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
L
Center E. Grossi Paoletti, Department of Pharmacological Sciences, University of Milano, 20133 Milano, Italy.
Tedeschi
G
G
Treu
C
C
Ronchi
S
S
Galbiati
D
D
Airoldi
S
S
Sirtori
C R
CR
Marcel
Y
Y
Franceschini
G
G
D000911:Antibodies, Monoclonal / Q000378:metabolism; D016632:Apolipoprotein A-I / Q000097:blood / Q000737:chemistry*; D015153:Blotting, Western; D019281:Dimerization; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D000939:Epitopes; D006801:Humans; D015151:Immunoblotting; D008075:Lipoproteins, HDL / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors
D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D004220:Disulfides; D000939:Epitopes; D008075:Lipoproteins, HDL; D011994:Recombinant Proteins