THE THYROXINE-BINDING SITE OF HUMAN APOLIPOPROTEIN-A-I - LOCATION IN THE N-TERMINAL DOMAIN

被引:15
作者
BENVENGA, S [1 ]
CAHNMANN, HJ [1 ]
ROBBINS, J [1 ]
机构
[1] NIDDKD,CLIN ENDOCRINOL BRANCH,BLDG 10,ROOM 8N315,BETHESDA,MD 20892
关键词
D O I
10.1210/endo-128-1-547
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
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 [I-125]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 [I-125]T4 in the presence of MAb or normal mouse IgG, the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent densitrometric quantitation of radioactivity associated with the 28.3-kDa band. Group IMAbs, 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 [I-125]T4 binding to isolated apoA-I with this order of potency: MAb 16 (-50% to -61%) > MAb 14 (-37% to -41%) > MAb 4 (-27% to -33%) > 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 [I-125]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.
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页码:547 / 552
页数:6
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