T. V. Bobik

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.

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity

Significance Biocompatible microfluidic double water-in-oil-in-water emulsion (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. The MDE–FACS platform we developed enabled highly sensitive single-cell selection of predesigned activity and exploration of pairwise interactions between target and effector cells without interference from other microbiota species. 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-to-cell 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 slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.

Heavy–light chain interrelations of MS-associated immunoglobulins probed by deep sequencing and rational variation ☆

The mechanisms triggering most of autoimmune diseases are still obscure. Autoreactive B cells play a crucial role in the development of such pathologies and, in particular, production of autoantibodies of different specificities. The combination of deep-sequencing technology with functional studies of antibodies selected from highly representative immunoglobulin combinatorial libraries may provide unique information on specific features in the repertoires of autoreactive B cells. Here, we have analyzed cross-combinations of the variable regions of human immunoglobulins against the myelin basic protein (MBP) previously selected from a multiple sclerosis (MS)-related scFv phage-display library. On the other hand, we have performed deep sequencing of the sublibraries of scFvs against MBP, Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), and myelin oligodendrocyte glycoprotein (MOG). Bioinformatics analysis of sequencing data and surface plasmon resonance (SPR) studies have shown that it is the variable fragments of antibody heavy chains that mainly determine both the affinity of antibodies to the parent autoantigen and their cross-reactivity. It is suggested that LMP1-cross-reactive anti-myelin autoantibodies contain heavy chains encoded by certain germline gene segments, which may be a hallmark of the EBV-specific B cell subpopulation involved in MS triggering.

Multiple sclerosis autoantigen myelin basic protein escapes control by ubiquitination during proteasomal degradation.

Background: Most proteins must be ubiquitinated prior to proteasomal degradation. Results: Myelin basic protein (MBP) is hydrolyzed by the 26S proteasome without ubiquitination in vitro and in mammalian cells. Conclusion: Proteasome-mediated hydrolysis of the multiple sclerosis autoantigen MBP is uncontrolled by the ubiquitination system. Significance: Results reveal the first example of an autoantigen degraded by the proteasome without ubiquitin. The vast majority of cellular proteins are degraded by the 26S proteasome after their ubiquitination. Here, we report that the major component of the myelin multilayered membrane sheath, myelin basic protein (MBP), is hydrolyzed by the 26S proteasome in a ubiquitin-independent manner both in vitro and in mammalian cells. As a proteasomal substrate, MBP reveals a distinct and physiologically relevant concentration range for ubiquitin-independent proteolysis. Enzymatic deimination prevents hydrolysis of MBP by the proteasome, suggesting that an abnormally basic charge contributes to its susceptibility toward proteasome-mediated degradation. To our knowledge, our data reveal the first case of a pathophysiologically important autoantigen as a ubiquitin-independent substrate of the 26S proteasome.

Role of κ→λ light-chain constant-domain switch in the structure and functionality of A17 reactibody.

Catalytic antibody variants with κ and λ light-chain constant domains show differences in their crystal structures which lead to subtle changes in catalytic efficiency and thermodynamic parameters as well as in their affinity for peptide substrates.

Immunoproteasome enhances intracellular proteolysis of myelin basic protein

300 Proteasome is a multisubunit protein complex that exhibits proteolytic activity and is present in the nuclei and cytoplasm of cells. The 20S proteasome, which has a molecular weight of 700 kDa and a sedimenta tion coefficient of 20S, is present as a proteolytic core in a more complex particle, the 26S proteasome [1]. The degradation of proteins in the cell is regulated by the ubiquitinylation system, which marks the old or defective protein molecules for their recognition by the proteasome and subsequent proteolysis [2]. One of the key biological functions of the proteasome is the hydrolysis of intracellular proteins to the antigenic peptides, which are then presented on the cell surface on the major histocompatibility complex class I mole cules [3]. Recent studies indicate the existence of a molecular mechanism by which the peptides gener ated by the proteasome can also be presented on the major histocompatibility complex class II molecules [4]. The catalytic activity of a constitutive proteasome is mediated by three subunits, β1, β2, and β5, which are constitutively expressed in cells. The proteasome, which contains corresponding immunosubunits β1i, β2i, and β5i the catalytic center, is called the immuno proteasome and is significantly different from the con stitutive proteasome in its activity and substrate speci ficity. The set of antigenic peptides produced by the immunoproteasome differs from the set of peptides produced by the constitutive proteasome [5, 6]. It was recently shown that immunoproteasome not only changes the degradation spectrum of antigenic pro teins but also ensures the maintenance of protein homeostasis under conditions of oxidative stress caused by the action of interferons on the cell [7]. The amount of immunoproteasome in cells increases in various diseases (hematologic malignancies [8], rheu matoid arthritis [9], autoimmune colitis [10], Alzhe imer’s disease [11], and Huntington disease [12]). One of the most common and socially significant autoimmune diseases is multiple sclerosis (MS), which is characterized by the destruction of the myelin sheath of nerve fibers. Myelin basic protein (MBP) is a major autoantigen in multiple sclerosis. At present, the molecular mechanisms underlying the develop ment of multiple sclerosis are being actively studied. Recent studies have demonstrated an important role of both the constitutive proteasome and the immuno proteasome in the development of this disease [13]. Earlier, we studied in vitro the proteolysis of MBP by the proteasome isolated from normal mice and mice with experimental autoimmune encephalomy elitis (EAE), an experimental model of MS [14]. Dur ing further development of this research, we created a model to study the intracellular proteolysis of MBP in mammalian cells. Here we show that the intracellular hydrolysis of MBP is significantly accelerated when the proteasome–immunoproteasome balance is shifted toward the latter. It is known that the expression of the myelin basic protein in mammals is detected solely in the central and peripheral nervous systems. This protein is local ized in the membrane of specialized cells, oligoden drocytes and Schwann cells, forming the myelin sheath of axons. Unfortunately, work with primary human neuronal cultures is associated with numerous experimental difficulties (first of all, the lack of mate rial and ethical concerns). In view of this, at the first step of this work, to study the proteolysis of MBP ex vivo we created a genetic construct that made it possible to express a recombinant human MBP in mammalian cells. For this purpose, a DNA fragment 546 bp long, encoding the full length MBP, was amplificated by PCR using specific overlapping oligo nucleotides and then cloned into the pBudCE4.1/EF Immunoproteasome Enhances Intracellular Proteolysis of Myelin Basic Protein

Expression of catalytic antibodies in eukaryotic systems

Expression of recombinant antibodies in mammalian cells is one of the key problems in immuno-biotechnology. Alternatively, expression of a broad panel of antibodies and of their fragments may be effectively performed in yeast cells. We obtained expression strains of the methylotrophic yeast Pichia pastoris producing single-chain human catalytic antibody A17 (A.17scFv), Fab-fragment (A.17Fab), and full-size light chain (A.17Lch). These antibodies were characterized in terms of functional activity. The capacity to specifically bind and transform organophosphorus compounds has been demonstrated for A.17scFv and A.17Fab. The loss of activity of the antibody light chain when expressed alone indicates that the active site is formed by both heavy and light chains of the antibody. We determined the reversible constant Kd and the first order constant (k2) of the reaction of the covalent modification of A.17scFv and A.17Fab by irreversible inhibitor of the serine proteases p-nitrophenyl 8-methyl-8-azobicyclo[3.2.1]phosphonate (phosphonate X). Calculated values indicate that activity of the antibodies expressed in yeast is similar to the full-size antibody A17 and to the single-chain antibody A.17 expressed in CHO and E. coli cells, respectively.

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.

Heterogeneous catalysis on the phage surface: Display of active human enteropeptidase

Enteropeptidase (EC 3.4.21.9) plays a key role in mammalian digestion as the enzyme that physiologically activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the recognition sequence D4K. The high specificity of enteropeptidase makes it a powerful tool in modern biotechnology. Here we describe the application of phage display technology to express active human enteropeptidase catalytic subunits (L-HEP) on M13 filamentous bacteriophage. The L-HEP/C122S gene was cloned in the g3p-based phagemid vector pHEN2m upstream of the sequence encoding the phage g3p protein and downstream of the signal peptide-encoding sequence. Heterogeneous catalysis of the synthetic peptide substrate (GDDDDK-β-naphthylamide) cleavage by phage-bound L-HEP was shown to have kinetic parameters similar to those of soluble enzyme, with the respective Km values of 19 μM and 20 μM and kcat of 115 and 92 s(-1). Fusion proteins containing a D4K cleavage site were cleaved with phage-bound L-HEP/C122S as well as by soluble L-HEP/C122S, and proteolysis was inhibited by soybean trypsin inhibitor. Rapid large-scale phage production, one-step purification of phage-bound L-HEP, and easy removal of enzyme activity from reaction samples by PEG precipitation make our approach suitable for the efficient removal of various tag sequences fused to the target proteins. The functional phage display technology developed in this study can be instrumental in constructing libraries of mutants to analyze the effect of structural changes on the activity and specificity of the enzyme or generate its desired variants for biotechnological applications.