STRUCTURAL AND FUNCTIONAL DOMAINS OF APOLIPOPROTEIN-A-I WITHIN HIGH-DENSITY-LIPOPROTEINS

We prepared discoidal and spherical model high density lipoprotein (HD L) with apolipoprotein A-I and 1-palmitoyl-2-oleoyl phosphatidylcholin e at various lipid:protein ratios and compared their reactivity with e xo- and endopeptidases to that of human HDL2 and HDL3. Limited proteol ysis with trypsin, Staphylococcus V8 protease, and elastase yielded a major stable peptide of 11,000-11,500 daltons under conditions which c ompletely degraded lipid-free A-I. By Western blotting this protease-r esistant fragment was shown to consist of the amino-terminal 90-100 re sidues of the A-I protein; the residues on the carboxyl side of this p eptide are therefore protease-sensitive and appear to correlate with t he putative ''hinge'' region, which is especially reactive with antibo dies. The amino terminus was very resistant to digestion with a variet y of aminopeptidases, whereas carboxypeptidases could remove up to 70 residues from the lipid-free A-I protein or 12-24 residues from A-I in various HDL. When these truncated forms of A-I, in combination with l ipid, were used to examine binding interactions with rat hepatic plasm a membranes, it was found that removal of up to 20-24 residues from th e carboxyl terminus had no significant effect on binding, whereas remo val of 70 residues completely eliminated specific binding to the membr anes. Taken together, our data indicate that there is a protease-resis tant domain constituted by the first 90 residues of A-I, which, in HDL , contain little of the class of amphipathic helix characteristic of t he rest of the molecule and most likely form a structure dominated by protein-protein interactions. At the carboxyl end of the protein, ther e is a functional domain constituted by residues 149-219 that possesse s the capacity to bind to proteins on hepatic membranes.