Cleavage of supercoiled plasmid DNA by autoantibody Fab fragment: application of the flow linear dichroism technique.

Abstract

A highly effective method consisting of two affinity chromatography steps and ion-exchange and gelfiltration chromatography steps was developed for purification of autoantibodies from human sera with DNA-hydrolyzing activity. Antibody Fab fragment, which had been purified 130-fold, was shown to catalyze plasmid DNA cleavage. The flow linear dichroism technique was used for quantitative and qualitative studying of supercoiled plasmid DNA cleavage by these autoantibodies in comparison with DNase I and EcoRI restriction endonuclease. The DNA autoantibody Fab fragment was shown to hydrolyze plasmid DNA by Mg2+-dependent single-strand multiple nicking of the sub-strate. Kinetic properties of the DNA autoantibody Fab fragment were evaluated from the flow linear dichroism and agarose gel electrophoresis data and revealed a high affinity (KmS = 43 nM) and considerable catalytic efficiency (k8caPp/KbS = 0.32 min-l'nM'1) of the reaction. Antibodies able to catalyze a variety of chemical transformations were developed in the last decade, using the strategy of raising antibodies to haptens that resemble the transition state of reactions, as was suggested by Jencks (1) (for review see refs. 2 and 3). An increasing number of autoantibodies with catalytic activity toward natural substrates (4-6), as well as antiidiotypic antibodies exhibiting a catalytic function (7), have also been described recently. To date, however, the antibodymediated catalysis is usually characterized by relatively low rate enhancements, indicating the existence of at least two very important problems. The first one, especially important in the case of naturally occurring antibodies, is the purity of the antibody preparation used for the assay, since even a trace enzyme contaminant may mimic a high antibody turnover number (8). The second problem is the development of sensitive, continuous, and accurate methods for detection and monitoring of such low activity (9, 10). Here we describe a reproducible method of purification of DNA-hydrolyzing autoantibodies from human sera. We also demonstrate the advantages of the flow linear dichroism (FLD) technique for quantitative and qualitative characterization of the interaction of these catalytic antibodies with supercoiled (sc) plasmid DNA. EXPERIMENTAL PROCEDURESChemicals. All chemicals were from Sigma and Merck. Enzymes were obtained from Boehringer Mannheim.Plasmid DNA. Plasmid pUC19 was isolated as described elsewhere (11). More than 95% of the isolated plasmid DNA was in the sc form, judging by 1% agarose gel electrophoresis (AGE). Antibody Isolation. Antibodies in 5 ml of serum were precipitated twice with 50% saturated ammonium sulfate; this was followed by chromatography on a Pharmacia HR 5/5 staphylococcal protein A fast-performance liquid chromatography (FPLC) column, as described in ref. 13 (p. 310).Antibody Purification. Samples isolated with protein A were dialyzed twice for 4 hr against 500 vol of buffer A (20 mM Tris HCl, pH 9.0) at 4°C and applied to a Pharmacia HR 5/5 Mono Q FPLC column...

Autoantibodies to myelin basic protein catalyze site-specific degradation of their antigen

“…The range of catalytic efficiency of the autoantibodies was lower than in the case of sequence-specific proteases; e.g., the catalytic efficiency of bovine enterokinase was 7.1⅐10 5 M Ϫ1 ⅐sec Ϫ1 for its natural protein substrate trypsinogen (19). However, the calculated velocities of catalysis by the MBP autoantibodies are among the highest values reported for the antibody catalysis (16,(20)(21)(22)(23) and, therefore, sufficient for their pathological effects during the slow development of neurodegeneration in the course of MS progression.…”

Catalytic Antibodies in Norm and Systemic Lupus Erythematosus

“…The affinity of NGTA3-pro-DNase for supercoiled DNA is about 3.5 orders of magnitude higher than affinity of scDNA for DNase I (K m = 46-58 µM [122].…”

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients

“…For the second pIgG preparation two pairs of K m (2.6 ± 0.1 and 4.4 ± 0.7 nM) and k cat values ((6.7 ± 0.1)×10 -2 and (29.6 ± 5.0)×10 -2 min -1 ) were revealed. Thus, the affinity the scDNA substrate for AIDS IgGs varied (in terms of K m values) in the range 2.6-53 nM, which correspond to typical K d values for Abantigen interactions and is about 3-4 orders of magnitude higher than affinity of scDNA for DNase I (K M = 46-58 M) (Gololobov et al, 1995). These K m values for scDNA are comparable with the K m for plasmid DNA (43 nM) reported previously for IgG from SLE patients (Gololobov et al, 1995).…”

“…Thus, the affinity the scDNA substrate for AIDS IgGs varied (in terms of K m values) in the range 2.6-53 nM, which correspond to typical K d values for Abantigen interactions and is about 3-4 orders of magnitude higher than affinity of scDNA for DNase I (K M = 46-58 M) (Gololobov et al, 1995). These K m values for scDNA are comparable with the K m for plasmid DNA (43 nM) reported previously for IgG from SLE patients (Gololobov et al, 1995). The catalysis mediated by artificial Abzs against reaction transition states is usually characterized by relatively low reaction rates: k cat values are 10 2 -10 6 -fold lower than for canonical enzymes (Keinan, 2005).…”

“…The catalysis mediated by artificial Abzs against reaction transition states is usually characterized by relatively low reaction rates: k cat values are 10 2 -10 6 -fold lower than for canonical enzymes (Keinan, 2005). The known k cat values for natural Abzs from AI patients vary in the range of 0.001-40 min -1 (Gololobov et al, 1995;Kalaga et al, 1995; ). There are only several exceptions.…”