Heavy and light chain variable single domains of an anti-DNA binding antibody hydrolyze both double- and single-stranded DNAs without sequence specificity
Heavy and light chain variable single domains of an anti-DNA binding antibody hydrolyze both double- and single-stranded DNAs without sequence specificity
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DOI：
10.1074/jbc.m600937200
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发表时间：
2006-06-02
影响因子：
4.8
通讯作者：
Kim, Yong-Sung
中科院分区：
文献类型：
--
作者：
Kim, Young-Rim;Kim, Jeong-Sun;Kim, Yong-Sung
抗DNA抗体（Abs）是生物医学的兴趣，因为它们与人类和小鼠的自身免疫性疾病有关。以前，我们从自身免疫易感的MRL-lpr/lpr小鼠中分离出抗DNA单克隆抗体3D 8。在这里，我们已经表征了DNA结合动力学和水解活性的重组单链可变片段（scFv）和单可变结构域的重链（VH）和轻链（VL）使用各种单链（SS）和双链（DS）DNA底物。所有的Ab都与ds-和ssDNA结合，而没有显著的优先序列特异性，显示出scFv更高的亲和力（K-D =与17-74 n（M）相似）（K-D =与2.4-8.4 mu（M）相似）和VL（K-D =类似于3.2-72 μ（M）），并且以Mg 2+依赖性方式有效地水解ds-和ssDNA而没有序列特异性，除了3D 8 scFv对ss-（dT）的活性差（40）。在3D 8 scFv的VH互补决定区（His-H35 -> Ala）和/或VL互补决定区（His-L94 -> Ala）上的基于晶体结构的His至Ala突变显著抑制了3D 8 scFv的催化活性，表明His残基参与了3D 8 scFv的催化机制。然而，单结构域VH和VL的DNA水解活性不受突变的影响，表明它们与3D 8 scFv的催化机制不同。我们的研究结果首次证实了单结构域抗体具有DNA酶活性，这可能为抗DNA抗体的底物识别和催化机制提供新的见解。
Anti-DNA antibodies (Abs) are of biomedical interest because they are associated with autoimmune diseases in human and mice. Previously we isolated an anti-DNA monoclonal Ab 3D8 from an autoimmune-prone MRL-lpr/lpr mouse. Here we have characterized DNA binding kinetics and hydrolyzing activities of the recombinant single chain variable fragment ( scFv) and the single variable domains of heavy chain (VH) and light chain (VL) using various single-stranded (ss) and double-stranded (ds) DNA substrates. All the Abs bound to both ds- and ssDNAs without significant preferential sequence specificity showing scFv higher affinities (K-D = similar to 17-74 n(M)) than VH (K-D = similar to 2.4-8.4 mu(M)) and VL (K-D = similar to 3.2-72 mu(M)), and efficiently hydrolyzed both ds- and ssDNAs without sequence specificity in a Mg2+-dependent manner, except for the poor activity of 3D8 scFv for ss-(dT)(40). Elucidated crystal structure-based His to Ala mutations on the complementarity determining regions of VH (His-H35 -> Ala) and/or VL (His-L94 -> Ala) of 3D8 scFv significantly inhibited the catalytic activities, indicating that the His residues are involved in the catalytic mechanism of 3D8 scFv. However, the DNA hydrolyzing activities of single domain VH and VL were not affected by the mutations, indicative of their different catalytic mechanisms from that of 3D8 scFv. Our results demonstrate single domain Abs with DNase activities for the first time, which might provide new insights into substrate recognition and catalytic mechanisms of anti-DNA Abs.