Marcel Y L, Provost P R, Koa H, Raffai E, Dac N V, Fruchart J C, Rassart E
Laboratory of Lipoprotein Metabolism, Clinical Research Institute of Montreal, Québec, Canada.
J Biol Chem. 1991 Feb 25;266(6):3644-53.
The definition of epitopes on apoA-I provides evidence for a very dense packing of the peptide chain and supports the proposed supersecondary structure, made of repetitive antiparallel helices. Screening with overlapping synthetic hexapeptides shows that only a few epitopes are continuous and that all continuous contact sequences identified within the epitopes coincide or are contiguous to a putative beta-turn, such as residues 1-8, 48-55, 98-104, 118-123, and 135-140. On the N-terminal half of apoA-I we identified 6 overlapping tertiary discontinuous epitopes, uniquely constituted by amino acids and discontinuous sequences on helical segments that are far apart and which define a particular region with a complex tertiary structure. Among these are the epitopes for antibody 5G6, which reacts with residues 45-51, 83-92, 119-126, and 136-143; for A16, which reacts with 14-19, 23-28, and 60-82; and for r-FC1, which reacts with 1-8, 29-35, 78-83, and 98-121. The very far apart discontinuous sequences included in these epitopes can be explained by the predicted turns and coiled domains, and thus provide evidence for such a tertiary structure. Alternatively, these results could also be explained by intermolecular epitopes involving the N-terminal region. In contrast, in the middle of apoA-I, all identified epitopes are shorter and discontinuous within the secondary structure and are constituted by residues forming a beta-turn with all or part of an adjacent alpha-helix. We hypothesize that these multiple epitopes, that are mostly limited to a single helix, reflect the existence of a very mobile domain, possibly with hinged pairs of adjacent alpha-helices. On the C-terminal half of apoA-I, several monoclonal antibodies react with overlapping epitopes located between residues 149 and 186, which probably reflects 2 antiparallel alpha-helices interrupted by a beta-turn.
载脂蛋白A-I上表位的定义为肽链的紧密堆积提供了证据，并支持了由重复的反平行螺旋构成的超二级结构。用重叠的合成六肽进行筛选表明，只有少数表位是连续的，并且在表位内鉴定出的所有连续接触序列都与推定的β-转角重合或相邻，如残基1-8、48-55、98-104、118-123和135-140。在载脂蛋白A-I的N端一半区域，我们鉴定出6个重叠的三级不连续表位，它们由螺旋片段上相距很远的氨基酸和不连续序列独特构成，这些序列定义了一个具有复杂三级结构的特定区域。其中包括与残基45-51、83-92、119-126和136-143反应的抗体5G6的表位；与14-19、23-28和60-82反应的A16的表位；以及与1-8、29-35、78-83和98-121反应的r-FC1的表位。这些表位中包含的相距很远的不连续序列可以通过预测的转角和卷曲结构域来解释，从而为这种三级结构提供了证据。或者，这些结果也可以通过涉及N端区域的分子间表位来解释。相比之下，在载脂蛋白A-I的中部，所有鉴定出的表位在二级结构内都较短且不连续，并且由与全部或部分相邻α-螺旋形成β-转角的残基构成。我们假设这些大多局限于单个螺旋的多个表位反映了一个非常灵活的结构域的存在，可能存在相邻α-螺旋的铰链对。在载脂蛋白A-I的C端一半区域，几种单克隆抗体与位于残基149和186之间的重叠表位反应，这可能反映了由一个β-转角中断的2个反平行α-螺旋。
