DNA hydrolysing IgG catalytic antibodies: an emerging link between psychoses and autoimmunity

It is not uncommon to observe autoimmune comorbidities in a significant subset of patients with psychotic disorders, namely schizophrenia (SCZ) and bipolar disorder (BPD). To understand the autoimmune basis, the DNA abyzme activity mediated by serum polyclonal IgG Abs were examined in psychoses patients, quantitatively, by an in-house optimized DNase assay. A similar activity exhibited by IgG Abs from neuropsychiatric-systemic lupus erythematosus (NP-SLE) patients was used as a comparator. Our data revealed that the IgG DNase activity of SCZ was close to that of NP-SLE and it was twofold higher than the healthy controls. Interestingly, the association between DNase activity with PANSS (positive, general and total scores) and MADRS were noted in a subgroup of SCZ and BPD patients, respectively. In our study group, the levels of IL-6 and total IgG in BPD patients were higher than SCZ and healthy controls, indicating a relatively inflammatory nature in BPD, while autoimmune comorbidity was mainly observed in SCZ patients.

Catalytic antibodies in the bone marrow and other organs of Th mice during spontaneous development of experimental autoimmune encephalomyelitis associated with cell differentiation

Exact mechanisms of autoimmune disease development are still yet unknown. However, it is known that the development of autoimmune diseases is associated with defects in the immune system, namely, the violation of the bone marrow hematopoietic stem cells (HSCs) differentiation profiles. Different characteristics of autoimmune reaction development in experimental autoimmune encephalomyelitis (EAE) prone Th mice characterizing T-lymphocytes response were analyzed using standard approaches. Profiles of several HSCs differentiation of bone marrow (BFU-E, CFU-E, CFU-GM, CFU-GEMM, T- and B-lymphocytes) of Th male and female mice during spontaneous development of EAE were noticeably different. Patterns of total lymphocytes, B- and T-cells proliferation in several different organs (bone marrow, blood, spleen, thymus, and lymph nodes) were also remarkably different. In addition, there were in time noticeable differences in their changes for some organs of male and female mice. Characters of changes in the profiles of CD4 and CD8 cells proliferation in some organs not always coincide with those for total T lymphocytes. The changes in the differentiation profiles of HSCs and the level of lymphocytes proliferation in the bone marrow and other organs were associated with the increase in the concentration of antibodies against DNA, myelin basic protein, and myelin oligodendrocyte glycoprotein, and catalytic antibodies hydrolyzing these substrates. Despite some differences in changes in the analyzed parameters, in general, the spontaneous development of EAE in male and female mice occurs to some extent in a comparable way.

A glycan FRET assay for detection and characterization of catalytic antibodies to the Cryptococcus neoformans capsule

Classic antibody functions include opsonization, complement activation, and enhancement of cellular antimicrobial function. Antibodies can also have catalytic activity, although the contribution of catalysis to their biological functions has been more difficult to establish. With the ubiquity of catalytic antibodies against glycans virtually unknown, we sought to advance this knowledge. The use of a glycan microarray allowed epitope mapping of several monoclonal antibodies (mAbs) against the capsule of Cryptococcus neoformans. From this, we designed and synthesized two glycan-based FRET probes, which we used to discover antibodies with innate glycosidase activity and analyze their enzyme kinetics, including mAb 2H1, the most efficient identified to date. The validity of the FRET assay was confirmed by demonstrating that the mAbs mediate glycosidase activity on intact cryptococcal capsules, as observed by a reduction in capsule diameter. Furthermore, the mAb 18B7, a glycosidase hydrolase, resulted in the appearance of reducing ends in the capsule as labeled by a hydroxylamine-armed fluorescent (HAAF) probe. Finally, we demonstrate that exposing C. neoformans cells to catalytic antibodies results in changes in complement deposition and increased phagocytosis by macrophages, suggesting that the antiphagocytic properties of the capsule have been impaired. Our results raise questions over the ubiquity of antibodies with catalytic activity against glycans and establish the utility of glycan-based FRET and HAAF probes as tools for investigating this activity.

A Glycan FRET Assay for Detection and Characterization of Catalytic Antibodies to the Cryptococcus neoformans Capsule

<p>Classical antibody functions include opsonization, complement activation, and enhancement of cellular antimicrobial function. Antibodies can also have catalytic activity, although the contribution of catalysis to their biological functions has been more difficult to establish. With the ubiquity of catalytic antibodies against glycans virtually unknown, we sought to advance this knowledge. The use of a glycan microarray allowed epitope mapping of several monoclonal antibodies (mAbs) against the capsule of <i>Cryptococcus neoformans</i>. From this, we designed and synthesized two glycan based Förster Resonance Energy Transfer (FRET) probes, which we used to discover antibodies with innate glycosidase activity and analyse their enzyme kinetics, including mAb 2H1, a polysaccharide lyase, and the most efficient glycosidase to date. The validity of the FRET assay was confirmed by demonstrating that the mAbs mediate glycosidase activity on intact cryptococcal capsules, as observed by a reduction in capsule diameter. Further the mAb 18B7, a glycosidase hydrolase, resulted in the appearance of reducing ends in the capsule as labelled by hydroxylamine-armed fluorescent (HAAF) probe. Finally, we demonstrate that exposing <i>C. neoformans </i>cells to catalytic antibodies results in changes in complement deposition and increased phagocytosis by macrophages — suggesting the anti-phagocytic properties of the capsule have been impaired. Our results raise questions over the ubiquity of antibodies with catalytic activity against glycans and establish the utility of glycan-based FRET and HAAF probes as tools for investigating this activity.</p>

A Glycan FRET Assay for Detection and Characterization of Catalytic Antibodies to the Cryptococcus neoformans Capsule

<p>Classical antibody functions include opsonization, complement activation, and enhancement of cellular antimicrobial function. Antibodies can also have catalytic activity, although the contribution of catalysis to their biological functions has been more difficult to establish. With the ubiquity of catalytic antibodies against glycans virtually unknown, we sought to advance this knowledge. The use of a glycan microarray allowed epitope mapping of several monoclonal antibodies (mAbs) against the capsule of <i>Cryptococcus neoformans</i>. From this, we designed and synthesized two glycan based Förster Resonance Energy Transfer (FRET) probes, which we used to discover antibodies with innate glycosidase activity and analyse their enzyme kinetics, including mAb 2H1, a polysaccharide lyase, and the most efficient glycosidase to date. The validity of the FRET assay was confirmed by demonstrating that the mAbs mediate glycosidase activity on intact cryptococcal capsules, as observed by a reduction in capsule diameter. Further the mAb 18B7, a glycosidase hydrolase, resulted in the appearance of reducing ends in the capsule as labelled by hydroxylamine-armed fluorescent (HAAF) probe. Finally, we demonstrate that exposing <i>C. neoformans </i>cells to catalytic antibodies results in changes in complement deposition and increased phagocytosis by macrophages — suggesting the anti-phagocytic properties of the capsule have been impaired. Our results raise questions over the ubiquity of antibodies with catalytic activity against glycans and establish the utility of glycan-based FRET and HAAF probes as tools for investigating this activity.</p>

Synthetic Methods of Phosphonopeptides

Phosphonopeptides are phosphorus analogues of peptides and have been widely applied as enzyme inhibitors and antigens to induce catalytic antibodies. Phosphonopeptides generally contain one aminoalkylphosphonic acid residue and include phosphonopeptides with C-terminal aminoalkylphosphonic acids and phosphonopeptides with a phosphonamidate bond. The phosphonamidate bond in the phosphonopeptides is generally formed via phosphonylation with phosphonochloridates, condensation with coupling reagents and enzymes, and phosphinylation followed by oxidation. Pseudo four-component condensation reaction of amides, aldehydes, alkyl dichlorophosphites, and amino/peptide esters is an alternative, convergent, and efficient strategy for synthesis of phosphonopeptides through simultaneous construction of aminoalkylphosphonic acids and formation of the phosphonamidate bond. This review focuses on the synthetic methods of phosphonopeptides containing a phosphonamidate bond.

Natural Catalytic IgGs Hydrolyzing Histones in Schizophrenia: Are They the Link between Humoral Immunity and Inflammation?

Schizophrenia is known to be accompanied not only with an imbalance in the neurotransmitter systems but also with immune system dysregulation and chronic low-grade inflammation. Extracellular histones and nucleosomes as damage-associated molecular patterns (DAMPs) trigger systemic inflammatory and toxic reactions by activating Toll-like receptors. In this work, we obtained the first evidence that polyclonal IgGs of patients with schizophrenia effectively hydrolyze five histones (H1, H2a, H2b, H3, and H4). Several strict criteria were used to demonstrate that histone-hydrolyzing activity is a property of the analyzed IgGs. The IgGs histone-hydrolyzing activity level, depending on the type of histone (H1–H4), was statistically significantly 6.1–20.2 times higher than that of conditionally healthy donors. The investigated biochemical properties (pH and metal ion dependences, kinetic characteristics) of these natural catalytic IgGs differed markedly from canonical proteases. It was previously established that the generation of natural catalytic antibodies is an early and clear sign of impaired humoral immunity. One cannot, however, exclude that histone-hydrolyzing antibodies may play a positive role in schizophrenia pathogenesis because histone removal from circulation or the inflamed area minimizes the inflammatory responses. Thus, it can be assumed that histone-hydrolyzing antibodies are a link between humoral immunity and inflammatory responses in schizophrenia.

Immunoglobulins with Non-Canonical Functions in Inflammatory and Autoimmune Disease States

Immunoglobulins are known to combine various effector mechanisms of the adaptive and the innate immune system. Classical immunoglobulin functions are associated with antigen recognition and the initiation of innate immune responses. However, in addition to classical functions, antibodies exhibit a variety of non-canonical functions related to the destruction of various pathogens due to catalytic activity and cofactor effects, the action of antibodies as agonists/antagonists of various receptors, the control of bacterial diversity of the intestine, etc. Canonical and non-canonical functions reflect the extreme human antibody repertoire and the variety of antibody types generated in the organism: antigen-specific, natural, polyreactive, broadly neutralizing, homophilic, bispecific and catalytic. The therapeutic effects of intravenous immunoglobulins (IVIg) are associated with both the canonical and non-canonical functions of antibodies. In this review, catalytic antibodies will be considered in more detail, since their formation is associated with inflammatory and autoimmune diseases. We will systematically summarize the diversity of catalytic antibodies in normal and pathological conditions. Translational perspectives of knowledge about natural antibodies for IVIg therapy will be also discussed.

IgGs from Human Milk Hydrolyze microRNAs

Mother’s milk provides breast-fed infants with various nutrients, including peptides, proteins, DNA, RNA, antibodies, and other bioactive components promoting neonatal growth and protecting infants from viral and bacterial infection. The functions of many human milk components regarding the nutrition and protection of newborns may be very different compared to those of various biological fluids of healthy adults. For example, human milk contains catalytic antibodies (abzymes) with protein, lipid, and oligosaccharide kinase activities, which are absent in the biological fluids of healthy people and autoimmune patients. Obviously, the nutrition of infants with fresh breast milk is a special phenomenon having a very specific and important role. Here, we have shown that mother’s milk IgGs effectively split homo-(pN)23, and four miRNAs: miR-137, miR-219a-5p, miR-219-2-3p, and miR-9-5p. It was shown that ribonuclease activity is a unique property of milk IgGs. On average, individual IgGs hydrolyze (pA)23, (pU)23, and (pC)23 nonspecifically and with comparable efficiency, whereas the hydrolysis of four miRNAs is predominately site-specific. The specific sites of the hydrolysis of four miRNAs by IgGs from the blood of schizophrenic (SCZ) patients and secretory immunoglobulins A (sIgAs) from human milk were found earlier. The sites of the hydrolysis of four miRNAs by milk IgGs and sIgA-abzymes are almost the same, but are significantly different in comparison with those for SCZ IgGs. In addition, in contrast to the SCZ IgGs, milk IgGs and sIgAs efficiently hydrolyzed miRNAs in the duplex regions formed by their terminal sequences.

Glycan FRET Probes for Screening Catalytic Antibodies Against Cryptococcus Neoformans Capsule

Catalytic monoclonal antibodies (mAbs) against the capsule of <i>Cryptococcus neoformans</i> have been identified but characterization of their Michaelis-Menten kinetics against oligosaccharides has so far not been possible. To address this, we report the design and synthesis of two glycan based Förster resonance energy transfer (FRET) probes that express a major structural unit of the cryptococcal polysaccharide. These probes allowed the kinetic analysis of four catalytic antibodies with glycosidase activity, including 2H1, an antibody which was not known previously to be catalytic. This is only the second report of an antibody with naturally occurring catalytic activity against glycans and the most efficient identified to date. The probe’s capability as a diagnostic for catalysis was demonstrated by accurately predicting glycosidase activity on the native capsule. Furthermore, we used molecular docking studies to reveal the antibody-glycan interactions, with the first structural insights into these interactions between anti-GXM mAbs and their epitopes. Through modelling we see no classical catalytic residues in the antigen binding site, signifying the possibility of further glycan hydrolyzing mechanisms yet to be discovered.