Benvenga S, Cahnmann H J, Robbins J
Clinical Endocrinology Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Endocrinology. 1991 Jan;128(1):547-52. doi: 10.1210/endo-128-1-547.
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.
我们测试了九种抗人载脂蛋白 - A - I（apoA - I）的单克隆抗体（MAb）抑制其与[125I]T4进行光亲和标记的能力。apoA - I是高密度脂蛋白（HDL）的主要载脂蛋白，分子量为28.3 kDa。评估了两种形式：分离的无脂apoA - I（Sigma或Calbiochem）和脂质复合的apoA - I [HDL2，（密度，1.063 - 1.125 g/ml）和HDL3（密度，1.125 - 1.210 g/ml）]。在用0.5 nM [125I]T4在单克隆抗体或正常小鼠IgG存在下进行标记后，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析产物，并随后对与28.3 kDa条带相关的放射性进行光密度定量。第一组单克隆抗体，即那些在apoA - I的N端部分具有表位的抗体，包括单克隆抗体16（表位在第1 - 16位残基）、4和14（第1 - 86位残基）以及18（第98 - 105位残基）；第二组包括单克隆抗体7、10、15和17（表位在第87 - 148位残基）；第三组包括单克隆抗体9（第149 - 243位残基）。所有第一组单克隆抗体以如下效力顺序抑制[125I]T4与分离的apoA - I的结合：单克隆抗体16（-50%至-61%）大于单克隆抗体14（-37%至-41%）大于单克隆抗体4（-27%至-33%）大于单克隆抗体18（-19%至-27%）。对于与脂质相关的apoA - I，层级模式是可变的，大概与已知的HDL明显多分散性质有关，但与分离的apoA - I的情况相反，一个恒定的特征是单克隆抗体4比单克隆抗体14更有效。第二组单克隆抗体在分离的和脂质复合的apoA - I中均产生不到3%的抑制。第三组单克隆抗体9要么未能抑制，要么产生18 - 2
