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J. Biol. Chem., Vol. 281, Issue 22, 15287-15295, June 2, 2006

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Heavy and Light Chain Variable Single Domains of an Anti-DNA Binding Antibody Hydrolyze Both Double- and Single-stranded DNAs without Sequence Specificity^*

Young-Rim Kim, Jeong-Sun Kim¹, Seung-Hyun Lee^¶, Woo-Ram Lee^¶, Jong-Nam Sohn, Yu-Chul Chung^||, Hye-Kyung Shim^||, Suk-Chan Lee^||, Myung-Hee Kwon², and Yong-Sung Kim^¶3

From the Department of Microbiology, Ajou University School of Medicine, San 5, Woncheon-dong, Yeongtong-gu, Suwon 443-749, Korea, ^¶Department of Molecular Science and Technology, Ajou University, San 5, Woncheon-dong, Yeongtong-gu, Suwon 443-749, Korea, Department of Chemistry, Chonnam National University, 300, Yongbong-dong, Buk-gu, Gwangju, 500-757, Korea, and ^||Department of Genetic Engineering, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, Korea

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 = 17–74 nM) than VH (K_D = 2.4–8.4 µM) and VL (K_D = 3.2–72 µM), 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.

Received for publication, January 31, 2006 , and in revised form, March 6, 2006.

^* This work was supported by Basic Research Program of the Korea Science & Engineering Foundation Grant R01-2006-000-10743-0, National R&D Program for Cancer Control, Ministry of Health & Welfare Grant 0520110-1 (to Y. S. K.), Basic Research Promotion Fund of Korea Research Foundation Grant KRF-2005-204-E00034 (to M. H. K.), and the "GRRC" Project of Gyeonggi Provincial Government, Republic of Korea. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 2GKI) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

¹ To whom correspondence on crystallography should be addressed. Tel.: 82-62-530-3384; Fax: 82-62-530-3389; E-mail: jsunkim{at}chonnam.ac.kr. ² To whom correspondence may be addressed. Tel.: 82-31-219-5074; Fax: 82-31-219-5079; E-mail: kwonmh{at}ajou.ac.kr. ³ To whom correspondence may be addressed. Tel.: 82-31-219-2662; Fax: 82-31-219-2394; E-mail: kimys{at}ajou.ac.kr.

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