TOPOLOGICAL MAPPING OF THE P1-ADHESIN OF MYCOPLASMA-PNEUMONIAE WITH ADHERENCE-INHIBITING MONOCLONAL-ANTIBODIES
TOPOLOGICAL MAPPING OF THE P1-ADHESIN OF MYCOPLASMA-PNEUMONIAE WITH ADHERENCE-INHIBITING MONOCLONAL-ANTIBODIES
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DOI：
10.1099/00221287-136-3-471
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发表时间：
1990-03-01
期刊：
影响因子：
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通讯作者：
JACOBS, E
中科院分区：
文献类型：
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作者：
GERSTENECKER, B;JACOBS, E
利用5种粘附抑制单克隆抗体（mab）对肺炎支原体169 kDa膜整合粘附素的结合位点进行拓扑定位。抗体结合位点用重叠合成的八肽进行表征。该蛋白的三个区域似乎与粘附有关：n端区域[N-reg，表位分别从氨基酸（aa） 1至aa 14和aa 231至aa 238开始]；约位于分子中间的结构域（D1）（从aa 851至aa 858和aa 921至aa 928开始）；和靠近c端的结构域D2（始于aa 1303至aa 1310， aa 1391至aa 1398和aa 1407至aa 1414）。每个单抗P1.26和P1.62与两个一级氨基酸序列反应。这两种抗体都与D1区结合，但单抗p1.62与n端附近的一个序列（aa 231至aa 238）结合，单抗P1.26与D2结构域的第二个表位（aa 1303至aa 1310）反应。这两种抗体的双重结合表明，在天然蛋白中，表位由两个序列组成，分别位于分子的两个不同位置（D1/N-reg和D1/D2）。在以天然肺炎支原体细胞为抗原的竞争性ELISA试验中，指向n端（aa 1至aa 14）的单抗和指向最靠近c端表位（aa 1391至aa 1398和aa 1407至aa 1414）的单抗都能阻止P1.26和P1.62单抗与天然细胞的结合。表位定位和抑制试验结果表明，N-reg、D1和D2结构域在形成一个功能活跃的粘附结合位点方面具有密切的空间关系。此外，所涉及的结构域的最外层表位似乎位于蛋白质分子折叠环的顶部。
Five adherence-inhibiting monoclonal antibodies (mAbs) were used for topological mapping of the binding sites of the 169 kDa membrane-integrated adhesin of Mycoplasma pneumoniae. Antibody binding sites were characterized using overlapping synthetic octapeptides. Three regions of the protein seem to be involved in adherence: the N-terminal region [N-reg, epitopes beginning at amino acid (aa) 1 to aa 14 and aa 231 to aa 238, respectively]; a domain (D1) approximately in the middle of the molecule (beginning at aa 851 to aa 858 and aa 921 to aa 928); and a domain (D2) closer to the C-terminus (beginning at aa 1303 to aa 1310, aa 1391 to aa 1398 and aa 1407 to aa 1414). Each of the mAbs P1.26 and P1.62 reacted with two primary amino acid sequences. Both antibodies bound to the D1 region, but mAb p1.62 showed additional binding to a sequence (aa 231 to aa 238) near the N-terminus, and mAb P1.26 reacted with a second epitope in the D2 domain (aa 1303 to aa 1310). Such dual binding by the two antibodies suggests that in the native protein the epitopes are composed of two sequences which are located on two different sites of the molecule (D1/N-reg and D1/D2, respectively). In a competitive ELISA test using native M. pneumoniae cells as antigen, both the mAb directed to the N-terminus (aa 1 to aa 14) and mAbs directed to those epitopes nearest to the C-terminus (aa 1391 to aa 1398 and aa 1407 to aa 1414) were able to prevent binding of mAbs P1.26 and P1.62 to the native cells. The results of epitope mapping and inhibition tests suggest a close spatial relationship of the domains N-reg, D1 and D2 in forming a functionally active adherence binding site. Furthermore, the outermost epitopes of the domains involved seem to be located on top of folded loops of the protein molecule.