Monoclonal antibodies to human apolipoprotein AI: probing the putative receptor binding domain of apolipoprotein AI.
We have used four monoclonal antibodies (MAbs) specific for human apolipoprotein (apo) AI, designated AI-1, AI-3, AI-4.1 and AI-4.2, to study the interaction between high-density lipoprotein HDL3 and rat liver plasma membranes. MAbs AI-1 and AI-3 recognize epitopes within residues 28-47 and 140-147 respectively of apoA-I [Allan, Tetaz and Fidge (1991) J. Lipid Res. 32, 595-601]. Two previously unreported MAbs, AI-4.1 and AI-4.2, were raised against purified CNBr fragment 4 (CF4) of apoAI, the C-terminal region. Using e.l.i.s.a. and immunoblotting techniques, we have demonstrated that all four MAbs recognize distinct epitopes within apoAI. Epitope mapping studies using endoproteinase cleavage peptides of CF4 showed that AI-4.1 binds to an epitope within residues 223-233, which is poorly exposed on apoAI molecules associated with lipid. Fab fragments derived from MAb AI-4.2 inhibited the binding of 125I-labelled HDL3 to rat liver plasma membranes, whereas Fab fragments from AI-4.1, AI-3 and AI-1 had little or no effect. In ligand blotting studies with purified CNBr fragments of apoAI and using apoAI-specific antibodies for detection, CF4 showed the highest capacity to recognize two HDL-binding proteins previously identified in rat liver plasma membranes. We propose that the specific interaction between HDL and liver plasma membranes is largely mediated through a binding domain in the C-terminus of apoAI, which is consistent with the involvement of specific receptors for the apolipoprotein moiety of HDL.
Allan
C M
CM
Protein Chemistry and Molecular Biology Unit, Baker Medical Research Institute, Prahran, Victoria, Australia.
Fidge
N H
NH
Morrison
J R
JR
Kanellos
J
J
D000818:Animals; D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I / Q000276:immunology / Q000378:metabolism*; D002352:Carrier Proteins; D002462:Cell Membrane / Q000378:metabolism; D003488:Cyanogen Bromide; D000939:Epitopes; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008075:Lipoproteins, HDL; D008099:Liver / Q000378:metabolism; D016601:RNA-Binding Proteins; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018110:Receptors, Lipoprotein
D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D002352:Carrier Proteins; D000939:Epitopes; D008024:Ligands; D008075:Lipoproteins, HDL; D016601:RNA-Binding Proteins; D011956:Receptors, Cell Surface; D018110:Receptors, Lipoprotein; C031616:high density lipoprotein receptors; C075197:high density lipoprotein binding protein; D003488:Cyanogen Bromide