Structural and functional domains of apolipoprotein A-I within high density lipoproteins.
We prepared discoidal and spherical model high density lipoprotein (HDL) with apolipoprotein A-I and 1-palmitoyl-2-oleoyl phosphatidylcholine at various lipid:protein ratios and compared their reactivity with exo- and endopeptidases to that of human HDL2 and HDL3. Limited proteolysis with trypsin, Staphylococcus V8 protease, and elastase yielded a major stable peptide of 11,000-11,500 daltons under conditions which completely degraded lipid-free A-I. By Western blotting this protease-resistant fragment was shown to consist of the amino-terminal 90-100 residues of the A-I protein; the residues on the carboxyl side of this peptide are therefore protease-sensitive and appear to correlate with the putative "hinge" region, which is especially reactive with antibodies. The amino terminus was very resistant to digestion with a variety 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 lipid, were used to examine binding interactions with rat hepatic plasma membranes, it was found that removal of up to 20-24 residues from the carboxyl terminus had no significant effect on binding, whereas removal of 70 residues completely eliminated specific binding to the membranes. Taken together, our data indicate that there is a protease-resistant domain constituted by the first 90 residues of A-I, which, in HDL, contain little of the class of amphipathic helix characteristic of the rest of the molecule and most likely form a structure dominated by protein-protein interactions. At the carboxyl end of the protein, there is a functional domain constituted by residues 149-219 that possesses the capacity to bind to proteins on hepatic membranes.
Dalton
M B
MB
Department of Biological Chemistry, Hahnemann University, Philadelphia, Pennsylvania 19102.
Swaney
J B
JB
D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D010450:Endopeptidases; D006801:Humans; D015151:Immunoblotting; D008075:Lipoproteins, HDL / Q000097:blood* / Q000302:isolation & purification; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010713:Phosphatidylcholines / Q000378:metabolism; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D008075:Lipoproteins, HDL; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010450:Endopeptidases; C028694:1-palmitoyl-2-oleoylphosphatidylcholine