steps reveals a methanol-soluble substance amounting to about 10% (by mass dry material) of the amyloid fibril material. A yellow substance in this fraction shows absorbance maxima (270, 280 and 430 nm) similar to those observed for transthyretin in normal serum. Gas chromatography/mass spectrometry of the methanol extract revealed the presence of saturated fatty acids (C14:0, C16:0 and C18:0 in the corresponding ratio 2:8:5) and polyunsaturated fatty acids (C16:1, C18:1, C18:2 and C20:4 in the corresponding ratio of 1:2:1:1) as further constituents of the amyloid fibril material.