EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity

ABSTRACT: BACKGROUND: The endoglycosidase EndoS and the cysteine proteinase SpeB from the human pathogen Streptococcus pyogenes are functionally related in that they both hydrolyze IgG leading to impairment of opsonizing antibodies and thus enhance bacterial survival in human blood. In this study, we further investigated the relationship between EndoS and SpeB by examining their in vitro temporal production and stability and activity of EndoS. Furthermore, theoretical structure modeling of EndoS combined with site-directed mutagenesis and chemical blocking of amino acids was used to identify amino acids required for the IgG glycan-hydrolyzing activity of EndoS. RESULTS: We could show that during growth in vitro S. pyogenes secretes the IgG glycan-hydrolyzing endoglycosidase EndoS prior to the cysteine proteinase SpeB. Upon maturation SpeB hydrolyzes EndoS that then loses its IgG glycan-hydrolyzing activity. Sequence analysis and structural homology modeling of EndoS provided a basis for further analysis of the prerequisites for IgG glycan-hydrolysis. Site-directed mutagenesis and chemical modification of amino acids revealed that glutamic acid 235 is an essential catalytic residue, and that tryptophan residues, but not the abundant lysine or the single cysteine residues, are important for EndoS activity. CONCLUSIONS: We present novel information about the amino acid requirements for IgG glycan-hydrolyzing activity of the immunomodulating enzyme EndoS. Furthermore, we show that the cysteine proteinase SpeB processes/degrades EndoS and thus emphasize the importance of the SpeB as a degrading/processing enzyme of proteins from the bacterium itself

TLR3 and TLR4 expression in the human endometrium and their implications in endometrial diseases

Toll-like Rezeptoren (TLRs) spielen eine entscheidende Rolle in der angeborenen Immunabwehr. Neben der Identifizierung von Pathogenen, sind diese auch an der Modulation der nachfolgenden Immunantwort beteiligt. Obwohl TLR3 und TLR4 bereits im humanen Endometrium nachgewiesen werden konnten, sind die Folgen der TLR-induzierten Zytokinproduktion auf das endometriale Gewebe und eine mögliche Einbindung in pathogenetische Prozesse unbekannt. Bisherige Studien zeigten, dass sowohl in der Entstehung der Endometriose als auch des endometrioiden Endometriumkarzinoms inflammatorische Zytokine wie Interleukine, Tumornekrosefaktor α oder der Transkriptionsfaktor Nuclear factor kappa B eine wichtige Rolle spielen. Da TLR3 und TLR4 in der Lage sind, diese Moleküle zu aktivieren, ist ein Beitrag an der Pathogenese beider endometrialer Erkrankungen anzunehmen. Im Rahmen der vorliegenden Arbeit wurde die TLR3- und TLR4-mRNA- (messenger Ribonukleinsäure) und Protein-Expression im gesunden Endometrium während des menstruellen Zyklus und im postmenopausalen Gewebe untersucht. Generell wurden hierbei höhere TLR4-mRNA-Expressionen im Vergleich zu TLR3 beobachtet. TLR3- und TLR4-mRNA-Level zeigten im menstruellen Zyklus keine signifikanten Expressionsunterschiede. Im Bezug auf die Proteinlokalisation waren TLR3- und TLR4-Proteine hauptsächlich im glandulären und luminalen Epithel zu finden. Des Weiteren konnte TLR4 auf CD14- (cluster of differentiation) und CD163-positiven Immunzellen nachgewiesen werden. Die Untersuchungsergebnisse aus gesundem Gewebe wurden daraufhin mit der TLR3- bzw. TLR4-Expression in peritonealer Endometriose, atypischer endometrialer Hyperplasie und endometrioiden Endometriumkarzinom verglichen. Hierbei zeigte sich im Vergleich zu gesunden Kontrollen im proliferativen eutopen Gewebe von Endometriose-Patientinnen eine signifikant erniedrigte TLR3- und TLR4-mRNA-Expression. Interessanterweise konnte im Vergleich hierzu in korrespondierenden ektopen Läsionen ein signifikanter TLR-Expressionsanstieg beobachtet werden. In endometrialer Hyperplasie und im Endometriumkarzinom wurden im Vergleich zu gesunden postmenopausalen Kontrollen erniedrigte TLR3- und TLR4-Expressionen gefunden. Niedrigste TLR-Transkriptionslevel konnten im undifferenzierten Endometriumkarzinom nachgewiesen werden. Zusammenfassend deuten die veränderten TLR-Expressionen auf eine Beteiligung von TLR3 und TLR4 an den endometrialen Erkrankungen Endometriose und Endometriumkarzinom hin. Zukünftige Studien sind nötig, um zu untersuchen, welche Rolle beide TLRs in der Pathogenese dieser Erkrankungen spielen.Toll-like receptors (TLRs) play an essential role in the innate immune system by initiating and directing immune response to pathogens. Although TLR3 and TLR4 are known to be expressed in the human endometrium, the consequences of TLR-induced cytokine production on the endometrial function and pathogenesis remain unclear. In this study, TLR3 and TLR4 expression was investigated during the menstrual cycle and in postmenopausal endometrium considering endometriosis, hyperplasia and endometrial adenocarcinoma specimens (G1 to G3) applying quantitative RT-PCR and immunolabelling of these proteins. TLR3 and TLR4 proteins were mostly localised to the glandular and luminal epithelium without significant changes in their mRNA levels and protein expression patterns. In addition, TLR4 was present on endometrial dendritic cells, monocytes and macrophages. In endometriosis, TLR3 and TLR4 mRNA expression decreased significantly in eutopic proliferative endometrium compared to healthy endometrium. Interestingly, endometriotic lesions showed a significant increase of TLR3 und TLR4 mRNA expression, compared to the corresponding eutopic tissues, indicating a local activation of the toll-like receptor pathway. As investigated in endometriotic tissues, endometrial hyperplasia and adenocarcinoma revealed significantly reduced receptor levels compared to postmenopausal controls. Within the different tumor stages, however, we could demonstrate a regain of TLR3 and TLR4 expression in G1, followed by a significant decrease in advanced carcinoma stages compared to hyperplasia. Our data suggest an involvement of TLR3 and TLR4 in endometrial diseases as we could demonstrate altered expression levels in endometriosis and endometrial cancer

Heme-Scavenging Role of alpha1-Microglobulin in Chronic Ulcers.

Chronic venous ulcers are characterized by chronic inflammation. Heme and iron, originating from blood cell hemolysis as well as extravascular necrosis, have been implicated as important pathogenic factors due to their promotion of oxidative stress. It was recently reported that the plasma and tissue protein alpha1-microglobulin is involved in heme metabolism. The protein binds heme, and a carboxy-terminally processed form, truncated alpha1-microglobulin, also degrades heme. Here, we show the presence of micromolar levels of heme and free iron in chronic leg ulcer fluids. Micromolar amounts of alpha1-microglobulin was also present in the ulcer fluids and bound to added radiolabeled heme. Truncated alpha1-microglobulin was found in the ulcer fluids and exogenously added alpha1-microglobulin was processed into the truncated alpha1-microglobulin form. Histochemical analysis of chronic wound tissue showed the presence of iron deposits, heme/porphyrins in infiltrating cells basement membranes and fibrin cuffs around vessels, and alpha1-microglobulin ubiquitously distributed but especially abundant in basement membranes around vessels and at fibrin cuffs. Our results suggest that alpha1-microglobulin constitutes a previously unknown defense mechanism against high heme and iron levels during skin wound healing. Excessive heme and iron, which are not buffered by alpha1-microglobulin, may underlie the chronic inflammation in chronic ulcers

TLR3 and TLR4 expression in healthy and diseased human endometrium

<p>Abstract</p> <p>Background</p> <p>Toll-like receptors (TLRs) play an essential role in the innate immune system by initiating and directing immune response to pathogens. TLRs are expressed in the human endometrium and their regulation might be crucial for the pathogenesis of endometrial diseases.</p> <p>Methods</p> <p>TLR3 and TLR4 expression was investigated during the menstrual cycle and in postmenopausal endometrium considering peritoneal endometriosis, hyperplasia, and endometrial adenocarcinoma specimens (grade 1 to 3). The expression studies applied quantitative RT-PCR and immunolabelling of both proteins.</p> <p>Results</p> <p>TLR3 and TLR4 proteins were mostly localised to the glandular and luminal epithelium. In addition, TLR4 was present on endometrial dendritic cells, monocytes and macrophages. TLR3 and TLR4 mRNA levels did not show significant changes during the menstrual cycle. In patients with peritoneal endometriosis, TLR3 and TLR4 mRNA expression decreased significantly in proliferative diseased endometrium compared to controls. Interestingly, ectopic endometriotic lesions showed a significant increase of TLR3 und TLR4 mRNA expression compared to corresponding eutopic tissues, indicating a local gain of TLR expression. Endometrial hyperplasia and adenocarcinoma revealed significantly reduced receptor levels when compared with postmenopausal controls. The lowest TLR expression levels were determined in poor differentiated carcinoma (grade 3).</p> <p>Conclusion</p> <p>Our data suggest an involvement of TLR3 and TLR4 in endometrial diseases as demonstrated by altered expression levels in endometriosis and endometrial cancer.</p

Apolipoprotein D is involved in the mechanisms regulating protection from oxidative stress

Producción CientíficaMany nervous system pathologies are associated with increased levels of apolipoprotein D (ApoD), a lipocalin also expressed during normal development and aging. An ApoD homologous gene in Drosophila , Glial Lazarillo, regulates resistance to stress, and neurodegeneration in the aging brain. Here we study for the first time the protective potential of ApoD in a vertebrate model organism. Loss of mouse ApoD function increases the sensitivity to oxidative stress and the levels of brain lipid peroxidation, and impairs locomotor and learning abilities. Human ApoD overexpression in the mouse brain produces opposite effects, increasing survival and preventing the raise of brain lipid peroxides after oxidant treatment. These observations, together with its transcriptional up-regulation in the brain upon oxidative insult, identify ApoD as an acute response protein with a protective and therefore beneficial function mediated by the control of peroxidated lipids. Key words: learning, lipid peroxidation, lipocalin, locomotor behavior, paraquat, oxidative stress

Pathological Conditions Involving Extracellular Hemoglobin: Molecular Mechanisms, Clinical Significance, and Novel Therapeutic Opportunities for alpha(1)-Microglobulin

Hemoglobin is the major oxygen-carrying system of the blood, but has many potentially dangerous side effects due to oxidation and reduction reactions of the heme-bound iron and oxygen. Extracellular hemoglobin, resulting from hemolysis or exogenous infusion, is shown to be an important pathogenic factor in a growing number of diseases. This review briefly outlines the oxidative/reductive toxic reactions of hemoglobin and its metabolites. It also describes physiological protection mechanisms that have evolved against extracellular hemoglobin, with a focus on the most recently discovered: the heme- and radical-binding protein α1-microglobulin (A1M). This protein is found in all vertebrates including man and operates by rapidly clearing cytosols and extravascular fluids of heme groups and free radicals released from hemoglobin. Five groups of pathological conditions with high concentrations of extracellular hemoglobin are described: hemolytic anemias and transfusion reactions, the pregnancy complication preeclampsia, cerebral intraventricular hemorrhage of premature infants, chronic inflammatory leg ulcers, and infusion of hemoglobin-based oxygen carriers as blood substitutes. Finally, possible treatments of these conditions are discussed, giving special attention to the described protective effects of A1M

Up-Regulation of A1M/α1-Microglobulin in Skin by Heme and Reactive Oxygen Species Gives Protection from Oxidative Damage

During bleeding the skin is subjected to oxidative insults from free heme and radicals, generated from extracellular hemoglobin. The lipocalin α1-microglobulin (A1M) was recently shown to have reductase properties, reducing heme-proteins and other substrates, and to scavenge heme and radicals. We investigated the expression and localization of A1M in skin and the possible role of A1M in the protection of skin tissue from damage induced by heme and reactive oxygen species. Skin explants, keratinocyte cultures and purified collagen I were exposed to heme, reactive oxygen species, and/or A1M and investigated by biochemical methods and electron microscopy. The results demonstrate that A1M is localized ubiquitously in the dermal and epidermal layers, and that the A1M-gene is expressed in keratinocytes and up-regulated after exposure to heme and reactive oxygen species. A1M inhibited the heme- and reactive oxygen species-induced ultrastructural damage, up-regulation of antioxidation and cell cycle regulatory genes, and protein carbonyl formation in skin and keratinocytes. Finally, A1M bound to purified collagen I (Kd = 0.96×10−6 M) and could inhibit and repair the destruction of collagen fibrils by heme and reactive oxygen species. The results suggest that A1M may have a physiological role in protection of skin cells and matrix against oxidative damage following bleeding

Human IgG/FcγR Interactions Are Modulated by Streptococcal IgG Glycan Hydrolysis

BACKGROUND: The human pathogen Streptococcus pyogenes produces an endoglycosidase, EndoS that hydrolyzes the chitobiose core of the asparagine-linked glycan on the heavy chain of human IgG. IgG-binding to Fc gamma receptors (FcgammaR) on leukocytes triggers effector functions including phagocytosis, oxidative burst and the release of inflammatory mediators. The interactions between FcgammaR and the Fc domain of IgG depend on the IgG glycosylation state. METHODOLOGY/PRINCIPAL FINDINGS: Here we show for the first time that EndoS hydrolyzes the heavy chain glycan of all four human IgG subclasses (IgG1-4), in purified form and in a plasma environment. An inactive form of EndoS, obtained by site-directed mutagenesis, binds IgG with high affinity, in contrast to wild type EndoS that only transiently interacts with IgG, as shown by Slot-blotting and surface plasmon resonance technology. Furthermore, EndoS hydrolysis of the IgG glycan influences the binding of IgG to immobilized soluble FcgammaR and to an erythroleukemic cell line, K562, expressing FcgammaRIIa. Incubation of whole blood with EndoS results in a dramatic decrease of IgG binding to activated monocytes as analyzed by flow cytometry. Moreover, the IgG bound to K562 cells dissociates when cells are treated with EndoS. Likewise, IgG bound to immobilized FcgammaRIIa and subsequently treated with EndoS, dissociates from the receptor as analyzed by surface plasmon resonance and Western blot. CONCLUSIONS/SIGNIFICANCE: We provide novel information about bacterial enzymatic modulation of the IgG/FcgammaR interaction that emphasizes the importance of glycosylation for antibody effector functions. Moreover, EndoS could be used as a biochemical tool for specific IgG N-glycan hydrolysis and IgG purification/detection, or as a potential immunosuppressing agent for treatment of antibody-mediated pathological processes

A novel high mobility group box 1 neutralizing chimeric antibody attenuates drug-induced liver injury and postinjury inflammation in mice

Acetaminophen (APAP) overdoses are of major clinical concern. Growing evidence underlines a pathogenic contribution of sterile postinjury inflammation in APAP‐induced acute liver injury (APAP‐ALI) and justifies development of anti‐inflammatory therapies with therapeutic efficacy beyond the therapeutic window of the only current treatment option, N‐acetylcysteine (NAC). The inflammatory mediator, high mobility group box 1 (HMGB1), is a key regulator of a range of liver injury conditions and is elevated in clinical and preclinical APAP‐ALI. The anti‐HMGB1 antibody (m2G7) is therapeutically beneficial in multiple inflammatory conditions, and anti‐HMGB1 polyclonal antibody treatment improves survival in a model of APAP‐ALI. Herein, we developed and investigated the therapeutic efficacy of a partly humanized anti‐HMGB1 monoclonal antibody (mAb; h2G7) and identified its mechanism of action in preclinical APAP‐ALI. The mouse anti‐HMGB1 mAb (m2G7) was partly humanized (h2G7) by merging variable domains of m2G7 with human antibody‐Fc backbones. Effector function‐deficient variants of h2G7 were assessed in comparison with h2G7 in vitro and in preclinical APAP‐ALI. h2G7 retained identical antigen specificity and comparable affinity as m2G7. 2G7 treatments significantly attenuated APAP‐induced serum elevations of alanine aminotransferase and microRNA‐122 and completely abrogated markers of APAP‐induced inflammation (tumor necrosis factor, monocyte chemoattractant protein 1, and chemokine [C‐X‐C motif] ligand 1) with prolonged therapeutic efficacy as compared to NAC. Removal of complement and/or Fc receptor binding did not affect h2G7 efficacy. Conclusion: This is the first report describing the generation of a partly humanized HMGB1‐neutralizing antibody with validated therapeutic efficacy and with a prolonged therapeutic window, as compared to NAC, in APAP‐ALI. The therapeutic effect was mediated by HMGB1 neutralization and attenuation of postinjury inflammation. These results represent important progress toward clinical implementation of HMGB1‐specific therapy as a means to treat APAP‐ALI and other inflammatory conditions. (Hepatology 2016;64:1699‐1710)