Molecular analysis of multicatalytic monoclonal antibodies

Molecular Immunology 47 (2010) 1747–1756

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Molecular analysis of multicatalytic monoclonal antibodies Haggag S. Zein a,c,∗ , Jaime A. Teixeira da Silva b , Kazutaka Miyatake c a

Department of Genetics, Faculty of Agriculture, Cairo University, 12613,12 Gamma Street, Giza, 12613, Egypt Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Miki-cho, Ikenobe 2393, Kagawa-ken, 761-0795, Japan c Department of Applied Biological Chemistry, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, 1-1, Gakuen-Cho, Sakai, Osaka, 599-8531, Japan b

a r t i c l e

i n f o

Article history: Received 16 January 2010 Accepted 25 February 2010 Available online 30 March 2010 Keywords: Monoclonal antibodies Cucumber mosaic virus Coat protein Catalytic antibody genes Affinity ELISA RNase DNase Protease

a b s t r a c t Recently, our first report demonstrated that Cucumber mosaic virus (CMV)-specific monoclonal antibodies (mAbs) possess DNase-like activity against DNA. In the present study, we show for the first time ever how one mAb (mAb-5) has polyreactive (protease, DNase, and RNase) catalytic activities (catAbs). Amino acid sequence analysis of the encoded variable-genes showed that the light chains of the hybridomas expressed the germline family genes V␬1A, bb1.1 and V␬II, bd2, whose protease and DNase catalytic activities have been reported, while the heavy chain genes were expressed in several germline families (eight of VH 1/J558, three of VH 5/VH 7183, and three of VH 8/VH 3609). Interestingly, these germline genes have been well studied in esterolytic antibodies. Here we present for the first time convincing evidence showing that highly purified mAb-5 catalyze both single- and double-stranded DNA and exhibit RNase and protease activity. The greatest therapeutic potential of catAbs could lie in selective prodrug activation. Furthermore, catAbs offer excellent or unique specificity for individual and defined antigenic targets. Therefore, the phenomenon of autoantibody catalysis can potentially be applied to isolate efficient catalytic domains directed against pathogenetically and clinically relevant autoimmune epitopes. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Naturally occurring catalytic antibodies (catAbs) with efficient protease and DNase activity have been isolated from the sera of healthy subjects as well as in patients with certain autoimmune diseases. CatAbs against vasoactive intestinal peptide (VIP) were first isolated from the sera of healthy subjects and in patients with asthma (Paul et al., 1989, 1991). Antibodies (Abs) with protease activity against thyroglobulin and prothrombin were found in the sera of patients with Hashimoto’s thyroiditis and with multiple myeloma, respectively (Thiagarajan and Paul, 2000). CatAbs that hydrolyze factor VIII were also shown to arise as alloantibodies in patients with severe hemophilia-A in response to infusion of homologous factor VIII (Lacroix-Desmazes et al., 1999, 2005). DNA- and RNA-hydrolyzing Abs were isolated from the sera of

Abbreviations: ELISA, enzyme-linked immunosorbent assay; FR, framework region; HRP, horseradish peroxidase; mAb, monoclonal antibody; catAbs, abzymes, catalytic antibodies PBS, phosphate-buffered saline; scFv, single chain Fv; VH , variable heavy chain; VL , variable light chain. ∗ Corresponding author at: Department of Genetics, Faculty of Agriculture, Cairo University, 12613,12 Gamma Street, Giza, 12613, Egypt. Tel.: +20 187676770/235685148; fax: +20 235717355/20739. E-mail addresses: [email protected], [email protected] (H.S. Zein). 0161-5890/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2010.02.024

patients with systemic autoimmune disorders, including systemic lupus erythematosus, scleroderma or rheumatoid arthritis (Shuster et al., 1992; Vlassov et al., 1998). The idiotypic network could play a key role in the appearance of DNase Abs (Izadyar et al., 1993; Avalle et al., 2000). Our recent report showed that Cucumber mosaic virus (CMV) coat protein (CP)-stimulated Abs possessed DNase-like activity (Zein et al., 2009). In addition to the DNA-binding activity, some anti-DNA Abs also possess DNA and/or RNA-hydrolyzing catalytic activity (Jang and Stollar, 2003; Gololobov et al., 1997; Kim et al., 2006). For anti-DNA Abs originating from humans, only some polyclonal Abs (pAbs), but not monoclonal Abs (mAbs), have been reported to have DNA-hydrolyzing activity (Baranovskii et al., 2001; Krasnorutskii et al., 2008). However, insight into the detailed biochemical and structural basis of how anti-DNA Abs can hydrolyze DNA and/or RNA are poorly understood. What is known, however, is that they have commonly multiple cationic amino acids, such as Arg and Lys, in the complementarity determining regions (CDRs) of VH and/or VL , particularly in VH-CDR3 (Jang and Stollar, 2003; Nevinsky and Buneva, 2002; Marion et al., 1997; Kim et al., 2009). The biological relevance of hydrolytic Abs to vasoactive intestinal peptide (VIP), DNA described in patients with asthma, or systemic lupus erythematosus, remains unclear. Indeed, it is not yet known whether proteolytic Abs are involved in the pathogenesis of the disease or result from altered immune

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mechanisms that are associated with the manifestations of the disease. The positions of the interacting key residues in the CDRs that interact with the phosphorus moiety show strong homology with our catAbs (Zein et al., 2009; Miyashita et al., 1994, 1997), and other reported catAbs with esterase/amidase activity, which were elicited by the phosphonate/phosphonamidate haptens from normal mice (Fujii et al., 1995). Further comparison of Abs elicited by the phosphorus haptens, such as DNA, RNA, phosphocholine, and phosphotyrosine, indicated that some of them had similarity in the sequence of the basic amino acids and their positions in the CDRs (Kim et al., 2006; Nevinsky and Buneva, 2002). The number of catAbs that can be prepared by normal immunization using ground-state peptide or proteins is of primary importance since they possess a vital role such as CPs of bacteria, viruses, cancer cells, etc. The recent identification (Durova et al., 2009) in multiple myeloma patients of an Ab VL that cleaves the human immunodeficiency virus (HIV) protein gp120 demonstrates that natural catAbs are not restricted to autoantigenic substrates. Hifumi et al. (2002) reported that mice were immunized by HIV-1 gp41 polypeptide, whose VL could enzymatically cleave the conserved region of the HIV-1 envelope, with all catAbs possessing at least one catalytic triad in the structure composed of Asp1 , Ser27a , and His93 , as suggested by conformational analysis. However, the catAbs pool contains different types, Abs to DNA or RNA (Nevinsky and Buneva, 2002; Andrievskaya et al., 2002), anti-idiotypic Ig to DNases, RNases and probably to other enzymes such as topoisomerases, or to their complexes with nucleic acids or with other ligands, including allosteric enzyme regulators. Some antigens may change their conformation when they associate with other proteins, and their structure in such a complex could mimic that of a transition state of the antigen’s reaction. The phenomenon of catalysis by Abs is extremely interesting and can potentially be applied to many different objectives, including new types of efficient catalysts (Ponomarenko et al., 2002). The increased occurrence of autoantigen-specific catAbs in autoimmune disease is accompanied by a decreased of polyreactive catAbs (Nevinsky and Buneva, 2002; Durova et al., 2009). This supports the hypothesis that the former are harmful and the latter are beneficial because catAbs have high efficiency and specificity. There is interest in their application for medical treatment i.e. utilizing DNA-cleaving Abs for construction of engineered drugs for therapeutic applications. Abzymology is a novel trend in basic immunology and enzymology and a new avenue of biomedical research and clinical practice. The Ab catalyzes itself and, regardless of the defined field of application, is a new area of research that is generating considerable interest in biologists, chemists and medical investigators. catAbs, like other Abs, are able to recognize canonical three-dimensional arrays found in diverse bioactive compounds. Thus, catAbs offer excellent or unique specificities for individual and defined antigenic targets. In general terms, the phenomenon of catAbs catalysis can potentially be applied to isolate efficient catalytic domains directed against pathogenetically and clinically relevant autoimmune epitopes (Ponomarenko et al., 2002). The aim of this work was to study the characteristics of multicatalytic activity of the mAbs which had been raised against CMV-CP. Study the nucleotide sequences mAb-4 heavy and light for multicatalytic activity of an Abs, therefore, the molecular modeling of the VH –VL could build the 3D structure of the Abs. The structural information from the theoretically modeled complex can help us to further understand the muticatalytic mechanism of anti-DNA antibodies. Until now, and to the best of our knowledge, this is the first report in the literature on the immunization of CMV-CP or other substances that can stimulate BALB/c mice whose multicatlytic Abs possess triple activities (DNase, RNase, and protease-like), which were investigated.

2. Materials and methods 2.1. Immunization Immunized eight-weeks old BALB/c mice (Nippon SLC Co., Japan) were injected subcutaneously with 100 ␮g of purified CMV strain pepo in 0.1 ml phosphate-buffered saline (PBS; 0.01 M phosphate and 0.015 M NaCl, pH 7.5), mixed with an equal volume of adjuvant (RIBI, Immunochem Research Inc.) containing monophosphoryl lipid A MPL (25 ␮g) plus trehalose dicorynomycolate TDM (25 ␮g) (RIBI, Immunochem Research Inc.) as described in (Zein et al., 2009). 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 in which lymphocytes from the spleens of the immunized mice were mixed at a 5:1 ratio with non-secreting P3X63-Ag8-U1 myeloma cells in polyethylene glycol 6000 (50%, w/v). The cells were distributed to 96-well plates at a concentration of 105 cells/well with HAT medium (100 ␮M hypoxanthine, 0.4 ␮M amino protein, 16 ␮M thymidine, 6 mM Hepes, and 200 ␮M ␤-mercaptoethanol). Clones that successfully secreted Abs specific to CMV were examined by both ELISA and western blotting. Furthermore, these 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 (Harlow and Lane, 1988). 2.2. Production of monoclonal antibodies mAbs were produced following the intraperitoneal (i.p.) injection of 107 hybridoma cells into i.p. cavities of BALB/c mice primed 2 weeks previously with 0.5 ml pristine (2,6,10,14tetramethylpentadecane), and the Abs were purified from the isolated ascitic fluid by affinity chromatography protein as described by (Zein et al., 2009). 2.3. Preparation of IgG Culture supernatants of the established hybridoma specific to CMV-CP (200 ml), ascitic fluid (5–10 ml), were precipitated with 50% saturated ammonium sulphate, then 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 solution of mAbs 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, then finally washed with 10 vol of binding buffer. The mAb was eluted (1-ml fraction) with elution buffer (0.1 M glycine, pH 2.6), and the eluted Abs were neutralized with collection buffer (1.0 M Tris, pH 9.0). The eluted mAbs were 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. The collected mAb fractions were homogenized by SDS-PAGE and the DNA-hydrolyzing activity toward supercoiled plasmid DNA verified the DNase activity, according to (Shuster et al., 1992). 2.4. SDS-polyacrylamide gel electrophoresis Purified mAbs were analyzed by SDS-PAGE using 10% polyacrylamide gels, which were mixed with 5 × protein loading buffer (80 mM Tris–HCl, 50% (v/v) glycerol, 10% (v/v) ␤-mercaptoethanol, 10% (w/v) SDS, pH 6.8) to a final concentration of 1× and heat-denatured by incubation for 5 min in a boiling water bath.

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SDS-PAGE was performed using 10% polyacrylamide gels in an electrophoresis apparatus (Bio-Rad) at 250 V with running buffer (Tris 125 mM, pH 8.3), glycine 960 mM, (w/v) 0.5% SDS until the bromophenol blue band ran off the gel. Gels were stained with Coomassie Brilliant Blue R-250 (Pierce). 2.5. cDNA synthesis, PCR amplification of immunoglobulin variable regions Total RNAs were prepared from about 107 hybridoma cells using ISOGEN (Nippon Gene Co., Tokyo, Japan). Chloroform was added, followed by vigorous agitation, and incubation at RT for 2–5 min, centrifugation, and the upper aqueous phase was procured and incubated with isopropanol at RT for 10 min to precipitate the RNA. The RNA pellet was washed with 75% ethanol, air-dried, and dissolved in 0.1% diethylpyrocarbonate water (Sigma). RNA concentration and purity were gauged using absorbance at OD260/280 . The mRNAs were isolated with Oligotex-dT30 (Super) columns (Takara, Kyoto, Japan) according to the manufacturer’s instruction. The primers used in the PCR amplification were based on previously published data (Huse et al., 1989): for VH these were 5 -AGGTCCAACTG-CTCGAGTCAGG-3 (forward primer) and 5 -AGGCTTACTAGTACAA-TCCCTGGGCACAAT-3 (reverse primer), where the underlined portion of the 5 primers incorporates an XhoI site and that of the 3 primer an SpeI restriction site. The primers for the kappa light chain (V␬) genes were 5 CCAGATGT-GAGCTCGTGATGACCCAGACTCCA-3 (forward primer) and 5 -GCGCCG-TCTAGAATTAACACTCATTCCTGTTGAA-3 (reverse primer) where the underlined portion of the 5 primers incorporate a SacI restriction site and that of the Reverse primers an XbaI restriction site for amplification of the Fd and ␬Lc regions, respectively. First-strand cDNA was synthesized from mRNA template with a Moloney murine leukemia virus M-MLV Reverse Transcriptase kit (Takara, Kyoto, Japan) using oligo-dT20 primers (Pharmacia Biotech). The variable regions of VH and V␬ were amplified from first-strand cDNA using Ex-Taq DNA polymerase with 30 cycles of PCR (1 cycle of 1 min at 94 ◦ C, 1 min at 55 ◦ C, and 2 min at 72 ◦ C) in 50 ␮l of the following reaction mixture: 78 mM Tris–HCl (pH 8.8), 17 mM (NH4 )2 SO4 , 10 mM ␤-mercaptoethanol, 2 mM MgCl2 , 0.05% W-1 detergent (Takara, Kyoto, Japan), 0.2 mg of BSA/ml, 200 mM each of dATP, dCTP, dGTP, and dTTP, 1 mM of each primer, 10 ng of cDNA, and 2.5 U of Ex-Taq DNA polymerase (Takara, Kyoto, Japan). The PCR products were analyzed on a 2% low-meltingpoint agarose-Tris acetate-EDTA (TAE) gel and visualized with ethidium bromide (final concentration = 0.5 ␮g/ml). PCR products of an expected size of about 650 bp were excised from the gel and purified with a QIAGEN gel extraction kit as specified by the manufacturer. The amplified fragments were cloned into separate vectors pGEM-T Easy Vector (Promega Biotech), while PCR products were ligated into plasmid pGEM-T Easy with a molar ratio of 3:1–10:1 PCR products to the vector) respectively of a ligation kit (Takara, Kyoto, Japan), for the purpose of transfer into competent E. coli DH5␣ cells. 2.6. Sequencing of V regions The target DNA fragments cloned into pGEM-T Easy were propagated and purified from E. coli DH5␣ cells by alkaline lysis and sequenced directly with Sequenase (ABI PRISM 310 genetic Analyzer). Cyclic sequencing of these DNAs was performed in both directions using a commercial kit (Thermo Sequence kit, Amersham Pharmacia Biotech) and the M13 forward (5 -CACGACGTT-GTAAAAACGAC-3 ) and reverse (5 GGATAACAATTTCAC-ACAGG-3 ) primers set (Pharmacia Biotech) using an ABI PRISM BigDye Primer Cycle Sequencing Kit.

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2.7. Sequence analysis Sequence analysis was performed using GENETIX-WIN software. Sequences were initially aligned and checked for stop codons with reference to the IMGT standard for codon numbering (Lefranc and Lefranc, 2001). Clustal W analysis was performed on each Ab gene to determine the relationship between sequences. Since there is the possibility of PCR crossover error, especially with the slower proof reading polymerase used in this study, the alignments were studied carefully to see if any of the sequences could have arisen by PCR crossover. If there was any doubt as to the validity of a sequence after this scrutiny, the sequence was excluded from the results. The variability between the amino acid sequences within each subgroup was calculated using the method of (Kabat et al., 1991). The percentage similarity between the VH and VL sequences with an open reading frame (ORF) and the closest matched mice sequences was “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. The CDR position and numbering scheme adopted matched the Kabat numbering (Martin, 1996) and a CDR definition was adopted from Andrew’s web site (www.bioinf.org.uk/abs/). 2.8. DNA, RNA-hydrolysis assays Conditions for DNA and RNA hydrolysis were first optimized as described previously (16,26). Reaction mixtures (20 ␮l) for analysis of RNA-hydrolyzing activity contained optimal concentrations of standard components: 20 mM Hepes pH 7.49, 50 mM NaCl, 1 mM MgCl2 , 1 mM MnCl2 , 2.5 ␮g supercoiled-pUC18 plasmid or ␭ DNA or 2.5 ␮g bacterial RNA, and 1–5 ␮g of Ab and incubated at 37 ◦ C for 1–3 h. Hydrolysis was assessed by 1% AGE of the reaction products and 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. 2.9. In situ RNA-hydrolyzing activity of antibodies in the gel RNA-hydrolyzing activity of Abs in gel was estimated by SDSPAGE (5–20% gradient polyacrylamide gel containing 10 ␮g/ml bacterial RNA). After separation of the light and heavy chain of mAb-5 in the gel, the latter was washed free of SDS with 7 M urea at 37 ◦ C for 1 h then soaked in water (10 portions of water for 5–7 min each). mAb-5 activity was restored when the gel was immersed in 20 mM Tris–HCl, pH 7.5, containing 5 mM MgCl2 and 1 mM EDTA for 16 h at 37 ◦ C, and then stained with ethidium bromide. The gel portion where RNA was cleaved was revealed as a dark spot on a uniformly UV-fluorescing background. Positions of the protein bands were determined by staining of the gel with Coomassie Blue R-250. 2.10. Analysis of proteolytic activity of mAbs Protease activity of the mAbs was quantified by the azocasein assay (Reichard et al., 1990). Azocasein (Sigma) was dissolved at a concentration of 2% in a 50 mM Tris (pH 7.5) buffer. The reaction mixture (azocasein solution (200 ␮l), 200 ␮l of mAb-5 (100 ␮g/ml), and 200 ␮l of 50 mM Tris (pH 7.5) was incubated at 37 ◦ C overnight. The reactions were stopped by adding 400 ␮l of 20% trichloroacetic acid, and the reaction mixtures were allowed to stand at ambient temperature for 30 min. Tubes were then centrifuged for 3 min at 8000 × g, the absorbance of the supernatant was measured at A366 nm of released azo dye with a spectrophotometer. Protease inhibitors, namely serine protease inhibitors,

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Table 1 Chromogenic peptide sequence substrate.

P1 P2 P3 P4 P5 P6

Peptide sequence

Protease-like activity

H-D-Iie-Pro-Arg-pNA H-D-Val-Leu-Lys-pNA Bz-L-Arg-pNA Suc-Ala-Ala-Pro-Leu-pNA Suc-Ala-Ala-Pro-Phe-pNA Gle-Phe-pNA

tPA Plasmin Trypsin Elastase chymotrypsin

diisopropyl fluorophosphates and phenylmethylsulfonyl fluoride (PMSF) were preincubated at 0.5 mM for 30 min at 37 ◦ C, and then added to the substrate assay. The proteolytic activity of the mAbs was determined against chromogenic peptide substrates (Table 1) according to (Gabibov et al., 2002). The appropriate concentration of each Ab was incubated with 5–25 ␮M of chromogenic peptide in 0.1 M Tris–HCl, pH 8.6. The enzymatic reaction was monitored by the increase in optical density at 410 nm. 3. Results 3.1. Purity of the mAbs To prove that hydrolyzing activity is an intrinsic property of mAbs and is not due to copurifying enzymes, we applied some rigid criteria previously proposed by (Paul et al., 1989; Andrievskaya et

al., 2002), the most important of which were: [1] electrophoretic homogeneity of IgG by silver staining (Fig. 1); [2] immunoprecipitation of protease activity by anti-IgG Abs; [3] complete adsorption of protease activity by anti-IgG sepharose and its elution with buffer of low pH from the adsorbent; [4] during this elution the protease activity coincided exactly with the IgG peak and there were no other peaks of activity; [5] gel-filtration of IgG at pH 2.6 did not lead to disappearance of activity and the peak of activity coincided exactly with 150 kDa IgG; [6] IgGs from the sera of 50 healthy donors did not hydrolyze protein. The majority of natural catAbs are pAbs which are produced in microgram amounts and are products of different immuno-competent cells. The origin of our Abs is mAbs that are much easier to purify than serum pAbs. In addition, mAbs can be produced in milligram amounts from different sources i.e., culture supernatants or ascitic fluids. We applied several aspects for high purity Abs as suggested by (Nevinsky and Buneva, 2005). However, 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 an Abs with a preparation purity of >99%, shown in SDS-PAGE and western blotting (Fig. 1B and C) under reducing conditions as only two bands, Hc and Lc, with expected sizes of 50 and 25 KDa, respectively. In contrast, under non-reducing conditions, only one band appeared for the whole Abs. DNA-hydrolyzing activity of the gel-filtration fraction samples

Fig. 1. Chromatography of IgG-5 specific to CMV-CP on Sephadex-200 and its relative DNase activity against plasmid DNA (A). Purification of mAbs on 10% SDS-PAGE under reducing conditions (␤-mercaptoethanol) after eluted from gel-filtration column fractions (58, 59, 60, and 61) (B). Western blot analysis for detection of mAb under reducing conditions (C).

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Fig. 3. DNase and RNase activity of CMV-specific mAbs. (A) Relative activity against single-stranded ␭ DNA with mAbs-(4, 5, 52, 6, 7, M2–1, M2–3, and M2–4) = lanes 1–8, respectively. (B) RNase activity of mAbs-specific CMV-CP; RNA catalytic activity was stimulated with Mg2+ mAbs-(4, 5, 52, 6, 7, 8, M2–4; lanes 1–7, respectively), incubated with E. coli RNA. Lane M is control (incubation of RNA or DNA without Ab).

3.3. DNase and RNase hydrolyzing activity

Fig. 2. Protease and peptidase catalytic activity of CMV-specific mAbs. (A) Relative activity of mAb5 against azocasein substrate measured at 366 nm. (B) Chromogenic peptide hydrolysis with different mAbs, absorbance at 405 nm.

containing Abs were incubated with supercoiled (sc) plasmid DNA pUC18 (Fig. 1A).

3.2. ss- and dsDNA hydrolysis by mAbs The mAbs demonstrated different catalytic activity against double- and single-stranded DNA. High DNase catalytic activity showed against both ss- and dsDNA disappeared after incubation with mAb-5 for 75 min (Fig. 4C, lanes 5–9) or mAbs-(5, 52, 6, 7) against ␭ DNA (Fig. 2A, lanes 2–5). In contrast, a low catalytic activity caused only a single break in the plasmid DNA—thus showing the DNA to be linear (Fig. 3C, lanes 2–4), and ␭ DNA (Fig. 2A, lanes 1 and 7–9) .The properties of the DNase-like activity of the our mAbs distinguish it from other known DNases in autoimmune diseases. Interestingly, these mAbs demonstrated a different combination of endo- and exonuclease activity with non-specific sequences. The optimum pH was 4–5.3 and was metal-independent (Zein et al., 2009), which was in agreement with a report on the DNA-hydrolysis of autoAbs (Nguyen et al., 2003). Plasmid DNA disappeared in most mAbs after incubation with Abs, suggesting that carboxylates in the antigen-binding site are involved in the catalytic mechanism.

There were two groups of CMV-specific mAbs. The relative DNase and RNase activities of the CMV-specific mAbs against ss ␭ DNA with the first group of five mAbs-(4, 5, 52, 6, and 7) were determined after migration in agarose gel electrophoresis (Fig. 2A, lanes 1–6). These five mAbs were derived from the germline gene V␬II bd2 of the light chain variable region (VL ) while another three mAbs (M2–1, M2–3, and M2–4) were derived from another germline gene V␬IA bb1.11 of the light chain (Fig. 2A, lanes 7–9). Interestingly, this is the first report to prove that the mAbs derived from germline gene V␬II bd2 showed both high DNase and RNase catalytic activity against genomic ␭ DNA and bacterial RNA (Fig. 2B), Therefore for the first time these CMV-specific mAbs are shown to possess double catalytic activity by the same mAb. On the other hand, all three mAbs-(M2–1, M2–3, and M2–4) showed very low DNase activity against ss ␭ DNA and there was no RNase activity against bacterial RNA (Fig. 2A and B, lanes 7–9) Moreover, RNase activity was stimulated by Mg2+ . The mAbs of non-specific CMV-CP (control) did not hydrolyze DNA (Fig. 3B, lanes 1–9). These data indicate clearly that the hydrolyzing activity is an intrinsic property of the mAbs and is not due to copurifying enzymes (Andrievskaya et al., 2002). Here we present studies on IgG RNase, which provide further evidence in support of this conclusion. We also used a new in situ RNase assay in SDS-PAGE gels (Andrievskaya et al., 2002) which was established to exclude any hypothetical traces of contaminating RNases. Briefly, after incubating a gel containing RNA to allow Abs renaturation and staining with EtBr, a sharp dark band at the position of RNAhydrolyzing polypeptide was revealed on a fluorescent background of RNA-bound EtBr (Fig. 3A). This assay showed RNase activity as a single band which corresponded to whole IgG (Fig. 3A, lane 1). After dissociation of IgG-5 by ␤-mercaptoethanol, interestingly the RNase activity was revealed only in the light chains not with heavy chain (Fig. 3A, lane 2).

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Fig. 4. In situ gel assay of RNAse activities of catalytic IgG-5 in non-reducing conditions or of separated chains (lane 1) and by SDS-PAGE in reducing conditions in a gel containing RNA. RNase activity was revealed by ethidium bromide staining as a sharp dark band on a fluorescent background. Separated L and H chains; activity revealed by the light chain as described by (26). DNase activity of mAb-5 against supercoiled pUC-18 plasmid DNA over time (15, 30, 45, 60, 75, 90, 105, and 120) min after incubation at 37 ◦ C for 2 h (lanes 2–9, respectively). (B) Negative control: anti-Rubisco large subunit mAbs incubated with plasmid DNA (A).

3.4. Molecular sequences of the CMV-specific antibody light chain

3.5. Proteolytic activity of the mAbs

The remarkable propertyof the sequence analyses of PCRamplified cDNA derived from 14 hybridomas showed that VL was derived from the germline V␬II gene bd2 (Zein et al., 2009) while the heavy chains’ variable region were derived from different germline genes (Figs. 4 and 5). The germline V␬1A gene bb1 showed a typical light chain sequence specific to ssDNA (1NBV L) (Gololobov et al., 1997), while the mAb BV04-01 possessed DNase-like activity by the light chain-dependent variable region as well as an RNA (1MRF L)-specific Ab. Moreover, the V␬1A germline is common to a relatively large population of Abs that bind a large number of antigens, including proteins, DNA, steroids, peptides, and small haptens. Thus, the polyspecificity intrinsic to V␬1 may contribute to the ability of the germline repertoire to bind a wide array of chemical structures (Romesberg et al., 1998).

In the present study, mAb-5 showed the highest level of proteolytic activity compared to other mAbs (Fig. 6A). However, the proteolytic activity of mAb-5 was susceptible to a number of serine protease inhibitors Table 2 (DFP and PMSF at 0.5 mM) and incubation with Ab decreased the activity compared with non-inhibitors that existed in the reaction at 10.2 and 22.5% respectively, while 2,2 -bipyridyl (5 mM) and chymostatin (0.5 mg/ml) were slightly inhibited by 84.2 and 87.6%, respectively. Furthermore, there was no detectable inhibition of proteolytic activity of the mAb-5 with other protease inhibitors (Table 2).

Table 2 The relative protease activity of mAb-5 against azocasein substrate with different protease inhibitors.

1 2 3 4 5 6 7 8 9 10 11 12

Inhibitor

Con.

%RA

DFP PMSF 2,2 -Bipyridyl Chymostatin ACA EDTA E-64 Phenantorolin SBTI Pepstatin A SSI TPCK

0.5 mM 5.0 mM 5.0 mM 0.5 mg/ml 5.0 mM 7.8 mM 0.5 mg/ml 5.0 mM 0.5 mg/ml 0.5 mM 0.5 mg/ml 0.5 mM

10.2 22.5 84.2 87.6 102.8 109.7 116.4 116.6 117.9 118.3 124.9 144.2

3.6. Chromogenic peptide hydrolysis Several chromogenic peptides (Table 2) were examined as Ab substrates. The mAbs were able to cleave the amide bond between the carbonyl peptides leaving group p-nitroanilide (pNA). Interestingly, most mAbs showed catalytic activity with peptides containing pNA linked to a basic residue with different specificity (Table 1). Otherwise, there was no hydrolytic activity with nonbasic amino acid conjugates with chromogenic compounds. mAb-5 showed hydrolytic activity with three peptides P2 > P1 > P3 depending on the absorbance after incubation at pH 8.5 (Fig. 6B), while other mAbs showed different specific substrate specificity: mAb-8 with peptide-1 at pH 9.0, mAb–M2–4 with peptide-2 at pH 8.5, and mAb-4 and -6 with peptide-1 at pH 8.5 (Fig. 6B). CMV-CP has a high variability of amino acid residues most of which are basic or neutral except for the highly conserved ␤H–␤I loop (Fig. 7) and amino acid sequence NH2 -DDKLEKDE-COOH, corresponding to positions 191–198 of the CMV-M2 capsid protein, which is a highly acidic residue reported to be an immunodominant epitope of the virus CP (Fig. 7) (Liu et al., 2002). We suppose that this ␤H–␤I loop and sequence of CMV-CP mimicking proteins

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Fig. 5. Alignment of amino acid sequences of the 14 VH variable regions of the Abs specific to CMV-CP. A dash in the individual sequences denotes a deletion. The framework region (FWR) and complementarity determining region (CDR) are indicated above the appropriate sequence segments in the figure. The amino acid residues are numbered according to Kabat et al. (1991). Amino acids are identified by a single-letter code.

Fig. 6. Alignment of the amino acid sequences of VH mAb-5 with the highest homology sequences of the catalytic antibodies submitted in GenBank database as accession nos. 2DTM, 2DQU, 1LO0, 1KNO, 1YED. It is an optimal global alignment produced by the CLUSTAL W program. A dash in the individual sequences denotes a deletion. The framework region (FWs) and complementarity determining regions (CDRs) are indicated above the appropriate sequence segments in the figure. The amino acid residues numbered according to Kabat et al. (1991). Amino acids are identified by a single-letter code.

immune response to CMV-CP due to their induction by Abs which bear structures (mimotopes) that mimic DNA and stimulate catAbs. Furthermore, the relative catalytic activity might depend on both light and heavy chain germline genes. The light chains are highly homologous (>90% identity) with catAbs, as well as with anti-hapten catAbs, which have been well studied (Fujii et al., 1998). Although there are no reports that revealed the important role of VH in natural catAbs, the amino acid sequence of the VH specific CMV-CP was highly homologous with several anti-hapten catAbs VH (Fig. 4). Remarkably, mAbs-specific CMV-CP have different heavy chain genes that combine with one VL gene and showed different DNase catalytic activity; therefore we speculate that the heavy chain gene could increase or decrease catalytic activity depending on the germline genes (Fig. 8).

Fig. 7. Phylogenetic tree based on multiple alignments of the amino acid sequences of VH mAb-5 (accession no. ABR32158) with the highest homology sequences of the catalytic antibodies submitted in GenBank database with accession nos. 2DTM, 2DQU, 1LO0, 1KNO, 1YED.

stimulated the electrostatic environment of the phosphate backbone of the DNA. However, the origin of anti-DNA Abs remains speculative. Nissen et al. (2000) revealed that some of these Abs may arise inadvertently in nature during the course of a normal

3.7. In situ RNA-hydrolyzing activity In situ activity of the mAbs is significant irrefutable proof for the activity of the mAbs not being a contaminated enzyme, as shown in (Fig. 3A), where Abs activity was detected in situ after protein separation by SDS-PAGE of IgG in a RNA-containing gel under nonreducing condition; activity was observed only in the gel portions corresponding to the positions of protein bands of the initial IgG (Fig. 3A, lane 1).

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Fig. 8. The 3D Structure of highly conserved ␤H–␤I loop of the CMV coat protein (CP), amino acid sequence 191-NH2 -DDKLEKDE-COOH-198. It is a unique sequence, a highly acidic residue and an immunodominant epitope of the CMV-CP (Liu et al., 2002).

The reducing condition of only the positions of Ab VL (Fig. 3A, lane 2)—activity was absent in the heavy—undoubtedly indicating that RNA catalytic activity is an intrinsic property of the VL of IgG. However, the VL alone or whole IgG showed an ability to hydrolyze RNA, while the heavy chain alone did not show any catalytic activity (Fig. 3A). One of the important aspects of VL and VH amino acid sequences is the study of the structural analysis of antigen-binding loops by molecular modeling and simulation of molecular dynamics (research currently in progress). Through these findings, it will be suggested that amino acid residues may play a crucial role in the antigen–Ab interaction. 4. Discussion Erhan and Greller (1974) suggested that the light chain of the Ab could function as a peptidase/protease by itself based on the homology between Ab light chains and serine proteases. It has been reported that the active site composed of a catalytic dyad or triad of the catAb can function to hydrolyze antigens. Kolesnikov et al. (2000) reported that an Ab possessing the catalytic dyad (His35 and Ser99 ) in the heavy chain is the active site that hydrolyzes the acetylthiocholine molecule. The detection of RNase activity in the gel region corresponding only to IgG and its light chain, together with the absence of any other bands of RNase or protein, provides direct evidence that IgG possesses RNA-hydrolyzing activity. DNase activity of IgGs was revealed for the first time by Gabibov et al. (2002) as well as our recent report (Zein et al., 2009) and most of the above criteria of Ab catalytic activity were satisfied. Andrievskaya et al. (2002) proposed that two DNase and RNase activities reside in the same catAb. The frequency of B-cell clones producing catAbs also increased in NZB/W, MRL-lpr/lpr and SJL/J mice, which are classical animal models for human autoimmune diseases (Tawfik et al., 1995). RNase activity is associated with IgGs and IgMs from the sera of patients with systemic lupus erythematosus (SLE) but not with those from the sera of normal humans (Andrievskaya et al., 2002). Importantly, RNase catalytic activity has been revealed with highly purified SLE IgG, its F(ab) fragments and separated L-chains. On the

other hand, SLE IgGs may contain a mixture of different catAbs, which hydrolyze either only DNA or only RNA, or both substrates simultaneously, like monoclonal SLE IgGs (Nevinsky and Buneva, 2005). In agreement with the first finding that monoclonal Lupus IgGs show both reactivity and DNase and RNase activity (Andrievskaya et al., 2002), our mAb-5 not only showed DNase and RNase activity but also protease activity. In other words, three different catalytic activities were shown by a single mAb which is the first time ever (to the authors’ knowledge) that such a finding has not been reported in the literature. Therefore, it can be concluded that CMV-specific Abs have multifunctional and/or polyreactive catalytic activity. Interestingly, the light chain germline V␬II gene bd2 has been reported as a partner of a number of different Abs raised in autoimmune diseases such as anti-DNA, -RNA, Sm, and Histone, as well as HIV and HCV human virus-specific Abs (Hifumi et al., 2002; Gololobov et al., 1997; Jang and Stollar, 2003) which conclude that this germline gene might play a vital and dominant function in autoimmune diseases as well as in viral infection. Durova et al. (2009) reported that some light chains play a fundamental role in an extra defense mechanism against foreign antigens. Hifumi et al. (2002) reported that immunizing ground-state peptides or proteins stimulated super catAbs possessing serine protease-like characteristics. Further, they succeeded in preparing super catAbs that destroy the HIV-1 envelope protein gp41. The catalytic function of an Ab mostly resides in its light chain. From mouse V␬ germline analysis, it became clear that super catAbs are generated the light chain from some discrete germlines such as bb1, cr1, cs1, bl1, bj2 and bd2. In these V␬ germlines, at least one catalytic triad composed of three amino acid residues, namely, Asp1 , Ser27a and His93 , is encoded. Interestingly, CMV-CP stimulated from five different fusions highly restricted light germline V␬II, gene bd2, and germline V␬1A gene bb1 had a very high homology (98–100%) germline (Zein et al., 2009). Recently, Durova et al. (2009) investigated a different approach which could lead to the production of “catalytic vaccines” against HIV-1 gp120. Their deleterious role, due to their catalytic efficiency, was first suspected on the basis of their association with diseases and then confirmed in the case of haemophilia (LacroixDesmazes et al., 1999; Ponomarenko et al., 2006), multiple sclerosis (Ponomarenko et al., 2006), and HIV-1-related immune thrombocytopenia (Nardi et al., 2001). In contrast, the presence of circulating catAbs was also demonstrated to be linked to a protective effect for survival in severe sepsis (Lacroix-Desmazes et al., 2005) or against chronic allograft nephropathy (Wootla et al., 2008). catAbs may thus be seen as natural efficient catalysts that can be elicited in response to a perturbation in metabolism or pathological disturbance. These results suggest that CMV-specific mAbs showed serine protease-like activity, which is in agreement with a previous report (Mitsuda et al., 2004). Successfully raising Ab-protease may seriously enforce the potential of neutralizing Abs for passive vaccination. Several examples with gp120-targeted catAbs have been reported during this decade (Paul et al., 2004; Ponomarenko et al., 2006; Planque et al., 2004). Further, numerous evidence for antigen-specific and non-specific Ab-mediated proteolysis in different human pathologies have been demonstrated (Matsuura et al., 1994). A catAb can be a tool for fulfilling the function of catalytic vaccines (Ponomarenko et al., 2006). Various innate and adaptive immune mechanisms have been documented as important in defense against microbial infection. A number of reports have described catAbs performing other specific functions directed against proteins, including the formation of cyclic peptides (Smithrud et al., 2000), catalysis of peptidyl-prolyl cis–trans isomerization in protein folding (Ma et al., 1998), and development of a novel enzymatic activity cleaving the bacterial protein HPr (Liu et al., 1998). Some catAbs have

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been described that require cofactors for activity similar to standard enzymes (Iverson and Lerner, 1989). The existence of protein-specific catAbs is tightly related with different pathological processes in humans and animals (Ponomarenko et al., 2006). Production de novo of Ab-protease remains one of the intriguing tasks needed for the development of a “catalytic vaccine”. This problem is partly solved by induction of anti-idiotypic Abs against a protease active site (Ponomarenko et al., 2007). Why did CMV-CP stimulate catAbs? Protease activity of this VL germline has been extensively studied and been shown to possess DNase-like activity (Zein et al., 2009). The findings high acidic charge epitope (Fig. 7) agree well with several previous reports in which catAb 41S-2 was raised against the peptide sequence RGPDRPE-GIEEEGGERDRD of gp41HIV-1 (Uda et al., 2000). The 41S-2 Ab strongly recognizes the GIEEE sequence of the gp41 peptide (Uda et al., 1995). Because gp120 has an EEE sequence [gp120: 267–269; Muesing et al., 1985], the mAb and L chain might slightly cross-react with gp120. The catalytic L chain of mAb 41S-2 essentially displayed similar binding to gp160 and gp41 as intact 41S-2 mAb. Importantly, CMV-specific mAbs could stimulate DNAhydrolyzing catAbs, which have been reported in human IgG in the case of autoimmune disease and model mice; Moreover, DNAse catalytic activity has been reported with whole IgG or VL alone but not with the heavy chain. These data are in agreement with the literature in which the activity of natural catAbs contributed to the variable region of the VL (Nevinsky and Buneva, 2005) as well as some catAbs which were stimulated with hapten, or a transition-state analog which contributed to VL (Fujii et al., 1998; Takahashi et al., 2001). We could prove that Abs which possess both DNase and RNase might be stimulated with an antigen such as CMV-CP. The phenomenon of catalysis by catAbs is extremely interesting and can potentially be applied to many different objectives, including new types of efficient catalysts, evaluation of the functional roles of catAbs in innate and adaptive immunity, and understanding certain aspects of self-tolerance and of the destructive responses in autoimmune diseases (Nevinsky and Buneva, 2002). References Andrievskaya, O.A., Buneva, V.N., Baranovskii, A.G., Gal’vita, A.V., Benzo, E.S., Naumov, V.A., Nevinsky, G.A., 2002. Catalytic diversity of polyclonal RNAhydrolyzing IgG antibodies from the sera of patients with systemic lupus erythematosus. Immunol. Lett. 81, 191–198. Avalle, B., Debat, H., Friboulet, A., Thomas, D., 2000. Catalytic mechanism of an abzyme displaying a ␤-lactamase-like activity. Appl. Biochem. Biotechnol. 83, 163–169. 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., Nevinsky, G.A., 2001. Catalytic heterogeneity of polyclonal DNA-hydrolyzing antibodies from the sera of patients with multiple sclerosis. Immunol. Lett. 76, 163–167. Durova, O.M., Vorobiev, I.I., Smirnov, I.V., Reshetnyak, A.V., Telegin, G.B., Shamborant, O.G., Orlova, N.A., Genkin, D.D., Bacon, A., Ponomarenko, N.A., Friboulet, A., Gabibov, A.G., 2009. Strategies for induction of catalytic antibodies toward HIV-1 glycoprotein gp120 in autoimmune prone mice. Mol. Immunol. 47, 87–95. Erhan, S., Greller, L.D., 1974. Do immunoglobulins have proteolytic activity? Nature 251, 353–355. Fujii, I., Fukuyama, S., Iwabuchi, Y., Tanimura, R., 1998. Evolving catalytic antibodies in a phage-displayed combinatorial library. Nat. Biotechnol. 16, 463–467. Fujii, I., Tanaka, F., Miyashita, H., Tanimura, R., Kinoshita, K., 1995. Correlation between antigen-combining-site structures and functions within a panel of catalytic antibodies generated against a single transition state analog. J. Am. Chem. Soc. 117, 6199–6209. Gabibov, A.G., Friboulet, A., Thomas, D., Demin, A.V., Ponomarenko, N.A., Vorobiev, I.I., Pillet, D., Paon, M., Alexandrova, E.A., Telegin, G.B., Reshetnyak, A.V., Grigorieva, O.V., Gnuchev, N.V., Malishkin, K.A., Genkin, D.D., 2002. Antibody proteases: induction of catalytic response. Biochemistry (Moscow) 67, 1168–1179. Gololobov, G.V., Rumbley, C.A., Rumbley, J.N., Schourov, D.V., Makarevich, O.I., Gabibov, A.G., Voss Jr., E.W., Rodkey, L.S., 1997. DNA hydrolysis by monoclonal

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