Heavy and light chain variable single domains of an anti-DNA binding antibody hydrolyze both double- and single-stranded DNAs without sequence specificity.
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 (KD = approximately 17-74 nm) than VH (KD = approximately 2.4-8.4 microm) and VL (KD = approximately 3.2-72 microm), 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.
Department of Microbiology, Ajou University School of Medicine, San 5, Woncheon-dong, Yeongtong-gu, Suwon 443-749, Korea.
Young-Rim
YR
Kim
Jeong-Sun
JS
Kim
Seung-Hyun
SH
Lee
Woo-Ram
WR
Lee
Jong-Nam
JN
Sohn
Yu-Chul
YC
Chung
Hye-Kyung
HK
Shim
Suk-Chan
SC
Lee
Myung-Hee
MH
Kwon
Yong-Sung
YS
Kim
D019943:Amino Acid Substitution; D000818:Animals; D000974:Antibodies, Antinuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D018360:Crystallography, X-Ray; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D019463:Mice, Inbred MRL lpr; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000974:Antibodies, Antinuclear; D004277:DNA, Single-Stranded; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D004247:DNA