Endocrinology, Vol 127, 2241-2246, Copyright © 1990 by Endocrine Society

ARTICLES

Localization of the thyroxine binding sites in apolipoprotein B-100 of human low density lipoproteins

S Benvenga, HJ Cahnmann and J Robbins
Clinical Endocrinology Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.

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.

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