Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M600937200 on March 20, 2006

J. Biol. Chem., Vol. 281, Issue 22, 15287-15295, June 2, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/22/15287    most recent
M600937200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, Y.-R.
Right arrow Articles by Kim, Y.-S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, Y.-R.
Right arrow Articles by Kim, Y.-S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

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{ddagger}, Jeong-Sun Kim§1, Seung-Hyun Lee, Woo-Ram Lee, Jong-Nam Sohn{ddagger}, Yu-Chul Chung||, Hye-Kyung Shim||, Suk-Chan Lee||, Myung-Hee Kwon{ddagger}2, and Yong-Sung Kim3

From the {ddagger}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

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 = ~17–74 nM) than VH (KD = ~2.4–8.4 µM) and VL (KD = ~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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Anti-DNA antibodies (Abs)4 are naturally present in healthy humans, but are preferentially found in patients with autoimmune diseases, particularly systemic lupus erythematosus and multiple sclerosis (1, 2). Naturally occurring anti-DNA Abs generally do not exhibit sequence specificities (1, 2). Instead, they can be classified as specific for single-stranded (ss) DNA (3–5), double-stranded (ds) DNA (6, 7), or both ssDNA and dsDNA (5, 8) with a preference for certain DNA sequences, such as poly(dT) or poly(dG-dC) sequences (1, 2). Recently natural anti-DNA Abs have been reported showing sequence specificity for ssDNA (9, 10) or dsDNA (11). However, only a few studies characterized detailed binding kinetics and specificities of anti-DNA Abs (5, 12, 13).

Since Shuster et al. (14) reported that some autoantibodies derived from patients with systemic lupus erythematosus possessed DNA nicking activities, some anti-DNA Abs from many autoimmune and viral diseases have also shown DNA and/or RNA hydrolyzing catalytic activities (15, 16). The origin of DNA-hydrolyzing catalytic Abs, so called "DNA-abzymes," mainly belonging to immunoglobulin M (IgM) or G (IgG) class, have been proposed to be anti-idiotypic Abs to active sites of nucleases, Abs produced against DNA or nucleoprotein complexes, and/or Abs existing in germ line cells even before somatic mutations (15–18). A number of polyclonal DNA-abzymes have been described, but the detailed biochemical and structural basis of catalytic mechanisms of monoclonal Abs (mAbs) have not been extensively characterized (1, 2, 15). An exception is BV04-01, which binds to and hydrolyzes both ss- and dsDNA with preferential cleavages for T-rich ssDNA and CG-rich dsDNA (8). In addition to intact IgG, IgM, and IgA Abs, their fragments of Fab, scFv, and/or light chains have been attributed to DNase activities (15, 18–20). However, no studies have been reported for single variable domain Abs of heavy chain (VH) and light chain (VL) with DNase activities.

Previously we have isolated an anti-DNA mAb 3D8 from the spleen cells of the MRL-lpr/lpr mouse, which spontaneously develops an autoimmune syndrome that resembles human systemic lupus erythematosus (21). In the present study, we aim to characterize DNA binding kinetics and hydrolyzing activities of 3D8 scFv, VH, and VL proteins in detail using supercoiled plasmids and various synthetic ss- and ds-oligodeoxynucleotides as substrates. We found that 3D8 scFv, VH, and VL bound to and hydrolyzed both ss- and dsDNAs in the presence of Mg2+ without significant sequence specificities. Furthermore, we determined the crystal structure of 3D8 scFv and performed site-directed mutagenesis studies based on the structural information to gain some insights into catalytic mechanism(s) of the proteins.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Restriction enzymes, T4 DNA ligase, and high fidelity DNA polymerase were purchased from New England Biolabs. All other chemicals and solvents used were of analytical grade. The following 40-bp oligodeoxynucleotides were synthesized from Genotech (Seoul, Korea) in unlabeled and labeled (5'-digoxigenin and/or 3'-biotin) forms: ss-(dT)40, ss-(dA)40, ss-(dG-dC)20, ss-(dC-dG)20, ss-(dN)40 (n = 5'-CCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAAC-3') and ss-(dN')40 (N'= the reverse complementary sequence of N). dsDNAs were generated by annealing equimolar amounts of the labeled and its unlabeled reverse complementary ssDNAs in water at 65 °C for 5 min, cooling to 20 °C at a rate of 2 °C/min, and then chilling on ice (22). The preparation of dsDNAs was confirmed by hypochromicity at 260 nm (22).

Plasmid Constructions—The 3D8 scFv gene was derived from the hybridoma cell line producing mAb 3D8 IgG, originating from an autoimmune-prone MRL-lpr/lpr mouse (21). The VH (NCBI accession number AAF79128 [GenBank] ) and VL (NCBI accession number AAF79129 [GenBank] ) genes were cloned into the pGEM®-T Easy vector (Promega Corp.), then subcloned into the bacterial expression vector pIg20 (23), resulting in pIg20-3D8 scFv. This vector encodes a (G4S)3 flexible linker between the VH and VL sequences (VH-(G4S)3-VL), a thrombin cleavage site followed by the Staphylococcal protein A (SPA) tag at the C terminus, and a N-terminal bacterial alkaline phosphatase signal peptide for targeting protein expression to the periplasm under control of the T7 promoter. Each gene of the VH and VL single domains was also subcloned into the pIg20, generating pIg20-VH and pIg20-VL, respectively. The constructs were confirmed by sequencing and transformed into Escherichia coli BL21(DE3) pLysE cells (Novagen).

Bacterial Expression and Purification of the Proteins—The transformed cells were grown at 37 °C to an A600 of ~0.8 in 1-liter of Luria-Bertani medium containing 100 µg/ml ampicillin and 20 µg/ml chloramphenicol, and then induced for 16 h at 30 °C by adding 0.5 mM isopropyl 1-thio-beta-D-galactopyranoside. The induced cells were harvested and the fusion proteins of 3D8 scFv-SPA, VH-SPA, and VL-SPA were purified from the culture supernatant with an IgG-SepharoseTM affinity column (Amersham Biosciences), as described (21, 23). The SPA tag was removed from the fusion proteins by thrombin cleavage and then the cleaved proteins of scFv, VH, and VL were further purified using the IgG-SepharoseTM column as described (21, 24). Proteins were >99% pure as judged by Coomassie Blue-stained SDS-PAGE (Fig. 1). Protein concentrations were determined using extinction coefficients of 1.995 for scFv, 2.325 for VH, and 1.674 for VL in units of mg ml–1 cm–1 at 280 nm, which were calculated from the respective amino acid sequence.

Size Exclusion Chromatography (SEC) Analyses of Proteins—SEC analyses for the purified 3D8 scFv, VH, VL, and Fv (non-covalently associated form of VH and VL) were performed on a Agilent 1100 high performance liquid chromatography system using a TSK G3000SWXL size exclusion column (7.8 x 300 mm, TosoHaas, Japan), with a mobile phase of 50 mM sodium phosphate, pH 7.4, plus 150 mM NaCl at a flow rate of 0.7 ml/min. Chromatograms were obtained by monitoring absorbance at 280 nm. The injection amount ranged between 5 and 20 µM of proteins in a volume of 20 µl. A set of molecular mass standard markers (Sigma) ranging from 13.7 to 66 kDa was used.

DNA Binding Assay by Enzyme-linked Immunosorbent Assay (ELISA)—Ninety six-well polystyrene microtiter plates (Nunc, Invitrogen Ltd.) were coated with 100 µl of oligodeoxynucleotide substrates at 10 µg/ml in 50 mM Tris-Cl, pH 7.5, 50 mM NaCl ("TBS") for 1 h at 37 °C, and washed (3 times) with TBS containing 0.05% Tween 20 ("TBST"), then blocked with TBS containing 3% (w/v) bovine serum albumin (BSA) (Sigma) for 1 h at 37 °C (5, 22). Then, proteins (100 µl of 20 µg/ml) were added and incubated for 1 h at 37 °C. After washing the wells with TBST, the wells were incubated with rabbit IgG (100 µl of 1 µg/ml) (Pierce Biotechnology) and then with alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (100 µl of 1:10,000 dilution) (Pierce). Each incubation step was performed for 1 h at 25 °C, followed by washing (3 times) with TBST. Finally, p-nitrophenyl phosphate (Sigma) solution (1 mg/ml in 0.1 M glycine, 1 mM ZnCl2, and 1 mM MgCl2, pH 10.3) was added to each well and absorbance was read at 405 nm in a microplate reader. To investigate effects of ionic strength on the binding activities of proteins, proteins were incubated on the substrate-coated wells in 50 mM Tris-Cl, pH 7.5, containing various concentrations of NaCl (0–0.8 M) for 1 h at 37 °C.

Surface Plasmon Resonance (SPR) Assays—Kinetic measurements of protein-protein interactions were performed at 25 °C using a Biacore 2000 SPR biosensor (Amersham Biosciences). The measured SPR values are expressed in arbitrary response units. For measurements of protein-protein interactions, ~0.5–1.0 mg/ml of proteins in a coupling buffer (10 mM sodium acetate buffer, pH 4.0) were immobilized onto the carboxymethylated dextran surface of a CM5 sensor chip at a level of 800–1000 response units by an amine coupling method according to the manufacturer's instructions (25). A reference surface was generated simultaneously under the same conditions, and BSA (100 µM) was injected as a control to correct for instrument and buffer artifacts. Each protein prepared in concentrations from 5 nM to 20 µM by serial dilutions with PBS buffer (50 mM sodium phosphate, pH 7.2, plus 100 mM NaCl) was injected into the flow cells (association phase), which was followed by PBS buffer (dissociation phase), both at a flow rate of 30 µl/min. The sensor surface was regenerated between assays by injecting 30 µl of a wash buffer containing 50 mM NaOH and 25 mM NaCl, to remove bound proteins (25).

For measurements of protein-DNA interactions, the following 3'-biotin-labeled ss- and dsDNAs were used as substrates: ss-(dT)40, ds-(dT: dA)40, ss-(dG-dC)20, ds-(dG-dC:dC-dG)20, ss-(dN)40, and ds-(dN: dN')40. To immobilize the substrates to the streptavidin-coated sensor chip SA (Amersham Biosciences) (5, 13), 0.1 µM substrate in HES buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) was injected at a rate of 5 µl/min over each flow cell, resulting in immobilized levels of 700–1000 response units. 3D8 scFv (5–200 nM), VH (0.2–50 µM), and VL (1.6–200 µM) prepared by serial dilutions with HES were injected over the flow cells at 50 µl/min for 3 min, followed by a constant flow of HES buffer at 50 µl/min for 3 min to observe dissociation of bound proteins. BSA (100 µM) and monoclonal anti-c-Myc 9e10 antibody (100 µM) (Sigma) were used as negative controls. At the end of each cycle, bound proteins were removed by injection of 50 mM NaOH containing 1 M NaCl for 60 s each to regenerate the chip. Comparison between sensorgrams was carried out by subtracting the responses in the control flow cell. All kinetic parameters were determined by nonlinear regression analysis according to a 1:1 binding model using the BIAevaluation version 3.2 software provided by manufacturer (25). The dissociation constant KD was calculated using the formula KD = koff/kon, where koff and kon are the dissociation and association rate constants, respectively.

DNA Hydrolyzing Assay by Agarose Gel Electrophoresis—The supercoiled plasmids of M13mp18 and pUC19 used as substrates were purified by a plasmid miniprep kit (Intron Inc., Korea). More than 95% of the isolated plasmid DNAs existed as the supercoiled form, judged by 0.8% agarose gel electrophoresis. DNA hydrolyzing experiments were initiated by mixing proteins (scFv (0.06–0.8 µM), Fv (5 µM), VH (5 µM), and VL (5 µM)) with the substrates (2.2 nM) in TBS containing 2 mM MgCl2. If necessary, 2 mM MgCl2 was replaced with 2–10 mM CaCl2, 2–10 mM MnCl2, or 50 mM EDTA in the buffer, which was specified "Results." In all cases, the total ionic strength was maintained at 150 mM by adjusting NaCl concentrations in the TBS buffer. After reactions were performed at 37 °C for 1 or 12 h and then terminated by incubating with trypsin protease (20 µg/ml) (Sigma) for 1 h at 37°C, samples were analyzed on 0.8% agarose gels by electrophoresis. The agarose gels were stained with ethidium bromide.

DNA Hydrolyzing Assay by Affinity-linked Oligonucleotide Nuclease Assay (ALONA)—ALONA was performed by following the protocol of Mouratou et al. (22). As substrates, 5'-digoxigenin and 3'-biotin-labeled oligodeoxynucleotides, ss-(dT)40, ds-(dT:dA)40, ss-(dN)40, ds-(dN:dN')40, ss-(dG-dC)20, and ds-(dG-dC:dC-dG)20, were used. Briefly, after immobilizing the labeled substrates to the streptavidin-coated microplate and subsequent washing (3 times) with TBST, each protein of 3D8 scFv (0.8 µM), Fv (5 µM), VH (5 µM), and VL (5 µM) was incubated for 10 h at 37 °C in TBS containing either 2 mM MgCl2 or 50 mM EDTA. After washing (3 times) with TBS, uncleaved labeled substrates were detected using anti-digoxigenin Fab conjugated to alkaline phosphatase (Hoffman-Roche Inc.) followed by incubation with p-nitrophenyl phosphate substrate and subsequent reading absorbance at 405 nm (22). As positive controls, bovine pancreatic DNase I (New England Biolabs) and S1 nuclease (New England Biolabs) were used in the buffers provided by the manufacturer.

Protein Crystallization and Structure Determination—The purified 3D8 scFv protein was concentrated to 10 mg/ml in TBS buffer. Initial screening for crystallization with a sparse matrix sampling method (26) was performed at 18 °C using a hanging drop vapor-diffusion method by mixing 1 µl of the protein with an equal volume of reservoir solution. Hexagonal-shaped crystals were obtained within 3 days in 2 M lithium sulfate and 0.1 M HEPES, pH 7.0, with a maximum dimension of 0.2 x 0.2 x 0.5 mm3. Crystals belonged to space group P6122 with unit cell dimensions of a = b = 179.74 Å, c = 184.28 Å, {alpha} = beta = 90.0°, {gamma} = 120.0°, and the asymmetric unit might contain six to two molecules with 40% to more than 80% of solvent content. Before cryocooling, crystals were briefly immersed in the reservoir solution containing 5–10% 2,4-methylpentanediol as a cryoprotectant. Diffraction data were collected at –173 °C at the HFMX4A at the Pohang Light Source (PLS at POSTECH, Korea), processed, merged, and scaled using the HKL2000 package (27). The phasing problem was solved by the molecular replacement program Phaser-1.3 (28) using the Phage Library-derived scFv Fragment (Protein Data Bank code 1DZB) (29). The model was built using the program O (30) and refined with the CNS package (31).

Site-directed Mutagenesis—Oligodeoxynucleotides were synthesized corresponding to the region covering two His residues (His-35 on VH and His-94 on VL in Kabat numbering). Site-specific mutants were generated by the QuikChange site-directed mutagenesis kit (Stratagene) and confirmed by sequencing.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression and Purification of the Recombinant Proteins—Each gene of 3D8 scFv, VH, and VL subcloned into pIg20 vector was expressed well (usually >0.5–1 mg from l-liter cultures) and purified from bacterial culture supernatants, as described under "Experimental Procedures." When analyzed by reducing SDS-PAGE, the proteins of 3D8 scFv, VH, and VL were migrated as single bands at the expected positions corresponding to each calculated molecular mass, i.e. 28 kDa for scFv, 15 kDa for VH, and 14 kDa for VL (Fig. 1).


Figure 1
View larger version (66K):
[in this window]
[in a new window]
  		FIGURE 1.
Reducing SDS-PAGE analysis of the purified 3D8 sbFv, VH, and VL Abs. About 2 µg of each protein was analyzed on 12% SDS-PAGE, which was stained with Coomassie Blue. The molecular mass markers are indicated in kDa.

 


Figure 2
View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 2.
Association of VH and VL. A, SEC elution profiles of 3D8 scFv (20µM, solid line), VH (20 µM, dashed line), VL (20 µM, dash-dotted line), and Fv (5 µM, dotted line) monitored at 280 nm. The arrows indicate the retention time of molecular size markers, i.e. ovalbumin (45 kDa), chymotrypsinogen A (23.2 kDa), and ribonuclease A (13.7 kDa). B, determination of the interactions between VH and VL by SPR at 25 °C. SPR sensograms were obtained from injections of VH at 200, 100, 50, 25, and 12.5 nM (from top to bottom) over a VL-immobilized surface. The injection of BSA (100 µM) gave no specific responses as shown by dashed lines.

 
Protein-Protein Interactions—To determine the size distribution of proteins under non-denaturing conditions, the proteins of scFv, VH, and VL were analyzed by SEC. SEC elution profile of each protein showed single peaks that correspond to an apparent molecular mass of each protein, demonstrating that the proteins were monomeric in solution at micromolar concentrations (Fig. 2A). When VH and VL (each 5 µM) were mixed at the equimolar ratio, they migrated as a single peak corresponding to an apparent molecular mass of ~26 kDa, which is close to the sum of molecular mass of VH and VL (Fig. 2A). This demonstrated that VH and VL spontaneously assembled to form Fv (VH: VL) in solution by noncovalent interactions.

The association affinity between VH and VL was quantified by SPR techniques at 25 °C (Fig. 2B). When the VH domain was flowed over a VL-immobilized surface chip, the apparent dissociation constant (KD) was 24 ± 4 nM with an association rate constant (kon) of 2.60 ± 0.11 x 105 M–1 s–1 and a dissociation rate constant (koff) of 6.19 ± 0.09 x 10–3 s–1. In a reciprocal experiment, where the VL domain was injected into a VH-immobilized surface chip, the KD was 14 ± 2nM with kon = 4.35 ± 0.27 x 105 M–1 s–1 and koff = 6.16 ± 0.50 x 10–3 s–1. No homomeric interactions of 3D8 scFv, VH, and VL were detected up to 50 µM concentration by SPR (data not shown), which was in good agreement with the SEC data.

DNA Binding Activities—Our previous ELISA result has shown that 3D8 IgG has a DNA binding activity to ds-poly(dT:dA) and ds-poly(dG: dC) (21). In the present study, we have employed SPR techniques to quantify the interactions of 3D8 scFv, VH, and VL with the various synthetic 40-bp oligodeoxynucleotides, including ss-(dT)40, ds-(dT: dA)40, ss-(dN)40, ds-(dN:dN')40, ss-(dG-dC)20, and ds-(dG-dC:dC-dG)20 (Table 1). 3D8 scFv, VH, and VL efficiently bound to all kinds of the substrates in a concentration-dependent manner, regardless of ss- and dsDNA forms and their specific sequences (Table 1). For 3D8 scFv, the kinetic binding parameters of kon and koff values ranged ~5.27–15.5 x 104 M–1 s–1 and ~1.98–4.85 x 10–3 s–1, respectively, resulting in KD values of ~17–74 nM (Table 1), which were within the ranges of those observed for other anti-DNA IgG, Fab, and scFv Abs (3–5, 13, 32). The binding affinity of 3D8 scFv against the ds form of each sequence was slightly higher than that for the corresponding ss form, mainly because of the slightly faster association kinetics (Table 1). Compared with 3D8 scFv, 3D8 VH bound less tightly with ~100-fold lower affinities for both ds- and ssDNA substrates. Contrary to 3D8 scFv, however, 3D8 VH exhibited slightly higher affinities with ssDNA forms (KD = ~2.4–7.5 µM) of each sequence than the corresponding dsDNA forms (KD = ~4.7–8.4 µM), mainly because of faster association kinetics (Table 1). The affinity of 3D8 VL (KD =~3.2–72 µM) for the substrates was ~103- and 10-fold lower than those of 3D8 scFv and VH, respectively. Unlikely with 3D8 scFv and VH, 3D8 VL exhibited a preferential binding for ds-(dT:dA)40 (KD =~3.2 µM) over the other substrates. For negative controls, BSA (100 µM) and anti-c-Myc 9e10 mAb (100 µM) gave no responses when passed over the DNA-immobilized surfaces (data not shown).


View this table:
[in this window]
[in a new window]
  		TABLE 1
Kinetic binding parameters for the interactions of 3D8 scFv, VH, and VL Abs with various synthetic oligodeoxynucleotides as substrates, which were determined by SPR

Each value represents the mean ± S.D. of two independent experiments. In each experiment, at least 5 data sets were used in the determination of the kinetic constants.

 
Effects of Ionic Strength on DNA Binding Activities—Effects of ionic strengths on the DNA binding activities of the proteins were determined by ELISA as a function of NaCl concentration (0–0.8 M NaCl) using synthetic ds-(N:N')40 and ss-(dN)40 as substrates. Binding capacities of 3D8 scFv to both ds- and ssDNA were not significantly affected by up to 0.2 M NaCl, but decreased by ~50% at and above 0.4 M NaCl (Fig. 3). Compared with 3D8 scFv, binding activities of 3D8 VH and VL for both ds- and ssDNA substrates were much more sensitive to ionic strengths, showing dramatic inhibitions by ~60–90% in the presence of NaCl at concentrations above 0.2 M, except that VL binding to ds-(dN: dN')40 showed similar magnitudes to that of 3D8 scFv up to 0.4 M NaCl.


Figure 3
View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 3.
Effects of ionic strength on DNA binding activities of 3D8 scFv, VH, and VL measured by ELISA at 25 °C. The substrates used were ss-(dN)40 (open symbols) and ds-(dN:dN')40 (closed symbols). The symbols represent: scFv ({circ}), VH ({triangledown} and {blacktriangledown}), and VL ({square} and {blacksquare}). The relative bound fraction was calculated by comparison with the absorbance at 405 nm in 50 mM Tris-Cl buffer, pH 7.5, in the absence of NaCl, after subtracting the absorbance for the buffer control. The error bars represent the standard deviation for triplicate experiments.

 
DNA Hydrolyzing Activities—Some anti-DNA Abs derived from the sera of human and mice with autoimmune diseases have shown DNase activities to hydrolyze the phosphodiester bond of DNA (14, 15). Thus we investigated whether or not 3D8 scFv, VH, VL, and Fv (VH:VL) exhibit DNase activities. The supercoiled plasmid of M13mp18 (~2.2 nM) was incubated with the proteins at 37 °C for 1 or 12 h in the TBS containing 2 mM MgCl2 or 50 mM EDTA. Then reaction mixtures were analyzed by electrophoresis on agarose gels, which were stained with ethidium bromide. 3D8 scFv at 0.8 µM almost completely hydrolyzed the substrate in the presence of Mg2+ showing the trace of degraded fragments in a 1-h incubation (Fig. 4A). For the same period, Fv (5 µM), VH (5 µM), and VL (5 µM) showed DNA nicking activities that were monitored by the appearance of linear and relaxed circular forms of the plasmid with a concomitant decrease in the quantity of the supercoiled plasmid DNA. When 3D8 scFv ranging in concentrations from 0.06 to 0.3 µM with 0.04 µM intervals was incubated with the substrate under the same condition (37 °C for 1 h), 0.1 µM 3D8 scFv generated mainly linear and relaxed circular forms of the plasmid without significant degraded, smearing bands (data not shown). Thus 3D8 scFv possessed ~50-fold greater catalytic activity with the supercoiled plasmid, compared with Fv, VH, and VL. Prolonged incubation (12 h) of the plasmid with the proteins resulted in further plasmid DNA hydrolysis producing the smear bands below the original size of the plasmid (Fig. 4B). In the presence of EDTA, all of the proteins did not show any DNA hydrolyzing activities. The same hydrolyzing profiles were observed for all of the proteins when the supercoiled pUC19 plasmid was used as a substrate (data not shown).


Figure 4
View larger version (71K):
[in this window]
[in a new window]
  		FIGURE 4.
DNA hydrolyzing activities of the purified 3D8 scFv, Fv, VH, and VL for the plasmid of M13mp18 as a substrate monitored by agarose gels. The supercoiled plasmid of M13mp18 (~2.2 nM) was incubated with 3D8 scFv (0.8 µM), Fv (5 µM), VH (5 µM), and VL (5 µM) for 1 h at 37°C (A) and 12 h (B) in the TBS buffer, pH 7.5, containing 2 mM MgCl2 (indicated as "Mg") or 50 mM EDTA (indicated as "E"). The reaction mixtures were analyzed by electrophoresis on agarose gels, then stained with ethidium bromide. The arrows indicate supercoiled (sc), linear (lin), and relaxed circular (rc) DNAs. The samples incubated with only buffer alone and molecular mass markers were designated as B and M, respectively.

 
Substrate Specificities—To determine substrate specificities of the DNase activity, the proteins were incubated with a variety of synthetic 40-bp oligodeoxynucleotides, including ss-(dT)40, ds-(dT:dA)40, ss-(dN)40, ds-(dN:dN')40, and ds-(dG-dC:dC-dG)20. DNase activity was monitored by ALONA, in which hydrolytic activities of proteins were inversely proportional to the absorbance at 405 nm (22). For positive controls, bovine pancreatic DNase I and S1 nuclease cleaved all of the substrates with an exception of DNase I, which showed only weak activity for ss-(dT)40 (Fig. 5). This observation was in good agreement with the previous result (22). In contrast to the negative control incubated with the buffer alone, 3D8 scFv, Fv, VH, and VL efficiently cleaved the synthetic DNAs in the presence of Mg2+ (Fig. 5), with an exception of 3D8 scFv for ss-(dT)40, for which it showed only weak activity, like DNase I. The metal-chelating agent EDTA (50 mM) completely inhibited the DNase activities for all of the proteins (data not shown), consistent with the observation for the supercoiled plasmids on agarose gels.

Metal Dependence for the Catalytic Activities—The kind of bivalent metal ions in most DNases is important for the catalytic activities (33). To determine whether Ca2+ and Mn2+ can replace Mg2+, 3D8 scFv, VH, and VL were incubated with ss-(dN)40 and ds-(dN:dN')40 as substrates at 37 °C for 12 h in the TBS containing CaCl2 (2–10 mM) and MnCl2 (2–10 mM). Their catalytic activities were monitored by ALONA. Compared with those in the presence of Mg2+, the catalytic activities of 3D8 scFv, Fv, VH, and VL were about 60–70% in the presence of Mn2+ in a concentration-independent manner. This behavior is consistent with bovine pancreatic DNase I (33). Unlikely with DNase I (33), however, the catalytic activities of 3D8 scFv, Fv, VH, and VL were not observed at all in the presence of Ca2+ (data not shown).


Figure 5
View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 5.
Substrate specificities of 3D8 scFv, Fv, VH, and VL determined by ALONA. As described in detail under "Experimental Procedures," the DNA hydrolyzing activity was represented by the relative absorbance at 405 nm compared with the just buffer control and was inversely proportional to absorbance at 405 nm. The numbers below each column represent the substrate as follows: 1, ds-(dN:dN')40; 2, ss-(dN)40; 3, ds-(dT: dA)40; 4, ss-(dT)40; 5, ds-(dG-dC:dC-dG)20. The proteins of 3D8 scFv (0.8 µM), Fv (5 µM), VH (5 µM), and VL (5 µM) were incubated with the substrates for 10 h at 37 °C in the TBS, pH 7.5, containing 2 mM MgCl2. Reactions for DNase I and S1 nuclease were carried out in the buffers recommended by the manufacturers. The error bars represent the standard deviation for triplicate experiments.

 
Crystal Structure of 3D8 scFv—We determined the tertiary structure of 3D8 scFv at 2.88-Å resolution with Rfac of 21.2% and Rfree of 24.2% (10% random data set) (Fig. 6A). The flexible linker of (G4S)3 to connect VH and VL domains could not be traced because of the weak electron density. The refined model showed that only two scFv molecules are located in the asymmetric unit, where the VH domain of one molecule interacts with the VL domain of a neighboring molecule to complete a presumably active scFv molecule (Fig. 6A). The other free VL and VH domains interact with the respective VH and VL domains of crystallographically related molecules.

To get an insight for the DNA binding and hydrolyzing mechanisms of 3D8 scFv, the elucidated structure was compared with the known complex structures of anti-DNA Fab Abs and ss-(dT)3, BV04–01/ss-(dT)3 (34), and DNA-1/ss-(dT)3 (35). 3D8 scFv superposed well with the complex structures at the central beta-sandwich regions, but exhibited large deviations at the hypervariable complementary determining regions (CDRs) with root mean square deviations of 1.7 Å for DNA-1 (Fig. 6B) and 3.8 Å for BV04-01 (data not shown). In the complex structures, the substrate binding pockets are at the interface of the VH and VL domains, specifically VL-CDR1 and VH-CDR3 for BV04-01/ss-(dT)3 and VL-CDR3 and VH-CDR3 for DNA-1/ss-(dT)3 (34, 35). Careful comparisons of the superposed structures revealed that the putative DNA binding cleft of 3D8 scFv might be more similar to that of DNA-1 than BV04-01 in that the possible DNA base stacking environments of 3D8 scFv were closer to DNA-1, along with the higher sequence homology of 3D8 scFv with DNA-1 (72%) than BV04-01 (49%). The critical base stacking Tyr residues of H100, H100a, L32, and L49 in the DNA-1/ss-(dT)3 complex were replaced by Tyr residues of H97, H100a, L32, and L49 in the putative DNA binding site of 3D8 scFv (Fig. 6B). Likewise, two His residues corresponding to H35 on VH-CDR1 and L91 on VL-CDR3 in DNA-1 Fab, located around the potential DNA binding pocket, were also conserved in H35 on VH-CDR1 (His-H35) and L94 on VL-CDR3 (His-L94) in the 3D8 scFv structure (Fig. 6B).


Figure 6
View larger version (49K):
[in this window]
[in a new window]
  		FIGURE 6.
Crystal structure of 3D8 scFv. A, two molecules in the asymmetric unit were colored in margenta and cyan, respectively. The aromatic residues at the contacting area and His residues were displayed as stick models. B, comparison of the putative DNA binding regions of 3D8 scFv with DNA-1/dT3. In B, the bound deoxynucleotides are represented as stick models and key residues as lines.

 
Site-directed Mutagenesis Studies—His residues frequently constitute the key active sites of many DNases and DNA-abzymes (8, 36). 3D8 scFv has only two His residues, His-H35 and His-L94. His-L94 was completely exposed toward the solvent region, whereas His-H35 was slightly buried in the interface of VH and VL (Fig. 6A). To address the possible role of the His residues of 3D8 scFv in catalytic activity, we constructed mutants with His-H35 mutation to Ala (His-H35 -> Ala) (designated as "VH*-VL"), His-L94 mutation to Ala (His-L94 -> Ala) (VH-VL*), and double mutations on both variable domains (VH*-VL*) and investigated their binding and catalytic activities. All of the mutants showed comparable binding activities to the wild type for both ss-(dN)40 and ds-(dN:dN')40 substrates (Fig. 7A). For the catalytic activity, the VH*-VL mutant efficiently degraded the substrate plasmid with comparable activity to that of the wild type, whereas VH-VL* and VH*-VL* mutants exhibited a substantial decrease in the hydrolyzing activities for the same period (Fig. 7B). Judged from the residual amount of the substrate, the order of hydrolyzing activities of scFv was wild type > VH*-VL > VH-VL* > VH*-VL*, which was also confirmed by ALONA with ss-(dN)40 and ds-(dN:dN')40 as substrates (data not shown). In the presence of EDTA, all of the mutants did not show any DNase activities (Fig. 7B).


Figure 7
View larger version (58K):
[in this window]
[in a new window]
  		FIGURE 7.
Effects of His mutations of 3D8 scFv on DNA binding activities assayed by ELISA (A) and DNA hydrolyzing activities monitored by agarose gel electrophoresis (B). In A, black and white bars designate ds-(dN:dN')40 and ss-(dN)40, respectively. The error bars represent the standard deviation for triplicate experiments. In B, 3D8 scFv wild type (0.8 µM) and mutants (0.8 µM) were incubated for 12 h at 37 °C with the supercoiled plasmid of M13mp18 (~2.2 nM) in the TBS buffer, pH 7.5, containing 2 mM MgCl2 (indicated as Mg) or 50 mM EDTA (indicated as E). VH*-VL, VH-VL*, and VH*-VL* designate the His-H35 -> Ala mutation on VH-CDR1, His-L94 -> Ala mutation on VL-CDR3, and both mutations in 3D8 scFv, respectively. The arrows indicate supercoiled (sc), linear (lin), and relaxed circular (rc) DNAs. The samples incubated with only buffer alone and molecular mass markers are designated as B and M, respectively.

 
Like the scFv mutants, the binding activities of single domain mutants, VH* (His-H35 -> Ala) and VL* (His-L94 -> Ala), were not affected significantly for both ss-(dN)40 and ds-(dN:dN')40 substrates (Fig. 8A). For the catalytic activity, strikingly enough, VH*, VL*, and Fv* (VH*: VL*) efficiently hydrolyzed the substrate in the presence of Mg2+ (Fig. 8B). Even VL* and Fv* apparently showed faster hydrolyzing activities compared with each wild type in both 1-(data not shown) and 12-h incubations (Fig. 8B). The presence of EDTA completely inhibited the DNA hydrolyzing activities of VH*, VL*, and Fv* (VH*:VL*).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
DNA Binding Activities of the Proteins—3D8 scFv bound to both ss- and dsDNAs of various sequences with KD values of ~17–74 nM. The KD values are within similar ranges of those for other anti-DNA Abs: ~76 nM for dC7 scFv binding to ss-(dC)65 (3); ~5–10 nM for DNA-1 Fab binding to ss-(dT)15 (4); ~16 nM for Z22 Fab binding to ds-(dG:dC)30 (13); and ~18 nM for V-88 IgG binding to ds-(dG:dC)25 (5). Single domains of 3D8 VH (KD =~2.4–8.4 µM) and VL (KD =~3.2–72 µM) exhibited about ~102- and 103-fold lower affinities than those of 3D8 scFv for each ss- and dsDNA substrates (Table 1), respectively, suggesting that the pairing of VH and VL cooperatively contributed to the high affinity of 3D8 scFv with the substrates. Previously, the dC7 VH domain (KD =~0.7 µM) showed about 10-fold lower affinity for ss-(dC)65 than its scFv form (3), but the Z22 VH domain bound to ss-Z-DNA with similar affinity (KD = ~17 nM) to that of the Fab (13). SEC and SPR analyses showed no homomeric interactions of 3D8 scFv, VH, and VL up to 50 µM concentrations (Fig. 2), suggesting that the anti-DNA Abs bind to DNA substrates most likely in the monomeric form. On the other hand, a VH domain spontaneously formed the homodimer, the formation of which was essential for the specific binding to dsDNAs with the CTGC motif (12).


Figure 8
View larger version (71K):
[in this window]
[in a new window]
  		FIGURE 8.
Effects of His mutations of VH and VL on DNA binding activities assayed by ELISA (A) and DNA hydrolyzing activities monitored by agarose gel electrophoresis (B). In A, black and white bars designate ds-(dN:dN')40 and ss-(dN)40, respectively. The error bars represent the standard deviation for triplicate experiments. In B, wild type (5 µM) and mutants (5 µM) of VH and VL were incubated for 12 h at 37 °C with the supercoiled plasmid of M13mp18 (~2.2 nM) in the TBS buffer, pH 7.5, containing 2 mM MgCl2 (indicated as Mg) or 50 mM EDTA (indicated as E). VH* and VL* designate the His-H35 -> Ala mutation on VH-CDR1 and His-L94 -> Ala mutation on VL-CDR3, respectively. The arrows indicate supercoiled (sc), linear (lin), and relaxed circular (rc) DNAs. The molecular mass marker was designated as M.

 
For anti-DNA Abs, VH domain played a more dominant role in DNA binding activity than VL domains (2, 15, 37). For example, the VH domain of mAb Z22 (13), dC7 (3), and 2C10 (7) bound to ds-Z-DNA, ss-(dC)65, and ds-DNA, respectively, whereas their respective VL domains alone did not bind to the substrates. Thus it has been proposed that VL alone could not bind to DNA, but instead modulates fine specificities and affinities of VH binding to DNA (2, 3, 7, 15, 38). However, our results demonstrated that 3D8 VL alone bound to ss- and dsDNAs of various sequences. It should be noted here that, to our knowledge, no VL domain alone has been reported to possess DNA binding activity.

Most anti-DNA Abs against either ss- or dsDNA substrates have shown preferential binding to a particular sequence by more than 10-fold affinity over other sequences (3–6). However, the substrate recognition patterns of 3D8 scFv, VH, and VL did not exhibit significant preferential bindings to any particular sequences in either ss or ds forms (Table 1). Exceptionally, 3D8 VL showed about 10-fold higher affinity to ds-(dT:dA)40 (KD = ~3.2 µM) over the other substrates tested. With this minor exception, it appears that the binding of 3D8 scFv, VH, and VL to both ss- and dsDNAs are sequence nonspecific. Furthermore, the appearance of smearing bands from the digestions of the supercoiled plasmids by the proteins (Figs. 4, 7B, and 8B), without certain degraded band patterns, also strongly supported that all of the proteins did not have any sequence specificity.

Many positively charged residues of Lys and Arg in CDR regions facilitate for anti-DNA Abs to interact with the phosphate backbone of DNA by compensating the negative charge of DNA molecules (1, 2, 38). Similarly, 3D8 scFv (pI = 9.15), VH (pI = 8.80), and VL (pI = 9.12) are basic proteins with many basic residues in the VH-CDRs and VL-CDRs. The binding activities of 3D8 scFv, VH, and VL dramatically decreased in parallel with ionic strength (Fig. 3), particularly for VH and VL domains, like other anti-DNA Abs (3, 4, 39). Therefore, the electrostatic interactions between basic residues of the proteins and phosphate backbones of DNA substrates might be a main stabilizing force, which is also supported by the protein bindings to both ss- and dsDNA substrates regardless of their specific sequences (Table 1). However, even at the highest ionic strength (0.8 M NaCl), 3D8 scFv, VH, and VL showed residual binding activities of ~50, 10, and 20% with the substrates (Fig. 3), respectively. Thus, additional interactions should be provided for the protein bindings to DNA substrates, which might modulate fine substrate recognition and specificity of the proteins. For example, sequence-specific ds-DNA binding Abs (11) and sequence nonspecific anti-ss/ds-DNA binding Abs (39) exhibited salt-independent binding activities. As stated earlier, the superposition of the 3D8 scFv structure with the known complex structure of BV04-01/ss-(dT)3 and DNA-1/ss-(dT)3 (34, 35) concluded that the putative DNA-binding environment of 3D8 scFv was much closer to that of DNA-1/ss-(dT)3 (Fig. 6B). The complex structure of DNA-1/ss-(dT)3 showed that the anti-DNA Ab binds the substrate primarily by sandwiching thymine bases between Tyr residues (Fig. 6B). The 3D8 scFv structure also has many aromatic residues around the putative substrate binding pockets, such as Tyr-H97, Tyr-H100a, and Tyr-H102 from VH-CDR3 and Tyr-L32, Tyr-L49, Trp-L50, Tyr-L92, Tyr-L93, and Tyr-L96 from VL-CDRs, including Tyr residues intriguingly at the exact same position as the DNA-1 structure (Fig. 6B). The possible base stacking interactions could be attributed to the residual activities of 3D8 scFv, VH, and VL even in the high ionic strength environments.

DNA Hydrolyzing Activities of the Proteins—3D8 scFv, Fv, VH, and VL efficiently hydrolyzed both ss- and dsDNAs without significant preference to a particular sequence, with an exception that 3D8 scFv exhibited poor hydrolyzing activity only for ss-(dT)40 (Fig. 5). The proteins also required the divalent metal ion of Mg2+ for their full catalytic activities, like other DNases and DNA-abzymes (8, 14, 15, 17). Exceptionally, an IgG from human milk exhibited metal-independent DNA hydrolyzing activity (20).

As stated earlier, SPR and SEC analyses showed no homomeric interactions of 3D8 scFv, VH, and VL up to 50 µM (Fig. 2), suggesting that the catalytic unit of the proteins is most likely the monomeric form, at least in the used protein concentrations (≤5 µM). The crystal structure of 3D8 scFv exhibited two molecules in the asymmetric unit, where the intermolecular pairing of VH and VL formed a putative active scFv molecule and the other VH and VL remained unpaired (Fig. 6A). Considering no homomeric interactions of 3D8 scFv up to 50 µM in solution (Fig. 2), the observed dimeric state in the crystal structure might be caused by highly concentrated proteins (~350 µM) used for crystallization or by the crystallization condition. However, the interface and interaction between VH and VL observed in the crystalline state might be considered to mimic those of a real active monomeric scFv, which was partially supported by our structure-based mutational studies (Fig. 7). Similar interactions between VH and VL of adjacent molecules in the asymmetric unit were observed in BV04-01 and DNA-1 (34, 35).

Previous studies of the subunits of IgG and IgA from mice or human, such as Fab, heavy and light chains, have shown that catalytic residues were preferentially positioned in light chains with no detectable catalytic activities on heavy chains, whereas DNA binding active sites were dominantly associated with heavy chains (15, 17, 18, 20, 40). However, both 3D8 VH and VL showed both DNA binding and hydrolyzing activities. 3D8 VH bound more tightly with ~10-fold higher affinities for the substrates than VL, except for only one substrate of ds-(dT:dA)40 (Table 1), suggesting that the DNA binding activity of 3D8 scFv is dominantly associated with its VH. 3D8 VH exhibited comparable hydrolyzing activities for the linear oligodeoxynucleotides (Fig. 5), but much poor hydrolyzing activities in the kinetic studies with the supercoiled plasmids, compared with those of VL (Figs. 4 and 8). Taken together, the relative catalytic activities of VH and VL apparently varied depending on whether the substrate was linear or supercoiled forms and did not strictly correlate with their substrate binding affinities (Table 1 and Fig. 5). It should be noted here that, to our knowledge, this is the first demonstration for single domain abzymes with DNase activities. For other abzymes, there was only one report of the single domain VL with protease activity (41).

Structural and mutational studies of many DNases have revealed that DNA cleavage occurs via the acid-base catalytic mechanism for the cleavage of phosphodiester bonds, where two His residues play important roles; one as a proton donor to a water molecule that acts as a nucleophile attacking the phosphate linkage with the help of bivalent metal ions and the other as a proton acceptor from the leaving oxygen anion (36, 42). 3D8 scFv protein has only two His residues over the full amino acid sequence and they are located at the domain interface in the crystal structure. More specifically, His-H35 of VH-CDR1 is hidden at the deep cleft and His-L94 of VL-CDR3 is exposed to the solvent accessible region without forming any interactions with the other protein residues (Fig. 6A). The catalytic activities of 3D8 scFv His mutants, i.e. VH*-VL, VH-VL*, and VH*-VL*, were significantly inhibited, but their substrate binding activities were not affected at all, compared with those of 3D8 scFv wild type (Fig. 7). This result strongly suggests that both residues participated at least partially in the catalytic mechanism of 3D8 scFv. However, the catalytic activity of scFv VH-VL* (His-L94 -> Ala) was much more significantly inhibited than scFv VH*-VL (His-H35 -> Ala) (Fig. 7), indicating that His-L94 is a key residue for the catalytic mechanism of 3D8 scFv. His-L94 could be rearranged by flipping the VL-CDR3 loop to be located nearer the phosphodiester bond of DNA for the catalysis when a DNA substrate binds to the active site (Fig. 6B). Similar reorientations of the hypervariable loops of the ligand-free BV04-01 and DNA-1 structures were observed upon DNA binding (34, 35).

Contrary to 3D8 scFv His mutants, DNA hydrolyzing activities of single domain of 3D8 VH* (His-H35 -> Ala) and VL* (His-L94 -> Ala) were not affected by the mutations (Fig. 8), indicating that the enzymatic mechanisms of 3D8 VH and VL are different from that of 3D8 scFv. Nonetheless, the His mutants of VH* and VL* showed Mg2+ dependence for the enzymatic activities, suggesting that the metal coordination is an essential constituent for their catalytic mechanism(s), like wild types. Structural information of each protein-DNA complex and subsequent mutational studies will elucidate the detailed catalytic mechanism for each protein.

3D8 scFv showed much higher binding affinities (Table 1) and faster hydrolyzing activities even at ~6-fold lower concentrations than the VH and VL (Figs. 4 and 7), suggesting that both VL and VH may be cooperatively involved in the recognition of the substrates and relevant organization of the catalytic sites in the scFv form. VH and VL spontaneously assembled each other to form Fv with KD values of ~14–24 nM. Previously the VH-VL associations of anti-Z-DNA mAb Z22 exhibited a similar KD value of ~54.7 nM measured by SPR (13). 3D8 Fv showed slightly slower hydrolyzing activities for the supercoiled plasmid (Fig. 4), but more efficient cleavage over the ss-(dT)40 (Fig. 5), compared with 3D8 scFv. Furthermore, contrary to the scFv VH*-VL* mutant, the catalytic activity of Fv* (VH*:VL*) was not inhibited at all by the His mutations (Fig. 8). These observations suggest that relative orientations of the two domains might be different between Fv and scFv.

In summary, here we have extensively characterized DNA-abzymes, 3D8 scFv, VH, and VL, for their substrate binding kinetics and catalytic activities, including the determination of the crystal structure of 3D8 scFv. Our results showed that 3D8 VH and VL domains alone bound to and hydrolyzed both ss- and dsDNA substrates in the presence of Mg2+ without significant sequence specificities, demonstrating a new source of single domain DNA-abzymes. Subsequent structural and functional studies of the abzymes should provide deep insights into their catalytic mechanisms and pathogenic origins of the autoimmune diseases.


   FOOTNOTES
 
* 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/). Back

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.

4 The abbreviations used are: Abs, antibodies; mAbs, monoclonal antibodies; Fab, antigen binding fragment; scFv, single chain variable fragment; Fv, variable fragment; VH, heavy chain variable domain; VL, light chain variable domain; CDR, complementarity determining region; SPR, surface plasmon resonance; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; SPA, Staphylococcal protein A; SEC, size exclusion chromatography; ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin; ALONA, affinity-linked oligonucleotide nuclease assay. Back


   ACKNOWLEDGMENTS
 
We thank Prof. D. H. Kim, Ajou University, for use of the high performance liquid chromatography system and K. W. Kim and H. S. Lee, Pohang Light Source, for helping with data collection.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Marion, T. N., Krishnan, M. R., Desai, D. D., Jou, N. T., and Tillman, D. M. (1997) Methods 11, 3–11[CrossRef][Medline] [Order article via Infotrieve]
  2. Jang, Y. J., and Stollar, B. D. (2003) Cell Mol. Life Sci. 60, 309–320[CrossRef][Medline] [Order article via Infotrieve]
  3. O'Connor, K. C., Nguyen, K., and Stollar, B. D. (2001) J. Mol. Recognit. 14, 18–28[CrossRef][Medline] [Order article via Infotrieve]
  4. Komissarov, A. A., and Deutscher, S. L. (1999) Biochemistry 38, 14631–14637[CrossRef][Medline] [Order article via Infotrieve]
  5. Eivazova, E. R., McDonnell, J. M., Sutton, B. J., and Staines, N. A. (2000) Immunology 101, 371–377[CrossRef][Medline] [Order article via Infotrieve]
  6. Kubota, T., Akatsuka, T., and Kanai, Y. (1986) Immunol. Lett. 14, 53–58[CrossRef][Medline] [Order article via Infotrieve]
  7. Jang, Y. J., and Sanford, D. (2001) Mol. Immunol. 38, 383–387[CrossRef][Medline] [Order article via Infotrieve]
  8. Gololobov, G. V., Rumbley, C. A., Rumbley, J. N., Schourov, D. V., Makarevich, O. I., Gabibov, A. G., Voss, E. W., Jr., and Rodkey, L. S. (1997) Mol. Immunol. 34, 1083–1093[CrossRef][Medline] [Order article via Infotrieve]
  9. Cleary, J., and Glick, G. D. (2003) Biochemistry 42, 30–41[CrossRef][Medline] [Order article via Infotrieve]
  10. Stevens, S. Y., and Glick, G. D. (1999) Biochemistry 38, 560–568[CrossRef][Medline] [Order article via Infotrieve]
  11. Di Pietro, S. M., Centeno, J. M., Cerutti, M. L., Lodeiro, M. F., Ferreiro, D. U., Alonso, L. G., Schwarz, F. P., Goldbaum, F. A., and de Prat-Gay, G. (2003) Biochemistry 42, 6218–6227[CrossRef][Medline] [Order article via Infotrieve]
  12. Jin, H., Sepulveda, J., and Burrone, O. R. (2004) Protein Sci. 13, 3222–3229[CrossRef][Medline] [Order article via Infotrieve]
  13. Polymenis, M., and Stollar, B. D. (1995) J. Immunol. 154, 2198–2208[Abstract]
  14. Shuster, A. M., Gololobov, G. V., Kvashuk, O. A., Bogomolova, A. E., Smirnov, I. V., and Gabibov, A. G. (1992) Science 256, 665–667[Abstract/Free Full Text]
  15. Nevinsky, G. A., and Buneva, V. N. (2002) J. Immunol. Methods 269, 235–249[CrossRef][Medline] [Order article via Infotrieve]
  16. Nevinsky, G. A., and Buneva, V. N. (2005) Natural Catalytic Antibodies-Abzymes, John Wiley & Sons, Weinheim, Germany
  17. Baranovskii, A. G., Ershova, N. A., Buneva, V. N., Kanyshkova, T. G., Mogelnitskii, A. S., Doronin, B. M., Boiko, A. N., Gusev, E. I., Favorova, O. O., and Nevinsky, G. A. (2001) Immunol. Lett. 76, 163–167[CrossRef][Medline] [Order article via Infotrieve]
  18. Dubrovskaya, V. V., Andryushkova, A. S., Kuznetsova, I. A., Toporkova, L. B., Buneva, V. N., Orlovskaya, I. A., and Nevinsky, G. A. (2003) Biochemistry (Mosc.) 68, 1081–1088[Medline] [Order article via Infotrieve]
  19. Gololobov, G. V., Chernova, E. A., Schourov, D. V., Smirnov, I. V., Kudelina, I. A., and Gabibov, A. G. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 254–257[Abstract/Free Full Text]
  20. Kanyshkova, T. G., Semenov, D. V., Khlimankov, D. Y., Buneva, V. N., and Nevinsky, G. A. (1997) FEBS Lett. 416, 23–26[Medline] [Order article via Infotrieve]
  21. Kwon, M. H., Lee, M. S., Kim, K. H., Park, S., Shin, H. J., Jang, Y. J., and Kim, H. I. (2002) Immunol. Investig. 31, 205–218[Medline] [Order article via Infotrieve]
  22. Mouratou, B., Rouyre, S., and Guesdon, J. L. (2002) J. Immunol. Methods 269, 147–155[CrossRef][Medline] [Order article via Infotrieve]
  23. Brigido, M. M., Polymenis, M., and Stollar, B. D. (1993) J. Immunol. 150, 469–479[Abstract]
  24. Chen, Y., and Stollar, B. D. (1999) J. Immunol. 162, 4663–4670[Abstract/Free Full Text]
  25. Lee, H. W., Lee, S. H., Lee, H. W., Ryu, Y. W., Kwon, M. H., and Kim, Y. S. (2005) Biochem. Biophys. Res. Commun. 330, 1205–1212[CrossRef][Medline] [Order article via Infotrieve]
  26. Jancarik, J., and Kim, S.-H. (1991) J. Appl. Crystallogr. 24, 409–411[CrossRef]
  27. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307–326[CrossRef]
  28. McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C., and Read, R. J. (2005) Acta Crystallogr. D Biol. Crystallogr. 61, 458–464[CrossRef][Medline] [Order article via Infotrieve]
  29. Ay, J., Keitel, T., Kuttner, G., Wessner, H., Scholz, C., Hahn, M., and Hohne, W. (2000) J. Mol. Biol. 301, 239–246[CrossRef][Medline] [Order article via Infotrieve]
  30. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, G. J. (1991) Acta Crystallogr. Sect. A 47, 110–119
  31. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. D Biol. Crystallogr. 54, 905–921[CrossRef][Medline] [Order article via Infotrieve]
  32. Cerutti, M. L., Centeno, J. M., Goldbaum, F. A., and de Prat-Gay, G. (2001) J. Biol. Chem. 276, 12769–12773[Abstract/Free Full Text]
  33. Junowicz, E., and Spencer, J. H. (1973) Biochim. Biophys. Acta 312, 72–84[Medline] [Order article via Infotrieve]
  34. Herron, J. N., He, X. M., Ballard, D. W., Blier, P. R., Pace, P. E., Bothwell, A. L., Voss, E. W., Jr., and Edmundson, A. B. (1991) Proteins 11, 159–175[CrossRef][Medline] [Order article via Infotrieve]
  35. Schuermann, J. P., Prewitt, S. P., Davies, C., Deutscher, S. L., and Tanner, J. J. (2005) J. Mol. Biol. 347, 965–978[CrossRef][Medline] [Order article via Infotrieve]
  36. Suck, D., and Oefner, C. (1986) Nature 321, 620–625[CrossRef][Medline] [Order article via Infotrieve]
  37. Barry, M. M., and Lee, J. S. (1993) Mol. Immunol. 30, 833–840[CrossRef][Medline] [Order article via Infotrieve]
  38. Radic, M. Z., Mackle, J., Erikson, J., Mol, C., Anderson, W. F., and Weigert, M. (1993) J. Immunol. 150, 4966–4977[Abstract]
  39. Kumar, S., Kalsi, J., Latchman, D. S., Pearl, L. H., and Isenberg, D. A. (2001) J. Mol. Biol. 308, 527–539[CrossRef][Medline] [Order article via Infotrieve]
  40. Nevinsky, G. A., Kanyshkova, T. G., Semenov, D. V., Vlassov, A. V., Gal'vita, A. V., and Buneva, V. N. (2000) Appl. Biochem. Biotechnol. 83, 115–129, 129–130, 145–153[CrossRef][Medline] [Order article via Infotrieve]
  41. Paul, S., Li, L., Kalaga, R., Wilkins-Stevens, P., Stevens, F. J., and Solomon, A. (1995) J. Biol. Chem. 270, 15257–15261[Abstract/Free Full Text]
  42. Clark, P., and Eichhorn, G. L. (1974) Biochemistry 13, 5098–5102[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Int ImmunolHome page
A. A. Andryushkova, I. A. Kuznetsova, I. A. Orlovskaya, V. N. Buneva, and G. A. Nevinsky
Nucleotide-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice
Int. Immunol., June 25, 2009; (2009) dxp060v1.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K.-J. Park, S.-H. Lee, T.-I. Kim, H.-W. Lee, C.-H. Lee, E.-H. Kim, J.-Y. Jang, K. S. Choi, M.-H. Kwon, and Y.-S. Kim
A Human scFv Antibody against TRAIL Receptor 2 Induces Autophagic Cell Death in Both TRAIL-Sensitive and TRAIL-Resistant Cancer Cells
Cancer Res., August 1, 2007; 67(15): 7327 - 7334.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/22/15287    most recent
M600937200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, Y.-R.
Right arrow Articles by Kim, Y.-S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kim, Y.-R.
Right arrow Articles by Kim, Y.-S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement