Nisonoff A, Oliveira T M, Sharon J
Department of Biology, Brandeis University, Waltham, MA 02254.
Int Immunol. 1993 Jan;5(1):1-9. doi: 10.1093/intimm/5.1.1.
Using two polyclonal (rabbit) and two monoclonal anti-idiotype (anti-Id) reagents, we investigated structural correlates of the Id of mAb 36-71, a somatically mutated member of the CRIA Id family that has an exceptionally high affinity for the p-azobenzenearsonate (Ars) hapten. The two monoclonal anti-Ids reacted principally with the L chain of 36-71. The polyclonal anti-Ids interacted with both the H and L chain. The amino acid sequences of the VH and VL regions of 36-71 differ in eight and 11 positions respectively from those of the anti-Ars mAb 36-65, an unmutated prototype of the CRIA family. In the presence of 36-71L only three substitutions in 36-65 VH, introduced by mutagenesis, sufficed to restore full expression of the 36-71 Id. The same three substitutions had previously been shown to increase the affinity of 36-65 by a factor of 200, to a level equivalent to that of 36-71. X-ray crystallography had indicated that two of these substitutions introduce conformational changes consistent with the increase in affinity. We propose that these conformational changes may also account for the critical role of the three amino acids in Id expression. We also found that 36-65 is a very poor inhibitor of the interaction of 36-71 with its polyclonal anti-Ids, despite identity of the hapten-contacting residues in the two mAbs and evidence (from hapten inhibition) that the hapten-binding region is part of an important Id. Again, a difference in conformation at the binding site of the two mAbs could account for these observations.
我们使用两种多克隆（兔源）和两种单克隆抗独特型（抗Id）试剂，研究了单克隆抗体36 - 71独特型的结构相关性。36 - 71是CRIA Id家族中一个发生体细胞突变的成员，对对氨基苯砷酸（Ars）半抗原具有极高的亲和力。两种单克隆抗Id主要与36 - 71的轻链反应。多克隆抗Id则与重链和轻链都相互作用。36 - 71的重链可变区（VH）和轻链可变区（VL）的氨基酸序列与CRIA家族未突变的原型抗Ars单克隆抗体36 - 65相比，分别在8个和11个位置上有所不同。仅在36 - 65的VH中通过诱变引入三个取代，在有36 - 71轻链存在的情况下，就足以恢复36 - 71独特型的完全表达。之前已表明，相同的这三个取代可使36 - 65的亲和力提高200倍，达到与36 - 71相当的水平。X射线晶体学表明，其中两个取代引入的构象变化与亲和力的增加一致。我们提出，这些构象变化也可能解释这三个氨基酸在独特型表达中的关键作用。我们还发现，尽管这两种单克隆抗体中与半抗原接触的残基相同，且有证据（来自半抗原抑制实验）表明半抗原结合区域是重要独特型的一部分，但36 - 65对36 - 71与其多克隆抗Id相互作用的抑制作用非常弱。同样，这两种单克隆抗体结合位点的构象差异可以解释这些观察结果。
