Limited proteolysis of a disulfide-linked apoA-I dimer in reconstituted HDL

The apolipoprotein A-I-Milano (apoA-I-M) is a molecular variant of apoA-I characterized by the Arg(173)--> Cys substitution, leading to the formation of homodimers A-I-M/A-I-M. Upon interaction with palmitoyloleoylphosphatidylcholine, A-I-M/A-I-M forms only two species of reconstituted HDL (rHDL) particles, with diameters of 7.8 and 12.5 nm, We used limited proteolysis to analyze the conformation of A-I-M/A-I-M in the two rHDL particles, in comparison with that of apoA-I in rHDL of similar size, ApoA-I in the small, 7.8-nm rHDL is degraded to a greater extent (50% after 6 h) than in the large rHDL (< 10% degraded after 6 h). The protease susceptibility of A-I-M/A-I-M in small and large rHDL is instead remarkably the same, with A-I-M/A-I-M being much more sensitive to proteolytic digestion (50% degraded after 10 min) than apoA-I, The identification of the proteolytic fragments by immunoblotting, N-terminal sequencing, and molecular mass determination, shows that the N-terminus of both proteins is resistant to proteolysis, with six cleavage sites located in the central and carboxy-terminal portions of the molecules. Cleavage in the middle of apoA-I occurs at distinct sites in 7.8-nm (Lys(118)) and 12.7-nm (Arg(123)) rHDL, indicating a different conformation in small and large rHDL particles. The A-I-M/A-I-M instead adopts a unique and identical conformation in small and large rHDL, with the carboxy-terminal portion of the molecule being remarkably more accessible to the proteases than in apoA-I, This suggests the presence of a novel carboxy-terminal domain in A-I-M/A-I-M, not organized in a compact structure and not shared by wild-type apoA-I, which may account for the unique functional properties of A-I-M/A-I-M. - Calabresi, L., G. Tedeschi, C. Treu, S. Ronchi, D. Galbiati, S. Airoldi, C. R. Sirtori, E Marcel, and G. Franceschini. Limited proteolysis of a disulfide-linked apoA-I dimer in reconstituted HDL.