England P, Nageotte R, Renard M, Page A L, Bedouelle H
Protein Engineering Group (Centre National de la Recherche Scientifique-URA 1129), Unité de Biochimie Cellulaire, Institut Pasteur, Paris, France.
J Immunol. 1999 Feb 15;162(4):2129-36.
The impact of somatic hypermutation on the affinity of Abs directed against protein Ags remains poorly understood. We chose as a model the secondary response Ab D1.3 directed against hen egg lysozyme. During the maturation process leading to this Ab, five replacement somatic mutations occurred. After reconstituting the germline Ab from which D1.3 originated, we assessed the energetic and kinetic importance of each of the somatic mutations, individually or combined, using the BIAcore apparatus. We found that the mutations induced an overall 60-fold improvement of affinity, principally due to a decrease in the kinetic rate of dissociation. We showed that their effects were additive and context independent; therefore, in the case of D1.3, the order in which somatic mutations were introduced and selected is unimportant. Interestingly, most of the affinity improvement was due to a single somatic mutation (Asn50-->Tyr in VL), involving a residue that belongs to the functional interface between Ab D1.3 and lysozyme. This replacement could either establish new Van der Waals contacts between the Ab and the Ag or help stabilize the conformation of a closely situated crucial residue of the Ab paratope. The four other mutations played only a marginal part in affinity maturation; potential reasons for which these mutations were nevertheless selected are discussed.
体细胞超突变对针对蛋白质抗原的抗体亲和力的影响仍知之甚少。我们选择针对鸡卵溶菌酶的二级反应抗体D1.3作为模型。在导致该抗体产生的成熟过程中，发生了五个置换性体细胞突变。从D1.3起源的种系抗体进行重构后，我们使用BIAcore仪器单独或组合评估了每个体细胞突变的能量和动力学重要性。我们发现，这些突变使亲和力总体提高了60倍，主要是由于解离动力学速率的降低。我们表明，它们的作用是累加的且与背景无关；因此，在D1.3的情况下，体细胞突变引入和选择的顺序并不重要。有趣的是，大部分亲和力的提高归因于单个体细胞突变（VL中的Asn50→Tyr），该突变涉及属于抗体D1.3与溶菌酶之间功能界面的一个残基。这种置换可能会在抗体与抗原之间建立新的范德华接触，或者有助于稳定抗体互补决定区中紧邻的关键残基的构象。其他四个突变在亲和力成熟中仅起很小的作用；文中讨论了这些突变仍被选择的潜在原因。
