key: cord-0683730-g3zf3i4a
authors: Zein, Haggag S.; Teixeira da Silva, Jaime A.; Miyatake, Kazutaka
title: Structure–function analysis and molecular modeling of DNase catalytic antibodies
date: 2010-03-10
journal: Immunol Lett
DOI: 10.1016/j.imlet.2010.01.004
sha: a230b1296c1e059a0e9e2cc0844c5d606b404ba6
doc_id: 683730
cord_uid: g3zf3i4a

There is great interest in the antibodies-to-DNA transformation, since this change is characteristic of autoimmune diseases and contributes to its pathology. After immunization and fusions, 14 hybridomas bearing DNA-hydrolysis activity against pUC19 plasmid DNA were obtained. Genes coding for V(H) and V(L) regions of the 14 monoclonal antibodies (mAbs) were cloned and sequenced. The sequences were compared with sequences of the Ig-Blast database to determine their germline and to identify potential mutations responsible for DNA binding and DNase activity. V genes of the H chains’ genes expressed four genes of the V(H)1/J558 family, three of V(H)5/V(H)7183, and three of V(H)8/V(H)3609. The genetic repertoire of these mAbs was examined by determining the nucleotide sequences of their H chain V regions. This V(H) and V(L) domain was most similar to an anti-ssDNA (DNA-1) antibody as well as to catalytic autoimmune mAb (m3D8). Computer-generated models of the three-dimensional structures of V(H) and V(L) (VHL4) of the IgG4 combinations were used to define the positions occupied by the important sequence motifs at the binding sites. The modeling structure showed that VHL4 binds to oligo (dT3) primarily by sandwiching thymine bases between Tyr L32, Tyr L49 and Tyr H97 side-chains. Superposing VHL4 with anti-nucleic acid m3D8 catAbs revealed a common ssDNA recognition module consisting of His L93, His H35 residues which are critical for DNA-hydrolyzing antibodies. This study demonstrates the potential usefulness of the protein DNA surrogate in the investigation of the origin of anti-DNA antibodies’ hydrolyzing activities.

Catalytic antibodies (catAbs) were first obtained in 1986 [1, 2] against transition state analogs. Amidase and peptidase activities were found in IgGs from the sera of patients with rheumatoid arthritis [3] , factor VIII-cleaving allo-Abs in the sera of patients with severe hemophilia [4] , and DNA-hydrolyzing, amidolytic and peptidolytic activities in Bence-Jones proteins from patients with multiple myeloma [5] . The multiple myeloma patients of an Ab light chain that cleaves the human immunodeficiency virus protein gp120 demonstrated that natural Abs are not restricted to autoantigenic substrates [6] . Anti-DNA antibodies play an impor- tant role in the pathogenesis of systemic lupus erythematosus (SLE) in humans [7] . It has been reported that some of the catAbs to DNA found in SLE patients have nuclease activity and catalyze hydrolysis of the DNA phosphodiester bond [7] . A natural catAbs was prepared by the immunization of mice with ground-state polypeptides or proteins such as Ab light chain-specific vasoactive intestinal peptide which has peptidase hydrolytic activity [8] . Also immunizing mice by human immunodeficiency virus (HIV)-1 gp41 polypeptide-stimulated Ab light chain enzymatically cleaved the conserved region of the HIV-1 envelope protein as well as the antigenic gp41 peptide [9] . Sequence analysis of anti-DNA mAbs from both patients with SLE and murine models of the disease showed that these high-affinity anti-dsDNA IgG contain a high proportion of somatic mutations in their V H and V L sequences [10, 11] . In many of these high-affinity anti-dsDNA IgG Abs, such somatic mutations lead to higher frequencies of certain amino acids, particularly arginine, asparagine, lysine, and tyrosine in the complementarity-determining regions (CDRs). It has been suggested that the structures of these amino acids allow them to form electrostatic interactions and hydrogen bonds with the negatively charged DNA phosphodiester backbone [11] . The aim of this work was to study mAbs and their DNA-hydrolyzing activities. The reactivity of the mAbs, to hydrolyse DNA, was intriguing enough to prompt us to further study their fine specificity and their catalytic mechanisms by analyzing the molecular sequence and structure of the V H and V L genes. Molecular modeling for stimulation with DNA catAb (m3D8) for predicting the catalytic mechanism of the variable region of mAb-4 (4 FV) and knowledge of the specific immunoglobulin genes used to target a common epitope may potentially be useful to identify protein DNA mimicry in the investigation of the origin of anti-DNA Abs catalytic activities.

Tobacco plants (Nicotiana tabacum cv. 'Xanthi-nc') and Nicotiana benthamiana plants at the five-leaf stage were used for inoculation. CMV was originally obtained from Cucurbita pepo in Japan; CMV propagated in tobacco was purified as described by Nitta et al. [12] .

Immunized 8-week-old BALB/c mice (Nippon SLC Co., Japan) were injected subcutaneously with 100 g of purified CMV (whole virus: coat protein contains RNAs) strain pepo in 0.1 ml phosphate-buffered saline (PBS; 0.01 M phosphate and 0.015 M sodium chloride, pH 7.5), which was mixed with an equal volume of adjuvant (RIBI; ImmunoChem Research, Inc., Hamilton, MT) and containing monophosphoryl lipid A MPL (25 g), trehalose dicorynomycolate TDM (25 g) and RIBI. Three injections were administered at 2-week intervals. Three days after the fourth injection, the mice were given a peritoneal injection of 200 g of virus in 0.2 ml PBS. The mice were sacrificed 3 days later and their spleens were harvested. Fusion experiments were carried out as previously described [13] . The positive hybridoma cells were subcloned by a limiting dilution method in the presence of thymocytes of BALB/c mice as feeder cells according to standard protocols [13] .

Ascites fluid (5-10 ml) was precipitated with 50% saturated ammonium sulfate, dialyzed twice for 4 h against 500 vol of (20 mM Tris-HCl, pH 8.0) at 4 • C; samples were diluted with the same amount of binding buffer (1.5 M glycine/3.0 M NaCl, pH 8.9) and the crude mAbs solution was applied to a protein A-agarose affinity chromatography column (1 ml), washed with 10 vol of binding buffer, followed by 10 vol of binding buffer containing 1% Triton X-100, and washed with 10 vol of binding buffer. The Ab was eluted (1-ml fraction) with elution buffer (0.1 M glycine, pH 2.6), and the eluant Abs were neutralized with collection buffer (1.0 M Tris, pH 9.0). The eluted mAb was dialyzed into 50 mM Tris-HCl (pH 7.5), followed by size-exclusion HPLC system chromatography on a Sephacryl-200 HR with 50 mM Tris-HCl (pH 7.5) at 4 • C according to the manufacturer's procedure.

Total RNAs were prepared from about 10 7 hybridoma cells using ISOGEN RNA extraction buffer (Nippon Gene Co., Tokyo, Japan). RNA concentration and purity were gauged using OD 260/280 . The mRNAs were isolated on Oligotex-dT30 (Super) columns (Takara, Kyoto, Japan), as specified by the manufacturer's instructions. The primers used in PCR amplification were based on data by Huse et al. [14] : for V H , 5 -AGGTCCAACTGCTCGAGTCAGG-3 and 5 -AGGCTTACTAGTACAATCC CTGGGCACAAT-3 , where the underlined portion of the 5 primers incorporates an XhoI site and that of the 3 primer an SpeI restriction site. Primers for the V genes were 5 -CCAGATGTGAGCTCGTGATGACCCAGACTCCA-3 and 5 -GCGCCGTCTAGAATTAACACTCTTCCTGTTGAA-3 where the underlined portion of the 5 primer incorporates a SacI restriction site and that of the 3 primer an XbaI restriction site for amplification of the Fd and Lc regions, respectively. First-strand cDNA was synthesized from mRNA template with the Moloney murine leukemia virus M-MLV Reverse Transcriptase kit (Takara, Kyoto, Japan) using oligo-dT20 primers (Pharmacia Biotech). V H and V L were amplified from first-strand cDNA as described by Zein et al. [15] . The amplified fragments were cloned into pGEM-T Easy Vector (1:1, 3:1, 10:1) according to the manufacturer's protocol (Promega, Biotech) and ligated with Ligation High Kit (Takara, Kyoto, Japan) for the purpose of transforming into competent Escherichia coli DH5␣ cells.

Direct sequencing of the treated DNA fragments was made using M13 primer and an ABI PRISM BigDye Primer Cycle Sequencing Kit reagent following the manufacturer's instructions (Applied Biosystems) and run on an ABI Prism 310 Genetic Analyzer (Applied Biosystems) using ABI Prism Sequencing Analysis 3.7 software for data analysis. The PCR product was analyzed and sequenced using M13 primer sequencing of the V regions. Fd or Lc sequences were "blasted" against the publicly accessible "Ig-Blast" database of mouse Ig sequences at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/igblast) to determine the closest germline gene of origin, and to identify potential mutations. CDR position and numbering adopted Kabat numbering [16] and CDR definition was adopted from Andrew's web site (http://www.bioinf.org.uk/abs/).

Assessment of catAbs' DNA-hydrolysis activities was carried out according to [15] . Briefly, an assay mixture containing 20 mM Hepes (pH 7.49), 50 mM NaCl, 1 mM MgCl 2 , 1 mM MnCl 2 , 2.5 g supercoiled pUC19 plasmid DNA, and 1-5 g of each one of the 14 mAbs clones (4, 5, 6, 7, 8, 9, 11, 52, 122 , 521, M21, M22, M23, M24) prepared in 20 l total volume was incubated for 1 h at 37 • C. Hydrolysis was assessed by 1% AGE of the reaction products; the gel was stained with ethidium bromide. Gels were photographed and scanned with Image J software. Molar ratios of reaction products were determined from the scanning data. To study the pH dependence of catalytic activity of Abs, the reactions were carried out in 50 mM acetate buffer (pH 4-5.3), 50 mM Tris-HCl (pH 7-9), carbonate buffer (pH 9.6), and 50 mM borate (pH 10) in the presence or not of 5 mM Mg 2+ .

3D structure models were constructed using the online Web Ab Modeling facility at the University of Bath, UK (http://www.bath.ac.uk/cpad/). Modeling is based on the AbM package using a combination of established theoretical methods together with the latest Ab structural information [16] . WAM predict was used to assign canonical classes and H-CDR3 C-terminal conformation. Structure analysis, superposition, and graphical renderings were carried out using PyMOL (Delano Scientific, San Carlos, CA). Electrostatic surface potentials were calculated using APBS [17] as a plugin (developed by Michael G. Lerner, University of Michigan) in the Pymol Molecular Graphics System (Warren L. DeLano, DeLano Scientific, San Carlos, CA, http://www.pymol. org). 

Immunization of BALB/c female mice with CMV whole virus (protein and RNAs)-stimulated Abs was intriguing: 14 mouse hybridoma cell lines secreting mAbs specific to CMV were well established. To prove that hydrolyzing activity is an intrinsic property of mAbs and is not due to copurifying enzymes, we applied some of the rigid criteria that have been previously proposed by Paul et al. [18] and regarding several aspects for high purity Abs as suggested by Nevinsky and Buneva [19] . Basically, three common steps (purification, precipitated with ammonium sulphate, and affinity chromatography) were used to remove non-specifically bound protein buffer containing 1% Triton X-100 and 0.15 M NaCl, followed by gel filtration, which resulted in Abs with a preparation purity of >99% [15] .

The V H and V regions of 14 CMV-specific mAbs generated from five different fusions of BALB/c mice were sequenced. These sequences were almost homologous with corresponding germline genes published in the GenBank database, outlined in Table 1 , which summarizes the V H , D, and J H fragments of V H genes, and V and J of V L genes. The nucleotide and deduced amino acid sequences of the expressed light chain germline gene were confidently assigned to a very restricted germline family V2, gene bd2 (10 mAbs), GenBank accession nos. (EF672211, EF672212, EF672213, EF672214, EF672215, EF672216, EF672217, EF672218, EF672219, and EF672220; Table 1 ). Four Abs belonged to germline family V1A, gene bb1.1. GenBank accession nos. (EF672221, EF672222, EF672223, and EF672224; Table 1 ). The identity of the V genes used was determined by searching the GenBank database for homologies to known V genes using the BLAST protocol [20] . Alternatively, the nucleotide and deduced amino acid sequences of the expressed V H genes of the 14 anti-CMV Abs are shown in Fig. 1 (Table 1 ). In addition, the V H genes of the IgG Abs were more somatically mutated. D segment usage also appears to be restricted with 7 mAbs of V H using the DSP2 segment, while 3 mAbs were used for another segment, DFL16 ( Fig. 1 and Table 2 ). On the other hand, it does not appear to be an obvious restriction in J H segment usage. Interestingly, most Abs could group into three sets based on their use of the same or highly similar V H and V L genes [21] . Gene rearrangement entails the joining of V H , D and J H germline genes followed by the joining of V L and J L genes. The heavy chains belong to three different families classified into three subgroups. The first includes four mAbs (4, 9, 11, and 521 ) and belongs to the V H J558 germline family with different genes; the homology of the amino acid sequences are V H 104B (99%), V H J558.45 (94%), V H J558.51 (89%) and V H J558.51 (93%) [22, 23] (Fig. 1E , F, G, respectively). However, the V H genes belong to germline family V H J558, gene V130.3, with 97, 97, 95, and 94% identity, respectively (Fig. 1B) [24] . D segments belong to DSP2.11 combined with J H 2 ( Table 1 ). The second subgroup includes three mAbs-(5, 8, and 52) ( Fig. 1C and D) whose V H gene segments are from the V H 7183 germline family [25] . The mAbs-(5 and 52) V H genes are derived from the same germline gene V H 7183.14 with 97 and 95% amino acid homology, respectively (Fig. 1D ) [26] . The third subgroup includes mAbs-(6, 7, and 122) V H genes which are derived from the same V H 3609 germline family, CB17H.10 gene [25] with 96, 96, and 95% homology, respectively ( Fig. 1A) (Table 1) .

Based on the sequence analyses of V genes in specific acquired immune responses to foreign antigens, somatic hypermutations were found to occur mainly in CDRs of V genes during the process of affinity maturation. The combined processes of immunoglobulin gene rearrangement and somatic hypermutation allowed for the creation of an extremely diverse Ab repertoire. V H -521 showed 16 mutations, five of which were silent, while 11 others led to the mutation of amino acid no. 6 (Fig. 1G) . In contrast, V H -11 revealed only two substitutions, the first in CDRH2 with Cys54 H Ser and the second in FW3 with Arg94 H Ile (Fig. 1E) . V H -5 revealed 7 mutants: 2 were silent and 5 were substitutions: Ser55 H Gly; Tyr56 H Ser; Arg75 H Lys; Arg83 H Lys; Lue89 H Met (Fig. 1D) . V H -52 revealed 10 mutations: . Germline precursors were identified as likely VH germline candidates, respectively, through a homology search of the Kabat database. Dots represent residues identical to the corresponding germline. A dash in the individual sequences denotes a deletion. The framework region (FW) and complementarity-determining regions (CDRs) are indicated above the appropriate sequence segments in the figure. The amino acid residue is numbered according to Kabat numbering [16] . Amino acids are identified by the single-letter code. 3 were silent and 7 were substitutions, 5 being typical as Fd-5 with two more substitutions; Thr50 H Tyr and Ser62 H Thr (Fig. 1D) . V H -6 has 10 mutants, 3 (Fig. 1A) . V H -122 showed 13 mutants, 3 silent and 10 substitutions, similar to Fd-72, except for Asn33 H Asp and Ser41 H Pro; Ala49 H Lue (Fig. 1A) . As the frequency of the PCR error used in this study was one in 5000-10,000 nucleotides, the intraclonal sequence heterogeneity observed here is most likely not derived from PCR errors.

The length of H-CDR3 varied from 27 nucleotides in mAb-4 to 51 nucleotides in mAb-6 ( Table 2 ). It has been suggested that the presence of Tyr and Trp residues in H-CDR3 confer flexibility upon the Ab molecule. Consequently, V H -(6, 7, and 122) (Fig. 1A) has five Tyr residues in this region, while the other V H has three (Table 2 ). There are different D and J H regions used in the CMV-specific V H and the number of N insertions between these regions (Table 2) 

The V H and V L gene families revealed high homology sequence with catAbs, and eight V H were derived from germline gene V H J588. V H - (4, 9) showed high homology sequence with different antigenspecific Abs, antinuclear Abs, hepatitis C virus neutralizing Abs [27] , and anti-P24 (HIV-1) [28] (Fig. 2) . In contrast, V H 8 showed sequence homology with anti-nucleic acid Abs [29] (Fig. 2) , while V H 11 had high homology sequence with anti-ssDNA Ab [30] and HIV-1 capsid protein (p24)-specific Abs [31] (Fig. 2) . V H -M2-(1, 2, 3, and 4) showed high homology sequence with coronavirus-neutralizing Abs [32] (Fig. 2) . Three mAbs (5, 52 and 521) are V H s derived from the V H 7183 family. The presumed V H 7183 germline encoding the heavy chain of this Ab has been reported in the IgM and IgG anti-DNA response in (NZB × NZW) F1 mice [33] . However, MAbs-(5 and 53) used the V H 7183.14 germline gene which showed high homology sequence with IgM polyreactive natural autoAbs [34] (Fig. 2 ) while mAb-8 shows high similarity with the heavy chain of influenza hemagglutinin Ha1 [35] (Fig. 2) . Three mAbs (6, 7, and 122) showed high homology with anti-sweetener heavy chain [36] and similarity with mimicry of cocaine by anti-idiotypic Abs. The nucleotides sequences of the different D and JH regions that are used in the hybridomas and the number of N insertions between these regions. Comprehensive analysis of the CDR3 regions of the heavy chain. D segments in each CDR3 region and a difference in D usage, N nucleotides contribution. 

The light chains of the CMV-specific Abs could be assigned to two major V groups, V2 or V1A (Fig. 3) , with sequence identity between the different light chains of each class ranging from 90 to 100% at the amino acid level. All 10 Abs use a V L region encoded by V 2-J1 or -J2 recombination; in addition, the Tyr residue was more frequently observed in 8 mAbs at the V-J joint (V L 96). This residue is encoded by J2, while the Gln residue was observed twice at position V L 96 while the Trp residue was observed once at the same position, V L 96. Interestingly, the V L 34 residue is an Asn germline code VII bd2 germline gene which is typical to Abs V L -specific CMV-CP while the V L -(4, 6, and 7), V L 34 Asn residue was substituted with Ser (Asn34Ser) (Fig. 3A) . Moreover, the V L gene was very restricted against CMV-CP, with high homology to numerous and different Abs raised against autoimmune diseases (anti-DNA, -RNA, -Sm, and -histone) as well as some human viruses (HIV-Gp41 and p24; Hepatitis B and C virus), and catAb proteolytic light chain, esterase-like catAb, and Ab catalysis of the cationic cyclization reaction (Fig. 3A) . Four Abs used another VIA-J4 (Fig. 3B ) which revealed high homology with the light chain against different specific antigens whose identity varied from 94 to 98% with light chains from the database i.e., influenza hemagglutinin neutralizing Ab, anti-ssDNA, -RNA, -fluorescein,polysaccharide, and -bisphenol-A (Fig. 3B) suggesting an intrinsic polyspecificity associated with the V L . In fact, V1 is common to a relatively large population of Abs that bind a large number of antigens, including proteins, DNA, steroids, peptides, and small haptens [37] . Thus, the polyspecificity intrinsic to V1 may contribute to the ability of the germline repertoire to bind to a wide array of chemical structures.

3.7. The relative activity of mAbs against pUC19 DNA Indeed, there are numerous reports regarding natural catAbs but databases of the germline sequence are actually rare and the catalytic domain is mostly revealed from the V L gene while the germline genes VIA bb1.1 and VII bd2 have been reported to possess DNase peptidase-like activity, respectively. Particularly, the Abs derived from germline gene VII bd2 showed higher relative activity than that derived from germline gene VIA bb1 (Fig. 4) . Furthermore, the relative DNAse catalytic activity might depend on the V H germline. In this case, in the presence of Mg 2+ , most mAbs showed high DNA catalytic activity within a varying pH range (Fig. 4) . Alternatively, the mAbs showed only a single break in linear DNA at pH 7-10 in the absence of Mg 2+ (Fig. 4B, D, F, H, and L) . In contrast, polyclonal antibodies (pAbs) illustrated a very restricted pH range, 7-7.5 ( Fig. 4I and G) . mAbs 5 and 6 revealed the disappearance of DNA in the presence of Mg 2+ (Fig. 4C and E) while mAbs 4 and M2-4 showed less activity than mAb-5 and -6 ( Fig. 4A and  G) . Notably, incubation of mAbs with CMV, polyglutamic acid, and dextrin sulphate efficiently inhibited DNase catalytic activity [15] . Remarkably, mAbs having different V H combining ability with one V L showed different DNase catalytic activity; therefore, we speculate that V H could increase or decrease catalytic activity depending on the germline genes ( Fig. 4 and Table 1 ).

A three-dimensional structure of 4-FV is built by means of homology modeling for predicting the DNA catalytic mechanism. The V L and V H sequences of the IgG4 Ab share a very high level of identity with known Abs for which a crystal structure has been reported [38] . Superposition of homology modeled 4-FV structure with the crystal structure of m3D8 scFv (PDB 2GKI) [38] . The alpha carbon traces of V H and V L domains of m3D8 and 4 are displayed in the indicated color code. Two critical residues for the catalysis (HisH35 and HisL93) with a similar orientation of key residues, potentially implied in the catalysis, were observed (Fig. 5 ) and putative DNA binding residues (Tyr residues at L32, L49, and H97) are highlighted as a stick model. The images were generated using PyMol software (DeLano Scientific LLC). The 3D structure similarity, added to the ability of 4-FV to hydrolyze DNA, suggest that the active sites of both catalysts probably have structural similarities. Superposition of the active sites of 4-FV to predict the binding site with the active sites of anti-DNA (m3D8) Ab indicates that Tyr [L32, L49 (green) and H97 (brown)] residues of 4-FV are equidistant to Tyr [L32, L49, L92 (turquoise), H97, and H100a (pink)] residues of the DNA catalytic m3D8 Ab (Fig. 6) . Furthermore, superposition of the active sites of 4-FV with the active sites of anti-ssDNA (DNA-1) Abs indicates that Tyr [L32, L49, and L92 (turquoise)], Tyr [H97, Fig. 6 . Superposition of homology modeled 4-FV structure with the crystal structure of m3D8 scFv the putative DNA binding residues (Tyr residues at H97, L32, and L49) are highlighted as a stick model. The images were generated using PyMol software (DeLano Scientific LLC). H100 and H100a (pink)] residues binding with dT3 (brown) are at similar distances to Tyr [L32, L49 (green), and H97 (brown)] (Fig. 7) . The structure shows that Ab binds oligo(dT) primarily by sandwiching thymine bases between Tyr side-chains, which allows the bases to make sequence-specific hydrogen bonds (Figs. 6 and 7 ). The 3D model is assessed by simulation of molecular dynamics to determine its stability and by comparison with those of known protein structures. The structural information from the theoretically modeled complex can help us to further understand the catalytic mechanism of anti-DNA Abs.

Using data from known Ab crystal structures and computer modeling, a series of linkers were designed and evaluated as potential candidates to genetically connect the V H and V L regions. The resulting scFv molecules were evaluated for their functional activities and relative affinity [39] . Very little molecular characterization of natural catalytic Abs with mAbs has been achieved so far. Due to the activation of the immune system as a response to a foreign antigen, maturation of the Ab response takes place, resulting in the production of specific, high-affinity Abs. Therefore, specific Abs can be selected using a relatively small, random combinatorial V gene library derived from an immunized donor [40] . The procedure included the isolation of the V H and V L of the murine mAb from mRNA of 14 hybridoma cells, followed by cloning, sequencing and characterization of the FV. V H gene usage was determined and compared to V H genes used by Ab fragments of a germline database. The V H and V regions of 14 anti-CMV mAbs generated from five different fusions of BALB/c mice were immunized with native CMV-CP, and the V H , D, J H , V, and J were determined ( Table 1 ). All the Abs were derived from distinct B cells because they had utilized diverse V H , D, and J H gene combinations, and because the length of the CDR3 region ranged from 7 to 17 amino acid residues (Table 2 ). An abundance of V H genes from the J558 family was observed (8/14) but each represented a separate member of the family ( Table 1 ). The V L is encoded by the V1 gene, which is common to a relatively large population of Abs that bind a large number of antigens including proteins, DNA, steroids, pep-tides, and small haptens [37] . Certain combinations of germline V genes (V, J and V H ) are polyspecific in nature and can be used to construct Ab-combining sites for structurally very distinct ligands. Germline Ab polyspecificity further expands the binding potential of the germline repertoire [37] . This polyspecificity may be general to several germline-encoded Abs and may have been selected for by the immune system to provide a mechanism for rapid generation of Abs of moderate to high affinity for a broad range of antigens [37] . CMV-CP is capable of inducing a variety of B cells that have distinct phenotypic and genotypic paratopes. Interestingly, the high DNase catalytic Abs were encoded by germline genes such as mAb-8 (Fig. 1C) . Furthermore, analysis of DNase catalytic activities and nucleotide sequences of the V H and V L showed a strong correlation with the germline heavy chains, in which mAb-(8) was derived from V H 7183, showing high DNase catalytic activity ( Fig. 4K and L) . Prominently, the result of the relative activity of the six different mAbs (4, 5, 6 and 8) showed diverse relative activities, although their light chain genes had high relative identity; therefore, the fact that V H domain can modulate catalytic activity is potentially important in these mAbs (Fig. 4) . One of the important aspects of V L and V H amino acid sequences is the study of the structural analysis of the antigen-binding loops by molecular modeling and simulation of molecular dynamics. Through these findings, amino acid His (H35 and L93) residues may play a crucial role in the DNA-Ab interaction (Fig. 5) . Tyr (L32, L49 and H97) side-chains that exist in the antigen combining site might be capable of mediating most of the contacts necessary for DNA recognition, and thus it seems likely that the overabundance of Tyr in natural antigen-binding sites is a consequence of the side chain being particularly well suited for making productive contacts with antigen [41] . Interestingly, the genes encoding the heavy chain variable region of these Abs displayed evidence of only minimal somatic hypermutation (Fig. 1C) . We consider that the negative charge on the acetate group in the CMV-CP was partially neutralized by a hydrogen bond with the phenolic hydroxyl group of tyrosine, which exists in HCDR3. Therefore, we speculate and expect that the HCDR3-peptide be used as tool for plant virus resistance depending on the peptide-neutralizing epitope.

We generated 14 mAbs raised by immunization with CMV that displayed DNase activity. Genes coding for V H and V L regions of all 14 mAbs were cloned and sequenced. The sequences were compared with sequences of the Ig-Blast database to determine their germline and to identify potential mutations responsible for DNA binding and DNase activity. Superposition of homology modeled 4-FV structure with the crystal structure of m3D8 scFv, two critical residues for catalysis (HisH35 and HisL93) and putative DNA binding residues (Tyr residues at L32, L49, and H97). Collectively our studies suggest that DNA binding and hydrolyzing activities of anti-CMV Abs are well conserved in both V H and V L , providing avenue to further studies of their biochemical and biological functions.

Selective chemical catalysis by an antibody

Catalytic antibodies

Amidase and peptidase activities of polyclonal immunoglobulin G present in the sera of patients with rheumatoid arthritis

Catalytic activity of antibodies against factor VIII in patients with hemophilia A

Does catalytic activity of Bence-Jones proteins contribute to the pathogenesis of multiple myeloma?

Natural catalytic immunity is not restricted to autoantigenic substrates: identification of a human immunodeficiency virus gp 120-cleaving antibody light chain

DNA hydrolyzing autoantibodies

Molecular cloning of a proteolytic antibody light chain

Targeted destruction of the HIV-1 coat protein gp41 by a catalytic antibody light chain

Immunoglobulin variable region sequences of human monoclonal anti-DNA antibodies

Genetic and structural evidence for antigen selection of anti-DNA antibodies

Comparative studies on the nucleotide sequence of Cucumber mosaic virus RNA3 between Y strain and Q strain

A laboratory manual

Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda

Monoclonal antibodies specific to Cucumber mosaic virus coat protein possess DNA-hydrolyzing activity

Accessing the Kabat antibody sequence database by computer PROTEINS: structure

Protein structure prediction and structural genomics

Catalytic hydrolysis of vasoactive intestinal peptide by human autoantibody

Natural catalytic antibodies-abzymes

Gapped BLAST and PSI-BLAST: a new generation of protein database search programs

Characteristics of the immunoglobulin Vkappa genes, pseudogenes, relics and orphons in the mouse genome

Allelic immunoglobulin VH genes in two mouse strains: possible germline gene recombination

Germline diversity of the expressed BALB/c VhJ558 gene family

Identification of novel VH1/J558 immunoglobulin germline genes of C57BL/6 (Igh b) allotype

Molecular characterization of a panel of murine monoclonal antibodies specific for the SARS-coronavirus

Sequences of four new members of the VH7183 gene family in BALB/c mice

Sequences in the hypervariable region 1 of hepatitis C virus show only minimal variability in the presence of antibodies against hypervariable region 1 during acute infection in chimpanzees

Evolutionary transition pathways for changing peptide ligand specificity and structure

Analysis of a nucleic-acid-binding antibody fragment: construction and characterization of heavy-chain complementarity-determining region switch variants

Repertoire diversification in mice with an IgHlocus-targeted transgene for the rearranged VH domain of a physiologically selected anti-ssDNA antibody

Food immunoassays: applications of polyclonal, monoclonal and recombinant antibodies

Interference of coronavirus infection by expression of immunoglobulin G (IgG) or IgA virus-neutralizing antibodies

Molecular signatures of antinuclear antibodies contributions of heavy chain CDR residues

Structural and affinity studies of IgM polyreactive natural autoantibodies

Structural evidence for induced fit as a mechanism for antibody-antigen recognition

The threedimensional structure of a complex of a murine Fab (NC10, 14) with a potent sweetener (NC174): an illustration of structural diversity in antigen recognition by immunoglobulins

Immunological origins of binding and catalysis in a Diels-Alderase antibody

Heavy and light chain variable single domains of an anti-DNA binding antibody hydrolyze both double and single-stranded DNAs without sequence specificity

Stability engineering of antibody single-chain Fv fragments

Making antibody fragments using phage display libraries

Antibody engineering by codon-based mutagenesis in a filamentous phage vector system

We are grateful to Professor Dr. Ikuo Fujii for his advice and helpful discussion. The authors would like to thank Dr. Yong-Sung Kim, Department of Genetic Engineering, Sungkyunkwan University Korea, for his grateful help in the antibody-docking.