Georgy B. Telegin

Catalytic antibodies in clinical and experimental pathology: human and mouse models

Most of the data accumulated through studies on natural catalytic autoantibodies indicate that production scales up markedly in pathological abnormalities. We have previously described an increased level of DNA-hydrolyzing autoantibodies in the sera of patients with various autoimmune disorders [systemic lupus erythematosus (SLE), rheumatoid arthritis, scleroderma], HIV infection and lymphoproliferative diseases accompanied by autoimmune manifestations. In the present study, we show that an increased level of catalytic activity of autoantibodies can be observed in the sera of autoimmune mice, thus providing a fundamental insight into the medical relevance of abzymes. Polyclonal autoantibodies purified from sera of NZB/W, MRL-lpr/lpr and SJL/J mice show proteolytic and DNA-hydrolyzing activities, as opposed to those harvested from non-autoimmune BALB/c mice. The expressiveness of the catalytic activity was strongly dependent on the age of the animal. The highest levels of catalytic activity were found in the sera of mice aged between 8 and 12 months; the lowest level was typical of younger animals whose age ranged from 6 to 8 weeks. Specific inhibition assays of the catalytic activities were performed to throw light on the nature of the abzyme activity. Within a cohort of aging animals, a strong correlation between marked autoimmune abnormalities and levels of catalytic activities has been established. Nonimmunized SJL/J mice revealed specific immune responses to myelin basic protein (MBP), skeletal muscle myosin (skMyo) and cardiac myosin (Myo), and highly purified antibodies from their serum show specific proteolytic attack against the target antigens. This finding prompted us to undertake a more detailed study of specific antibody-mediated proteolysis in diseased humans. A targeted catalytic response was originally demonstrated against MBP and Myo in multiple sclerosis and myocarditis patients, respectively.

Antibody Proteases: Induction of Catalytic Response

Most of the data accumulated throughout the years on investigation of catalytic antibodies indicate that their production increases on the background of autoimmune abnormalities. The different approaches to induction of catalytic response toward recombinant gp120 HIV-1 surface protein in mice with various autoimmune pathologies are described. The peptidylphosphonate conjugate containing structural part of gp120 molecule is used for reactive immunization of NZB/NZW F1, MRL, and SJL mice. The specific modification of heavy and light chains of mouse autoantibodies with Val-Ala-Glu-Glu-Glu-Val-PO(OPh)2 reactive peptide was demonstrated. Increased proteolytic activity of polyclonal antibodies in SJL mice encouraged us to investigate the production of antigen-specific catalytic antibodies on the background of induced experimental autoimmune encephalomyelitis (EAE). The immunization of autoimmune-prone mice with the engineered fusions containing the fragments of gp120 and encephalitogenic epitope of myelin basic protein (MBP89-104) was made. The proteolytic activity of polyclonal antibodies isolated from the sera of autoimmune mice immunized by the described antigen was shown. Specific immune response of SJL mice to these antigens was characterized. Polyclonal antibodies purified from sera of the immunized animals revealed proteolytic activity. The antiidiotypic approach to raise the specific proteolytic antibody as an “internal image” of protease is described. The “second order” monoclonal antibodies toward subtilisin Carlsberg revealed pronounced proteolytic activity.

Substrate specificity of catalytic autoantibodies in neurodegenerative processes.

61 Autoantibodies are acknowledged to play a pathological role in development of autoimmune processes [1]. The ability of autoantibodies to penetrate across the blood–brain barrier and to colocalize with neuronal antigens suggests that they may be involved in neurodegenerative processes. Recently, we have discovered the phenomenon of catalytic degradation of myelin basic protein (MBP) by antigen-specific autoantibodies isolated from blood serum of patients with multiple sclerosis (MS) and SJL mice developing experimental autoimmune encephalomyelitis (EAE) and assumed that autoantibodies may be involved in pathological demyelination [2, 3]. It was demonstrated that the catalytic activity of autoantibodies strongly correlates with the stage of progression of pathological process in MS, assessed according to the commonly used EDSS scale [4]. This consistent pattern was confirmed by other authors [5]; however, a number of principal questions regarding the discovered function of anti-MBP autoantibodies remained unanswered. In particular, it remains unclear (1) whether the catalytic autoantibodies to MBP are generated only in MS or in other neuronal pathologies as well and (2) what the specificity of MBP cleavage by autoantibodies is and whether the discovered reaction can be used as a diagnostic marker.

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

Peripherally Applied Synthetic Tetrapeptides HAEE and RADD Slow Down the Development of Cerebral β-Amyloidosis in AβPP/PS1 Transgenic Mice

Two tetrapeptides, HAEE and RADD, which are ionic-complementary to the primary zinc recognition site of amyloid-β (Aβ), have been reported to inhibit zinc-induced dimerization of the Aβ metal-binding domain and slow Aβ aggregation in vitro. In the present study, we investigate the impact of HAEE and RADD on the development of cerebral β-amyloidosis in a mouse model of Alzheimers disease. We have found chronic intravenous administration of each peptide results in significant decrease of amyloid plaque burden in the treated mice.

Exposure to the Epstein–Barr Viral Antigen Latent Membrane Protein 1 Induces Myelin-Reactive Antibodies In Vivo

Multiple sclerosis (MS) is an autoimmune chronic inflammatory disease of the central nervous system (CNS). Cross-reactivity of neuronal proteins with exogenous antigens is considered one of the possible mechanisms of MS triggering. Previously, we showed that monoclonal myelin basic protein (MBP)-specific antibodies from MS patients cross-react with Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1). In this study, we report that exposure of mice to LMP1 results in induction of myelin-reactive autoantibodies in vivo. We posit that chronic exposure or multiple acute exposures to viral antigen may redirect B cells from production of antiviral antibodies to antibodies, specific to myelin antigen. However, even in inbred animals, which are almost identical in terms of their genomes, such an effect is only observed in 20–50% of animals, indicating that this change occurs by chance, rather than systematically. Cross-immunoprecipitation analysis showed that only part of anti-MBP antibodies from LMP1-immunized mice might simultaneously bind LMP1. In contrast, the majority of anti-LMP1 antibodies from MBP-immunized mice bind MBP. De novo sequencing of anti-LMP1 and anti-MBP antibodies by mass spectrometry demonstrated enhanced clonal diversity in LMP1-immunized mice in comparison with MBP-immunized mice. We suggest that induction of MBP-reactive antibodies in LMP1-immunized mice may be caused by either Follicular dendritic cells (FDCs) or by T cells that are primed by myelin antigens directly in CNS. Our findings help to elucidate the still enigmatic link between EBV infection and MS development, suggesting that myelin-reactive antibodies raised as a response toward EBV protein LMP1 are not truly cross-reactive but are primarily caused by epitope spreading.

Hypoacylated LPS from Foodborne Pathogen Campylobacter jejuni Induces Moderate TLR4-Mediated Inflammatory Response in Murine Macrophages

Toll-like receptor 4 (TLR4) initiates immune response against Gram-negative bacteria upon specific recognition of lipid A moiety of lipopolysaccharide (LPS), the major component of their cell wall. Some natural differences between LPS variants in their ability to interact with TLR4 may lead to either insufficient activation that may not prevent bacterial growth, or excessive activation which may lead to septic shock. In this study we evaluated the biological activity of LPS isolated from pathogenic strain of Campylobacter jejuni, the most widespread bacterial cause of foodborne diarrhea in humans. With the help of hydrophobic chromatography and MALDI-TOF mass spectrometry we showed that LPS from a C. jejuni strain O2A consists of both hexaacyl and tetraacyl forms. Since such hypoacylation can result in a reduced immune response in humans, we assessed the activity of LPS from C. jejuni in mouse macrophages by measuring its capacity to activate TLR4-mediated proinflammatory cytokine and chemokine production, as well as NFκB-dependent reporter gene transcription. Our data support the hypothesis that LPS acylation correlates with its bioactivity.

Diagnostics of autoimmune neurodegeneration using fluorescent probing

The discovery of antibody-mediated catalysis was a breakthrough that showed antibody function is not limited to specific binding interactions, and that immunoglobulins (Igs) may also chemically transform their target antigens. Recently, so-called “natural catalytic antibodies” have been intimately linked with several pathologies, where they either protect the organism or contribute to the development of autoimmune abnormalities. Previously, we showed that myelin-reactive autoantibodies from patients with multiple sclerosis (MS) and mice with experimental autoimmune encephalomyelitis (EAE) exhibit the ability to recognize and hydrolyse distinct epitopes within myelin basic protein (MBP). Further, the antibody-mediated cleavage of encephalitogenic MBP peptide 81–103, flanked by two fluorescent proteins, can serve as a novel biomarker for MS. Here, we report the next generation of this biomarker, based on the antibody-mediated degradation of a novel chemically synthesized FRET substrate, comprising the fluorophore Cy5 and the quencher QXL680, interconnected by the MBP peptide 81–99: Cy5-MBP81–99-QXL680. This substrate is degraded upon incubation with either purified antibodies from MS patients but not healthy donors or purified antibodies and splenocytes from EAE but not from non-immunized mice. Data presented herein suggest the elaboration of potential specific, rapid, and sensitive diagnostic criteria of active progressive MS.

Intravenously Injected Amyloid-β Peptide With Isomerized Asp7 and Phosphorylated Ser8 Residues Inhibits Cerebral β-Amyloidosis in AβPP/PS1 Transgenic Mice Model of Alzheimer’s Disease

Cerebral β-amyloidosis, an accumulation in the patient’s brain of aggregated amyloid-β (Aβ) peptides abnormally saturated by divalent biometal ions, is one of the hallmarks of Alzheimer’s disease (AD). Earlier, we found that exogenously administrated synthetic Aβ with isomerized Asp7 (isoD7-Aβ) induces Aβ fibrillar aggregation in the transgenic mice model of AD. IsoD7-Aβ molecules have been implied to act as seeds enforcing endogenous Aβ to undergo pathological aggregation through zinc-mediated interactions. On the basis of our findings on zinc-induced oligomerization of the metal-binding domain of various Aβ species, we hypothesize that upon phosphorylation of Ser8, isoD7-Aβ loses its ability to form zinc-bound oligomeric seeds. In this work, we found that (i) in vitro isoD7-Aβ with phosphorylated Ser8 (isoD7-pS8-Aβ) is less prone to spontaneous and zinc-induced aggregation in comparison with isoD7-Aβ and intact Aβ as shown by thioflavin T fluorimetry and dynamic light scattering data, and (ii) intravenous injections of isoD7-pS8-Aβ significantly slow down the progression of institutional β-amyloidosis in AβPP/PS1 transgenic mice as shown by the reduction of the congophilic amyloid plaques’ number in the hippocampus. The results support the role of the zinc-mediated oligomerization of Aβ species in the modulation of cerebral β-amyloidosis and demonstrate that isoD7-pS8-Aβ can serve as a potential molecular tool to block the aggregation of endogenous Aβ in AD.

Phosphorylation of the Amyloid-Beta Peptide Inhibits Zinc-Dependent Aggregation, Prevents Na,K-ATPase Inhibition, and Reduces Cerebral Plaque Deposition

The triggers of late-onset sporadic Alzheimer’s disease (AD) are still poorly understood. Impairment of protein phosphorylation with age is well-known; however, the role of the phosphorylation in β-amyloid peptide (Aβ) is not studied sufficiently. Zinc-induced oligomerization of Aβ represents a potential seeding mechanism for the formation of neurotoxic Aβ oligomers and aggregates. Phosphorylation of Aβ by Ser8 (pS8-Aβ), localized inside the zinc-binding domain of the peptide, may significantly alter its zinc-induced oligomerization. Indeed, using dynamic light scattering, we have shown that phosphorylation by Ser8 dramatically reduces zinc-induced aggregation of Aβ, and moreover pS8-Aβ suppresses zinc-driven aggregation of non-modified Aβ in an equimolar mixture. We have further analyzed the effect of pS8-Aβ on the progression of cerebral amyloidosis with serial retro-orbital injections of the peptide in APPSwe/PSEN1dE9 murine model of AD, followed by histological analysis of amyloid burden in hippocampus. Unlike the non-modified Aβ that has no influence on the amyloidosis progression in murine models of AD, pS8-Aβ injections reduced the number of amyloid plaques in the hippocampus of mice by one-third. Recently shown inhibition of Na+,K+-ATPase activity by Aβ, which is thought to be a major contributor to neuronal dysfunction in AD, is completely reversed by phosphorylation of the peptide. Thus, several AD-associated pathogenic properties of Aβ are neutralized by its phosphorylation.