A DNA-nicking activity was detected in the sera of patients with various autoimmune pathologies and was shown to be a property of autoantibodies. The DNA hydrolyzing activity, which was purified by affinity and high-performance liquid chromatography, corresponded in size to immunoglobulin M (IgM) and IgG and had a positive response to antibodies to human IgG. The DNA hydrolyzing autoantibodies were stable to acid shock and yielded a DNA degradation pattern that was different from that of deoxyribonuclease (DNase) I and blood DNase.

Autoantibody-mediated tissue destruction is among the main features of organ-specific autoimmunity. This report describes ''an antibody enzyme'' (abzyme) contribution to the site-specific degradation of a neural antigen. We detected proteolytic activity toward myelin basic protein (MBP) in the fraction of antibodies purified from the sera of humans with multiple sclerosis (MS) and mice with induced experimental allergic encephalomyelitis. Chromatography and zymography data demonstrated that the proteolytic activity of this preparation was exclusively associated with the antibodies. No activity was found in the IgG fraction of healthy donors. The human and murine abzymes efficiently cleaved MBP but not other protein substrates tested. The sites of MBP cleavage determined by mass spectrometry were localized within immunodominant regions of MBP. The abzymes could also cleave recombinant substrates containing encephalytogenic MBP 85-101 peptide. An established MS therapeutic Copaxone appeared to be a specific abzyme inhibitor. Thus, the discovered epitope-specific antibodymediated degradation of MBP suggests a mechanistic explanation of the slow development of neurodegeneration associated with MS.

Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-tocell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slowgrowing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.ultrahigh-throughput screening | microfluidic encapsulation | butyrylcholinesterase | Staphylococcus aureus | cell-cell interactions T he ultrahigh-throughput (1, 2) technique of screening (uHTS) in a double emulsion was applied in directed enzyme evolution (3, 4) to investigate the idea that a universal genotypephenotype linkage was provided by compartmentalization. Artificial compartments of double emulsions were produced with high polydispersity by shear stress (5, 6), which significantly decreased the portion of uniform droplets and thereby reduced the sensitivity and the maximal sorting rate. By contrast, sophisticated custom sorters demonstrated the screening of precise monodisperse droplets of water-in-oil emulsions generated by microfluidic technology (1, 7). However, it is not always convenient to use custom devices, and the use of oil as a continuous phase limits the sorting rate. Alternatively, compartmentalization in microfluidic double emulsion (MDE) enables uHTS of >10,000 events/s using commercially available cell sorters (8, 9). Furthermore, biocompatible oil and water phases provide viability and proliferation of Escherichia coli cells (10) inside the microenvironment of a double emulsion.Here, we propose an MDE-FACS platform that combines the benefits of previously reported systems based on compartmentalization in MDE and FACS selection together with modern omics (Fig. 1). We succeeded in assembling this platform using commercially available parts, which included straightforward microfluidics (Fig. S1) for MDE generation, multiparametric FACS for uHTS, next-generation sequencing (NGS) for bioinformatic predictions, and mass spectrometry for proteome and secretome analysis. We demonstrated this idea with several single-cell methods (Fig. S2), including the selection of di...

The pathologic role of autoantibodies in autoimmune disease is widely accepted. Recently, we reported that anti-myelin basic protein (MBP) serum Abs from multiple sclerosis (MS) patients exhibit proteolytic activity toward the autoantigen. The aim of this study is to determine MBP epitopes specific for the autoantibodies in MS and compare these data with those from other neuronal disorders (OND), leading to the generation of new diagnostic and prognostic criteria. We constructed a MBP-derived recombinant “epitope library” covering the entire molecule. We used ELISA and PAGE/surface-enhanced laser desorption/ionization mass spectroscopy assays to define the epitope binding/cleaving activities of autoantibodies isolated from the sera of 26 MS patients, 22 OND patients, and 11 healthy individuals. The levels of autoantibodies to MBP fragments 48–70 and 85–170 as well as to whole MBP and myelin oligodendrocyte glycoprotein molecules were significantly higher in the sera of MS patients than in those of healthy donors. In contrast, selective reactivity to the two MBP fragments 43–68 and 146–170 distinguished the OND and MS patients. Patients with MS (77% of progressive and 85% of relapsing-remitting) but only 9% of patients with OND and no healthy donors were positive for catalysis, showing pronounced epitope specificity to the encephalitogenic MBP peptide 81–103. This peptide retained its substrate properties when flanked with two fluorescent proteins, providing a novel fluorescent resonance energy transfer approach for MS studies. Thus, anti-MBP autoantibody-mediated, epitope-specific binding and cleavage may be regarded as a specific characteristic of MS compared with OND and healthy donors and may serve as an additional biomarker of disease progression.

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...

The peptidoglycan recognition protein Tag7 is shown to form a stable 1:1 complex with the major stress protein Hsp70. Neither protein is cytotoxic by itself, but their complex induces apoptotic death in several tumorderived cell lines even at subnanomolar concentrations. The minimal part of Hsp70 needed to evoke cytotoxicity is residues 450 -463 of its peptide-binding domain, but full cytotoxicity requires its ATPase activity; remarkably, Tag7 liberated from the complex at high ATP is not cytotoxic. The Tag7-Hsp70 complex is produced by tag7-transfected cells and by lymphokine-activated killers, being assembled within the cell and released into the medium through the Golgi apparatus by a mechanism different from the commonly known granule exocytosis. Thus, we demonstrate how a heat shock protein may perform functions clearly distinct from chaperoning or cell rescue and how peptidoglycan recognition proteins may be involved in innate immunity and anti-cancer defense.

SignificanceAnalyzing complex microbial communities is the milestone of modern microbiology, calling for “deep functional profiling” techniques. While next generation sequencing revolutionized our understanding of microbiota communities, we still lack high-throughput technologies to precisely determine their functionality. Here we show how cultivation of individual bacteria inside droplets of microfluidic double water-in-oil-in-water emulsion enables us to isolate the clones with a desired activity. This approach allows us not only to select the potent antibiotic producer but also to discover a distinct mechanism of self-resistance as well as assess its efficiency on entire microbiomes. The outcome of this methodology shows that it could be effectively transferred to numerous applications in microbiology and biotechnology.

The concept of ''internal image'' of antiidiotypic antibodies has provided the basis for eliciting catalytic antibodies. A monoclonal IgM 9A8 that was obtained as an antiidiotype to AE-2 mAb, a known inhibitor of acetylcholinesterase, displayed esterolytic activity. Study of recombinant Fab fragments and separate light and heavy chains of 9A8 confirmed that the antibody variable domain encodes the catalytic function, whereas neither part of the primary sequence of the Fab exhibited homology with the enzyme. The specific modification of the 9A8 variable domain by an active site-directed covalent inhibitor revealed the presence of an active site Ser residue. A three-dimensional modeling suggests the existence of a functional catalytic dyad Ser-His. Comparison of active sites of 9A8 and 17E8 esterolytic abzyme raised against transitionstate analog revealed structural similarity although both antibodies were elicited by two different approaches.