The thyroxine-binding site of human apolipoprotein-A-I: location in the N-terminal domain.
We tested the ability of nine monoclonal antibodies (MAb) against human apolipoprotein-A-I (apoA-I), the 28.3-kDa major apoprotein of high density lipoproteins (HDL), to inhibit its photoaffinity labeling with [125I]T4. Two forms were evaluated: isolated lipid-free apoA-I (Sigma or Calbiochem) and lipid-complexed apoA-I [HDL2, (density, 1.063-1.125 g/ml) and HDL3 (density, 1.125-1.210 g/ml)]. After labeling with 0.5 nM [125I]T4 in the presence of MAb or normal mouse IgG, the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent densitometric quantitation of radioactivity associated with the 28.3-kDa band. Group I MAbs, namely those having epitopes in the N-terminal portion of apoA-I, include MAb 16 (epitopes at residues 1-16), 4 and 14 (residues 1-86), and 18 (residues 98-105); group II includes MAbs 7,10, 15, and 17 (epitopes at residues 87-148); group III includes MAb 9 (residues 149-243). All group I MAbs inhibited [125I]T4 binding to isolated apoA-I with this order of potency: MAb 16 (-50% to -61%) greater than MAb 14 (-37% to -41%) greater than MAb 4 (-27% to -33%) greater than MAb 18 (-19% to -27%). In the case of lipid-associated apoA-I, the pattern of hierarchy was variable, presumably related to the known markedly polydisperse nature of HDL, but a constant feature, in contrast to the case of isolated apoA-I, was that MAb 4 was more potent than MAb 14. Group II MAbs gave less than 3% inhibition in both isolated and lipid-complexed apoA-I. Group III MAb 9 either failed to inhibit or gave 18-27% inhibition (one preparation each of HDL2 and HDL3). We conclude that the T4 site of apoA-I is in the N-terminal domain of apoA-I, closer to the epitope for MAb 16 than to that for MAb 18, and that conformational changes occurring when apoA-I is associated with lipids in the HDL particle alter the spatial relationship between some epitopes and the T4 site. Our definition of the T4 site of apoA-I is consistent with another set of data showing that heparin failed to inhibit [125I]T4 binding to isolated apoA-I. Heparin is known to interact with clusters of basic residues, and these residues are concentrated in the midregion of apoA-I.
Benvenga
S
S
Clinical Endocrinology Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Cahnmann
H J
HJ
Robbins
J
J
D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites; D003198:Computer Simulation; D000939:Epitopes / Q000032:analysis; D006801:Humans; D007457:Iodine Radioisotopes; D008075:Lipoproteins, HDL / Q000378:metabolism*; D011487:Protein Conformation; D013974:Thyroxine / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D000939:Epitopes; D007457:Iodine Radioisotopes; D008075:Lipoproteins, HDL; D013974:Thyroxine