Li Y, Spellerberg M B, Stevenson F K, Capra J D, Potter K N
Department of Microbiology, The University of Texas, Southwestern Medical Center, Dallas, 75235, USA.
J Mol Biol. 1996 Mar 1;256(3):577-89. doi: 10.1006/jmbi.1996.0110.
Essentially all cold agglutinins (CA) with red blood cell I/i specificity isolated from patients with CA disease stemming from lymphoproliferative disorders utilize the VH 4-34 (VH 4-21) gene segment. This near universality of the restricted use of a single gene segment is substantially greater than that demonstrated for other autoantibodies. The monoclonal antibody 9G4 exclusively binds VH 4-34 encoded antibodies and serves as a marker for the VH 4-34 gene segment. Previous studies form our laboratory localized the 9G4 reactive area to framework region 1 (FR1). In the present study, the relative roles of VH FR1, heavy (H) chain complementarity determining region 3 (CDRH 3) and the light (L) chain in I antigen binding were investigated. Mutants containing FR1 sequences from the other VH families, CDRH 3 exchanges, and combinatorial antibodies involving L chain interchanges were produced in the baculovirus system and tested in an I binding assay. The data indicate that FR1 of the VH 4-34 gene segment and the CDRH 3 are essential for the interaction between CA and the I antigen, with the CDRH 3 being fundamental in determining the fine specificity of antigen binding (I versus i). Mutants with substantially altered CDRH 1 and CDRH 2 regions bind I as long as the FR1 is VH 4-34 encoded and the CDRH 3 has a permissive sequence. Light chain swaps indicate that even though antigen binding is predominantly mediated by the H chain, the association with antigen can be abrogated by an incompatible L chain. The necessity for VH 4-34 FR1 explains the almost exclusive use of the VH 4-34 gene segment in cold agglutinins. We hypothesize that, as a general phenomenon, the H chain FR1 of many antibodies may be important in providing the contact required for the close association of antibody with antigen, while the CDRH 3 dictates the fine specificity and strenght of binding.
基本上，从淋巴增殖性疾病引起的冷凝集素（CA）病患者中分离出的所有具有红细胞I/i特异性的冷凝集素都利用VH 4-34（VH 4-21）基因片段。单个基因片段的这种有限使用的近乎普遍性明显大于其他自身抗体所显示的普遍性。单克隆抗体9G4专门结合VH 4-34编码的抗体，并作为VH 4-34基因片段的标志物。我们实验室之前的研究将9G4反应区域定位到构架区1（FR1）。在本研究中，研究了VH FR1、重链互补决定区3（CDRH 3）和轻链在I抗原结合中的相对作用。在杆状病毒系统中产生了含有来自其他VH家族的FR1序列、CDRH 3交换以及涉及轻链互换的组合抗体的突变体，并在I结合试验中进行了测试。数据表明，VH 4-34基因片段的FR1和CDRH 3对于CA与I抗原之间的相互作用至关重要，其中CDRH 3在决定抗原结合的精细特异性（I与i）方面起关键作用。只要FR1由VH 4-34编码且CDRH 3具有允许序列，CDRH 1和CDRH 2区域发生实质性改变的突变体就能结合I。轻链互换表明，尽管抗原结合主要由重链介导，但不相容的轻链可消除与抗原的结合。VH 4-34 FR1的必要性解释了冷凝集素中VH 4-34基因片段几乎被唯一使用的现象。我们推测，作为一种普遍现象，许多抗体的重链FR1可能在提供抗体与抗原紧密结合所需的接触方面很重要，而CDRH 3决定结合的精细特异性和强度。
