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

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

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

Study of TLR3, TLR4 and TLR9 in breast carcinomas and their association with metastasis

<p>Abstract</p> <p>Background</p> <p>Toll-like receptors (TLRs) have garnered an extraordinary amount of interest in cancer research due to their role in tumor progression. By activating the production of several biological factors, TLRs induce type I interferons and other cytokines, which drive an inflammatory response and activate the adaptive immune system. The aim of this study was to investigate the expression and clinical relevance of TLR3, 4 and 9 in breast cancer.</p> <p>Methods</p> <p>The expression levels of TLR3, TLR4 and TLR9 were analyzed on tumors from 74 patients with breast cancer. The analysis was performed by immunohistochemistry.</p> <p>Results</p> <p>Samples of carcinomas with recurrence exhibited a significant increase in the mRNA levels of TLR3, TLR4 and TLR9. Tumors showed high expression of TLRs expression levels by cancer cells, especially TLR4 and 9. Nevertheless, a significant percentage of tumors also showed TLR4 expression by mononuclear inflammatory cells (21.6%) and TLR9 expression by fibroblast-like cells (57.5%). Tumors with high TLR3 expression by tumor cell or with high TLR4 expression by mononuclear inflammatory cells were significantly associated with higher probability of metastasis. However, tumours with high TLR9 expression by fibroblast-like cells were associated with low probability of metastasis.</p> <p>Conclusions</p> <p>The expression levels of TLR3, TLR4 and TLR9 have clinical interest as indicators of tumor aggressiveness in breast cancer. TLRs may represent therapeutic targets in breast cancer.</p

Redox properties of the lipocalin alpha(1)-microglobulin: Reduction of cytochrome c, hemoglobin, and free iron.

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Alpha-1-microglobulin, heme-binding protein, reductase and antioxidant

This thesis describes structural and functional studies of alpha-1-microglobulin (a1m), a 26 kDa plasma and tissue protein that is evolutionarily well-conserved and belongs to the lipocalin superfamily. The lipocalins are polypeptides of 160-190-amino acids that are folded into an 8-stranded beta-barrel forming a pocket with a hydrophobic interior. a1m carries heterogeneous yellow-brown chromophores covalently bound to one unpaired cysteinyl (C34) and three lysyl residues (K92, 118, 130) located at the entrance to the pocket. a1m is found both in free form and as a complex with IgA in blood and interstitial tissues. We show here that a1m is involved in heme metabolism. The protein binds to heme and, when exposed to hemoglobin or erythrocyte membranes, a processed form of a1m, t-a1m (t=truncated), with a free thiol group in position 34 and lacking the C-terminal tetrapeptide, is released from free a1m and the IgA-complex. The processed t-a1m has an intense yellow-brown colour and has properties suggestive of heme-degradation enzyme activity. The processed t-a1m-form is found in urine as well as in chronic ulcer fluids, where it is co-localized with heme. Chronic ulcers are characterized by long-standing inflammation and hemolysis with a continuous release of heme and iron, which are considered to be pathogenetic factors. These findings suggest that a1m is involved in the defence against heme-mediated oxidation. We also found that a1m has enzymatic reductase and dehydrogenase properties. Thus, a1m reduces cytochrome c, methemoglobin, and free iron to their non-oxidized forms, using the biological electron donors NADH and ascorbic acid as electron-supplying co-factors. Recombinant a1m-variants lacking C34 and/or the chromophore-carrying K92, 118 and 130 have less reducing activity, suggesting that the thiol group in position 34 as well as the nearby located lysyl groups are involved in the reaction. The protein has antioxidation properties, i.e. it inhibits the oxidation of biological targets as collagen and LDL, by the pro-oxidants heme, oxidized hemoglobin, hydrogen peroxide and oxidized iron. a1m can even remove some of the oxidation products on collagen and LDL after they have been formed. a1m has been found in LDL from human plasma, suggesting that a1m is a naturally occurring component of LDL. In vitro, a1m binds specifically to LDL-particles. a1m inhibits the hydroxyl radical-induced peroxidation of purified erythrocyte membranes. a1m can also inhibit the generation of intracellular reactive oxygen species in K562 cells after exposure of the cells to heme and hydrogen peroxide as well as in resting cells. In conclusion, a1m may be regarded as a heme-metabolising protein, a new regulator of oxidation and an extracellular antioxidant