Mockridge C I, Chapman C J, Spellerberg M B, Isenberg D A, Stevenson F K
Molecular Immunology Group, Tenovus Laboratory, Southampton University Hospitals, United Kingdom.
J Immunol. 1996 Sep 15;157(6):2449-54.
The V4-34 gene encodes the majority of autoanti-red cell Abs of I/i specificity. It also encodes a proportion of autoanti-DNA Abs found in patients with systemic lupus erythematosus. Nucleotide sequence analysis of mAbs that use this gene has indicated a role for CDR3 in discrimination between these autoantigens. Specifically, anti-DNA activity may require basic amino acids, such as arginine, found in this region. To investigate this requirement, we have expressed VH and VL sequences from a patient's IgG anti-DNA mAb, as Fab molecules at the surface of phage. Expressed Fab bound strongly to DNA, whereas control VH and VL pairs from an anti-red cell mAb did not. Replacement of the homologous mutated V4-34 sequence by germ-line sequence did not affect binding, indicating that somatic mutations in VH did not contribute significantly. In contrast, replacement of the basic CDR3 by an anti-red cell CDR3 abrogated anti-DNA activity, confirming its major role. However, an influence of VL was revealed by replacing homologous mutated V kappa IIIb by an unmutated V kappa IIIb sequence, reducing binding by approximately 50%. This influence was apparent only with homologous VH since the mutated V kappa was unable to generate anti-DNA activity when combined with anti-red cell VH. The 9G4 idiotope, which arises from FWR1, was expressed by all constructs. Substitution of Trp by Ser at position 7 in FWR11 caused complete loss of idiotope expression, with no effect on recognition of DNA, indicating no influence of idiotope expression on anti-DNA activity. Phage surface expression provides a powerful and rapid technique for assessing sequences relevant for Ab specificity or idiotope expression.
V4-34基因编码了大部分具有I/i特异性的自身抗红细胞抗体。它还编码了系统性红斑狼疮患者体内发现的一部分自身抗DNA抗体。对使用该基因的单克隆抗体进行核苷酸序列分析表明，互补决定区3（CDR3）在区分这些自身抗原中发挥作用。具体而言，抗DNA活性可能需要该区域中存在的碱性氨基酸，如精氨酸。为了研究这一需求，我们将患者IgG抗DNA单克隆抗体的重链可变区（VH）和轻链可变区（VL）序列作为Fab分子在噬菌体表面进行了表达。表达的Fab与DNA强烈结合，而来自抗红细胞单克隆抗体的对照VH和VL对则不结合。用种系序列取代同源突变的V4-34序列并不影响结合，表明VH中的体细胞突变没有显著贡献。相比之下，用抗红细胞CDR3取代碱性CDR3消除了抗DNA活性，证实了其主要作用。然而，将同源突变的VκIIIb替换为未突变的VκIIIb序列揭示了VL的影响，使结合减少了约50%。这种影响仅在同源VH存在时明显，因为突变的Vκ与抗红细胞VH结合时无法产生抗DNA活性。所有构建体都表达了源自框架区1（FWR1）的9G4独特型。在FWR11的第7位将色氨酸替换为丝氨酸导致独特型表达完全丧失，但对DNA识别没有影响，表明独特型表达对抗DNA活性没有影响。噬菌体表面表达为评估与抗体特异性或独特型表达相关的序列提供了一种强大而快速的技术。
