A. V. Kozyr

Novel functional activities of anti-DNA autoantibodies from sera of patients with lymphoproliferative and autoimmune diseases.

DNA-hydrolyzing activity of IgG autoantibodies from sera of patients with various types of lymphoproliferative diseases was investigated. The association of DNA-hydrolyzing activity with the antibody (Ab) fraction has been proved by newly developed affinity-capture assay. Study of abzyme incidence in blood tumors and systemic lupus erythematosis (SLE) revealed linkage of anti-DNA Ab catalysts to mature B-cell tumors, and increased probability of DNA-abzymes formation on the background of autoimmune manifestations. These data suggest possible similarity between mechanisms of abzyme formation in SLE and B-cell lymphomas. A new mechanism of formation of DNA-specific catalytic Abs has been proposed based on the increased crossreactivity of polyclonal DNA-abzymes to DNA-depleted nuclear matrix proteins. The possibility of the abzyme production as Ab to the energetically destabilized ground state of the antigen has been discussed. Preliminary results were obtained that indicate the complement-independent cytotoxicity of anti-DNA autoantibodies isolated from blood of patients with SLE and chronic lymphocytic leukemia.

Anti-DNA autoantibodies reveal toxicity to tumor cell lines.

Cytotoxicity of anti-DNA autoantibodies from sera of SLE and CLL patients was assayed on permanent cell lines L929, HL-60, Raji, and K562. L929 cells appeared to be the most sensitive to antibody treatment. DNA-hydrolyzing properties of the same autoantibody preparations were analyzed in parallel. The data obtained outlined the correlation between cytotoxicity and DNA-hydrolyzing properties of these autoantibodies. It was shown that treatment of the cells with cytotoxic anti-DNA autoantibodies induced internucleosomal DNA fragmentation and Annexin V binding to the cell surface characteristic of apoptotic pathway of cell death. A time-dependent profile of antibody-mediated toxicity to L929 cells suggested recruitment of at least two distinct mechanisms of cell death. The first peak of cell death observed in 3 h of incubation was completely inhibited by preincubation of cells with caspase inhibitor YVAD-CHO, while the second increase in cell mortality (18-30 h) persisted. Possible mechanisms for anti-DNA autoantibody cytotoxicity are discussed.

Autoantibodies to nuclear antigens: correlation between cytotoxicity and DNA-hydrolyzing activity.

Thecytotoxicity of DNA-specific autoantibodies from sera of patients with systemic lupus erythematosis (SLE) and with lymphoproliferative diseases, and from blood of healthy donors was examined on tumor-cell lines L929 and HL-60. DNA-binding IgG fractions from SLE and chronic lymphocytic leukemia (CLL) sera were cytotoxic at concentrations of up to 10−10 M. No detectable changes in cell viability were observed after incubation with antibodies devoid of DNA-binding activity and DNA-specific antibodies isolated from blood of healthy donors and patients with T-cell lymphoma, B-cell lymphosarcoma, and acute B-cell leukemia. There was good correlation between the cytotoxic activity and DNA-hydrolyzing activity of anti-DNA antibodies. The cytotoxic effect of DNA-binding antibodies presumably was complement-independent, because it was attributed only to the Fab fragment. The cytotoxic effect was completely inhibited by preincubation with double-stranded DNA (dsDNA). Both the cytotoxic effect and the DNA-hydrolyzing activity of anti-DNA antibodies were significantly increased in the antibody fraction that displayed cross-reactivity with nuclear matrix proteins. Possible mechanisms for the formation and pathogenicity of cytotoxic anti-DNA antibodies are discussed in this article.

Catalytic antibodies: balancing between Dr. Jekyll and Mr. Hyde

The immunoglobulin molecule is a perfect template for the de novo generation of biocatalytic functions. Catalytic antibodies, or abzymes, obtained by the structural mimicking of enzyme active sites have been shown to catalyze numerous chemical reactions. Natural enzyme analogs for some of these reactions have not yet been found or possibly do not exist at all. Nowadays, the dramatic breakthrough in antibody engineering and expression technologies has promoted a considerable expansion of immunoglobulins medical applications and is offering abzymes a unique chance to become a promising source of high‐precision “catalytic vaccines.” At the same time, the discovery of natural abzymes on the background of autoimmune disease revealed their beneficial and pathogenic roles in the disease progression. Thus, the conflicting Dr. Jekyll and Mr. Hyde protective and destructive essences of catalytic antibodies should be carefully considered in the development of therapeutic abzyme applications.

Purification of filamentous bacteriophage for phage display using size-exclusion chromatography

Phage display has proved itself as a powerful and convenient approach for rapid sampling of billions of protein-aceous structures against a ligand of interest. The most widespread system uses display of proteins and peptides as fusions to coat proteins III or VIII of the M13/fd phage. Efficient screening of phage display library requires high purity of the input phage particles. Double precipitation by polyeth-ylene glycol (PEG) with an average molecular weight of 8000 Da (PEG-8000) is a standard technique for purification of filamentous bacterio-phages (1,2). Certain contaminants could still remain in the PEG-purified phage preparation. Although efficient removal of contaminating RNases from bacteriophage preparation using treatment by ionic detergent followed by ultracentrifugation in CsCl gradient was reported (3), this method is costly, time-consuming, and is not applicable if the native state of the phage-displayed protein or peptide is affected by ionic detergents and high salt. Size-exclusion chromatography (SEC) has been used in preparation of pure eukaryotic viruses (4,5) and bacteriophage λ (6). We assumed that SEC can be the method of choice for efficient purification of filamentous phages under mild conditions. In this work we describe a rapid and simple technique for chromatographic purifi-cation of filamentous phage particles in Sephacryl™ S-500 resin (Amersham Biosciences, Piscataway, NJ, USA).The purity of phage preparations obtained after double PEG precipi-tation was assayed by electrophoresis in Tris-tricine (7). Phage particles displayed the single-chain variable fragment (scFv) of the MRL-4 anti-DNA monoclonal antibody (MAb) (8) fused to the C-terminal portion of the pIII minor phage coat protein (δpIII). Production of the scFv-δpIII fusion cloned into the pTScDisp phagemid (GenBank

Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches

Lethal factor (LF), a zinc-dependent protease of high specificity produced by Bacillus anthracis, is the effector component of the binary toxin that causes death in anthrax. New therapeutics targeting the toxin are required to reduce systemic anthrax-related fatalities. In particular, new insights into the LF catalytic mechanism will be useful for the development of LF inhibitors. We evaluated the minimal length required for formation of bona fide LF substrates using substrate phage display. Phage-based selection yielded a substrate that is cleaved seven times more efficiently by LF than the peptide targeted in the protein kinase MKK6. Site-directed mutagenesis within the metal-binding site in the LF active center and within phage-selected substrates revealed a complex pattern of LF-substrate interactions. The elementary steps of LF-mediated proteolysis were resolved by the stopped-flow technique. Pre-steady-state kinetics of LF proteolysis followed a four-step mechanism as follows: initial substrate binding, rearrangement of the enzyme-substrate complex, a rate-limiting cleavage step, and product release. Examination of LF interactions with metal ions revealed an unexpected activation of the protease by Ca2+ and Mn2+. Based on the available structural and kinetic data, we propose a model for LF-substrate interaction. Resolution of the kinetic and structural parameters governing LF activity may be exploited to design new LF inhibitors.

Role of Structure-Based Changes due to Somatic Mutation in Highly Homologous DNA-Binding and DNA-Hydrolyzing Autoantibodies Exemplified by A23P Substitution in the VH Domain.

Anti-DNA autoantibodies are responsible for tissue injury in lupus. A subset of DNA-specific antibodies capable of DNA cleavage can be even more harmful after entering the living cells by destroying nuclear DNA. Origins of anti-DNA autoantibodies are not fully understood, and the mechanism of induction of DNA-cleaving activity remains speculative. The autoantibody BV04-01 derived from lupus-prone mouse is the only DNA-hydrolyzing immunoglobulin with known 3D structure. Identification and analysis of antibodies homologous to BV04-01 may help to understand molecular bases and origins of DNA-cleaving activity of autoantibodies. BLAST search identified murine anti-DNA autoantibody MRL-4 with sequences of variable region genes highly homologous to those of autoantibody BV04-01. Despite significant homology to BV04-01, not only MRL-4 had no DNA-cleaving activity, but also reversion of its unusual P23 mutation to the germline alanine resulted in a dramatic loss of affinity to DNA. Contrary to this effect, transfer of the P23 mutation to the BV04-01 has resulted in a significant drop in DNA binding and almost complete loss of catalytic activity. In the present paper we analyzed the properties of two homologous autoantibodies and mutants thereof and discussed the implications of unusual somatic mutations for the development of autoantibodies with DNA-binding and DNA-hydrolyzing activity.

Caspase-Dependent Cytotoxicity of Anti-DNA Autoantibodies

The role of the catalytic function of natural antibodies in the organism is still unclear. It is commonly accepted that occurrence of natural antibodies is associated with the development of human autoimmune diseases (e.g., lupus erythematosus; LE) and autoimmune conditions in model mouse strains NZBxNZW and MRL/lpr. First of all, this dependence is characteristic of DNA-hydrolyzing antibodies. We discovered that the DNA-hydrolyzing antibodies are toxic for transplantable cell lines and analyzed pathway of cell death caused by these antibodies.

[Production of DNA-hydrolyzing antibody BV04-01 Fab fragment in methylotrophic yeast Pichia pastoris].

A system was developed for efficient production of recombinant Fab of catalytic DNA-hydrolyzing antibody BV04-01 in methylotrophic yeast Pichia pastoris. To stabilize Fab, the C ends of its chains were modified with dimerizing Jun and Fos, which are known to form a leucine zipper. The yield of functional recombinant Fab was 3 mg per liter culture. The catalytic efficiency of Fab was 1.8 · 106 M–1min–1 as inferred from the relaxation of supercoiled plasmid DNA.

Natural antibody catalytic activities in mice with autoimmune disorders.

Catalytic antibodies (abzymes) are a new type of nonenzymatic biocatalysts. The mechanism of their formation and possible role in immune disorders are now attracting researchers’ attention. A variety of catalytic antibodies with different activities have been revealed to date; however, the purpose-oriented search of and construction of efficient abzymes with certain specificities still remains a problem. There is evidence for spontaneous catalytic-antibody formation in the human body. This has often been observed in patients with systemic lupus erythematosus (SLE), rheumatoid arthritis, scleroderma, etc. [1–3]. Inbred lines of mice with autoimmune diseases serve as a model to study various aspects of autoimmunity in humans. MRL-lpr/lpr mice develop autoimmune disorders with age; these are similar to those observed in patients with SLE and rheumatoid arthritis [4]. The F 1 hybrids of New Zealand NZB/NZW mice spontaneously develop nephritis resembling that in patients with SLE [5]. In contrast, SJL/J mice are characterized by multiple autoimmune abnormalities, including experimental allergic encephalomyelitis, which is a model of human multiple sclerosis [6]. Of interest is a natural spectrum of catalytic antibodies in these animals. In this work, the catalytic activities of natural antibodies in MRL-lpr/lpr, SJL/J, and NZB/NZW F 1 mice were compared for the first time with those in BALB/c mice. The animals were kept under sterile conditions to minimize the interaction of their immune systems with foreign antigens (the Specific Pathogen-Free status). Polyclonal IgG antibodies were isolated from mouse blood serum using a previously developed chromatographic technique, precluding the contamination of the final preparation with serum enzymes [7]. The antibodies’ capacity to hydrolyze DNA, proteins, and esters was assessed because abzymes were previously shown to catalyze the hydrolysis of these substances [1, 8, 9]. To determine the DNA-hydrolyzing activity of antibodies, their effect on supercoiled plasmid DNA was estimated as described previously [7]. Serum antibodies of MRL-lpr/lpr mice exhibited the highest DNAhydrolyzing activity; they transformed supercoiled DNA not only into the relaxed circular form, but also into the linear form (Fig. 1). Both SJL/J and NZB/NZW F 1 mouse antibodies incubated with supercoiled DNA induced only single-strand nicks in some DNA molecules, converting supercoiled molecules into circular ones. Antibodies isolated from BALB/c mice exhibiting no autoimmunity were almost unable to hydrolyze DNA. The kinetic parameters of plasmid DNA hydrolysis by antibodies isolated from MRL-lpr/lpr mice were studied using the linear dichroism method as described in [10]; the efficiency of catalysis ( k cat / k M ) was 0.12 nM –1 min –1 , which is comparable with data previously obtained on the polyclonal antibodies isolated from the blood of an SLE patient ( k cat / k M = 0.32 nM –1 min –1 ) [10]. Note that, although the autoimmune processes are similar in the MRL-lpr/lpr and NZB/NZW F 1 mice, which have high titers of DNA-binding antibodies in the total pool of immunoglobulins [11], only the antibodies of MRL-lpr/lpr mice exhibit a high DNA-hydrolyzing activity. Proteolytic activity in antibody preparations was measured using both fluorescence [12] and enzymatic methods. The fluorescence method is based on the previously described phenomenon of quenching the fluorescence of a protein overlabeled with fluorophore, which enhanced the fluorescence after the initiation of nicks in a polypeptide chain. Bovine serum albumin overlabeled with fluoresceine isothiocyanate (BSA-FITC) served as a substrate for proteolysis in this test. The activity of antibodies was assayed by a time-dependent increase in fluorescence compared to antibody-free Natural Antibody Catalytic Activities in Mice with Autoimmune Disorders