The apolipoprotein A-IMilano (apoA-IM) is a molecular variant of apoA-I characterized by the Arg173→Cys substitution, leading to the formation of homodimers A-IM/A-IM. Upon interaction with palmitoyloleoylphosphatidylcholine, A-IM/A-IM 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-IM/A-IM 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-IM/A-IM in small and large rHDL is instead remarkably the same, with A-IM/A-IM 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 (Lys118) and 12.7-nm (Arg123) rHDL, indicating a different conformation in small and large rHDL particles. The A-IM/A-IM instead adopts a unique and identical conformation in small and large rHDL, with the carboxyterminal 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-IM/A-IM, not organized in a compact structure and not shared by wild-type apoA-I, which may account for the unique functional properties of A-IM/A-IM.
|Titolo:||Limited proteolysis of a disulfide-linked apoA-I dimer in reconstituted HDL|
|Autori interni:||RONCHI, SEVERINO|
|Parole Chiave:||Apolipoprotein A-IMilano; Limited proteolysis; Protein conformation|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
Settore BIO/14 - Farmacologia
|Data di pubblicazione:||2001|
|Appare nelle tipologie:||01 - Articolo su periodico|