Apolipoprotein A-IMilano. Detection of normal A-I in affected subjects and evidence for a cysteine for arginine substitution in the variant A-I

The A-I(Milano) contains a variant form of apolipoprotein A-I, which unlike normal A-I, has cysteine. We have characterized the A-I(Milano) isoforms in two affected subjects, D.M. and D.G. In these subjects, the two most prominent A-I(Milano) isoforms were displaced from the corresponding normal A-I isoforms by a single charge unit toward the anode, as determined by isoelectric focusing. The amino acid compositions of the purified isoforms, which were separated by either preparative isoelectric focusing or thiopropyl-Sepharose 6B chromatography, indicated that the A-I(Milano) from these subjects contained both a normal A-I (A-I(N)) and a cysteine-containing variant A-I (A-I(Cys)). Furthermore, amino acid analyses suggested that the A-I(Cys) differed from normal A-I by a single cysteine for arginine substitution, which was sufficient to account for the charge difference between the two proteins. Partial sequence analysis revealed that an arginine in normal A-I was replaced by cysteine in the variant A-I at residue 173. Consistent with the amino acid analyses, the cysteine-containing isoforms shifted one charge unit toward the cathode after modification of the cysteine residue with cysteamine. Quantitation of the relative amounts of the A-I(N) and A-I(Cys) in D.M. and D.G. by thiopropyl-Sepharose 6B chromatography revealed that the relative levels were different in each subject. The percentage of the total A-I represented by the A-I(N) in D.M. and D.G. was 16.1 and 25.7%, respectively. The demonstration of variable amounts of normal A-I in A-I(Milano) subjects raises some interesting questions regarding the genetics, regulation, and metabolism of apolipoprotein A-I