Localization of the thyroxine binding sites in apolipoprotein B-100 of human low density lipoproteins.
By photoaffinity labeling human low density lipoproteins (LDL) with [125I]T4 we confirmed our previous observation that of the three T4 binding sites of apolipoprotein B-100 (apoB-100) one is in its 26% NH2-terminal portion [apoB-26, the 140 kDa fragment, residues 1-1297] and two in the remaining 74% COOH portion (apoB-74, 410 kDa, residues 1298-4536). We now show that of these two sites one is in the NH2 portion of apoB-74 (apoB-44, 240 kDa, residues 1298-3249) and the other in the nonoverlapping COOH portion (apoB-30, 170 kDa, residues 3250-4536). ApoB-100 contains 13 binding sites for heparin, a known inhibitor of T4 binding to the major T4 carrier plasma proteins; however, heparin failed to inhibit T4 binding to apoB-100 and fragments thereof. The same failure was seen with three monoclonal antibodies (MAbs), 4G3, 5E11, and 43 that block totally or partially LDL binding to the LDL receptor [respective epitopes at residues 2980-3084 (apoB-44), 3441-3569, and 4027-4081 (apoB-30)]. Of the other 3 MAbs, all without effect on LDL binding to the LDL receptor, [1D1, 2D8, and 16, respective epitopes at residues 474-539 (apoB-26), 1438-1481 (apoB-44), and 4154-4189 (apoB-30)], only two (MAbs 1D1 and 2D8) inhibited T4 binding (21 to 39%). We conclude that the three T4-binding sites of apoB-100 are outside the LDL receptor binding domain, distant from the heparin binding sites and, assuming no allosteric effects, in the vicinity of residues 474-539 (T4 site of apoB-26), 1438-1481 (T4 site of apoB-44), and in the C terminal quarter of apoB-30.
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; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D013974:Thyroxine / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D008077:Lipoproteins, LDL; C055664:apolipoprotein B-74; D006493:Heparin; D013974:Thyroxine