The use of monoclonal antibodies to localize the low density lipoprotein receptor-binding domain of apolipoprotein B.
Human apolipoprotein (apo) B-100 is composed of 4536 amino acids. It is thought that the binding of apoB to the low density lipoprotein (LDL) receptor involves an interaction between basic amino acids of the ligand and acidic residues of the receptor. Three alternative models have been proposed to describe this interaction: 1) a single region of apoB is involved in receptor binding; 2) groups of basic amino acids from throughout the apoB primary structure act in concert in apoB receptor binding; and 3) apoB contains multiple independent binding regions. We have found that monoclonal antibodies (Mabs) specific for a region that spans a thrombin cleavage site at apoB residue 3249 (T2/T3 junction) totally blocked LDL binding to the LDL receptor. Mabs specific for epitopes outside this region had either no or partial ability to block LDL binding. In order to define the region of apoB directly involved in the interaction with the LDL receptor we have tested 22 different Mabs for their ability to bind to LDL already fixed to the receptor. A Mab specific for an epitope situated between residues 2835 and 2922 could bind to its epitope on LDL fixed to its receptor whereas a second epitope between residues 2980 and 3084 is inaccessible on receptor-bound LDL. A series of epitopes near residue 3500 of apoB is totally inaccessible, and another situated between residues 4027 and 4081 is poorly accessible on receptor-bound LDL. In contrast, an epitope that is situated between residues 4154 and 4189 is fully exposed. Mabs specific for epitopes upstream and downstream of the region 3000-4000 can bind to receptor-bound LDL with a stoichiometry close to unity. Our results strongly suggest that the unique region of apoB directly involved in the LDL-receptor interaction is that of the T2/T3 junction.
Milne
R
R
Laboratory of Lipoprotein Metabolism, Institut de Recherches Cliniques de Montréal, Quebec, Canada.
Théolis
R
R
Maurice
R
R
Pease
R J
RJ
Weech
P K
PK
Rassart
E
E
Fruchart
J C
JC
Scott
J
J
Marcel
Y L
YL
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000032:analysis; D001055:Apolipoproteins B / Q000276:immunology / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis; D006801:Humans; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000097:blood / Q000302:isolation & purification; D011487:Protein Conformation; D011973:Receptors, LDL / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001055:Apolipoproteins B; D000939:Epitopes; D008077:Lipoproteins, LDL; D011973:Receptors, LDL