New insights into thiocyanate oxidation by human myeloperoxidase.

Human myeloperoxidase (MPO) uses chloride and thiocyanate as physiological substrates at neutral pH. Oxidation of thiocyanate to hypothiocyanite mediated by the redox intermediate Compound I rapidly restores the ferric state of MPO. At low thiocyanate concentration and in the presence of hydrogen peroxide the observed reaction sequence is Compound I→ferric MPO→Compound II→MPO-cyanide complex, whereas at high thiocyanate concentrations and in the absence of H2O2 the only observed transition is Compound I→ferric MPO. The reaction of ferric MPO with hypothiocyanite directly forms the MPO-cyanide complex, whereas a transient product derived from the reaction between hypothiocyanite and hydrogen peroxide is demonstrated to mediate the conversion of ferric MPO to Compound II. Mechanisms for those reactions are discussed and proposed.

Lactoperoxidase as a potential drug target.

INTRODUCTION: Lactoperoxidase (LPO) belongs to the immunologically relevant mammalian heme peroxidases. The enzyme contributes in external secretions to the humoral immune defense against pathogens by oxidation of thiocyanate (SCN(-)) and iodide (I(-)). The generation of oxidized thiocyanate and/or iodine species is also important in numerous biotechnological applications of LPO. AREAS COVERED: In this review, we give an overview about the present knowledge of LPO concerning enzymatic structure, catalytic cycles and (pseudo-)halogenated species generated by the enzyme. Redox properties of LPO as well as kinetic aspects regarding the different enzymatic cycles are discussed in order to gain insights into the disturbance of the (pseudo-)halogenating enzyme activity under pathological conditions. Important structural features of LPO and crystallographic studies on the interaction and reaction of organic substrates with the enzyme are also summarized. A broad discussion is devoted to the binding and oxidation of substrates that either inhibit or promote LPO activity. EXPERT OPINION: On the basis of these data, different strategies to further optimize LPO functions in humoral defense of mucous surfaces and biotechnological applications are discussed. In particular, hydrophobic organic substrates with a 3,4-dihydroxyphenyl partial structure considerably enhance the (pseudo-)halogenating activity of LPO. Their application provides, thus, a new strategy to enhance the anti-microbial activity of this enzyme.

Formation of cyanogen iodide by lactoperoxidase.

The haem protein lactoperoxidase (LPO) is an important component of the anti-microbial immune defence in external secretions and is also applied as preservative in food, oral care and cosmetic products. Upon oxidation of SCN(-) and I(-) by the LPO-hydrogen peroxide system, oxidised species are formed with bacteriostatic and/or bactericidal activity. Here we describe the formation of the inter(pseudo)halogen cyanogen iodide (ICN) by LPO. This product is formed when both, thiocyanate and iodide, are present together in the reaction mixture. Using (13)C nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry we could identify this inter(pseudo)halogen after applying iodide in slight excess over thiocyanate. The formation of ICN is based on the reaction of oxidised iodine species with thiocyanate. Further, we could demonstrate that ICN is also formed by the related haem enzyme myeloperoxidase and, in lower amounts, in the enzyme-free system. As I(-) is not competitive for SCN(-) under physiologically relevant conditions, the formation of ICN is not expected in secretions but may be relevant for LPO-containing products.

Differences in innate immune response between man and mouse.

Mouse strains are frequently used to model human disease states, to test the efficiency of drugs and therapeutic principles. However, the direct translation of murine experimental data to human pathological events often fails due to sufficient differences in the organization of the immune system of both species. Here we give a short overview of the principle differences between mice and humans in defense strategies against pathogens and mechanisms involved in response to pathogenic microorganisms and other activators of the immune system. While in human blood mechanisms of immune resistance are highly prevailed, tolerance mechanisms dominate for the defense against pathogenic microorganisms in mouse blood. Further on, species-related differences of immune cells mainly involved in innate immune response as well as differences to maintain oxidative homeostasis are also considered. A number of disease scenarios in mice are critically reflected for their suitability to serve as a model for human pathologies. Due to setbacks in these studies, novel mouse models were created to bridge the immune system of both species: humanized mice. Accordingly, a special section of this review is devoted to new results applying humanized mouse models taking limitations and prospects into account.

Mechanism of reaction of chlorite with mammalian heme peroxidases.

This study demonstrates that heme peroxidases from different superfamilies react differently with chlorite. In contrast to plant peroxidases, like horseradish peroxidase (HRP), the mammalian counterparts myeloperoxidase (MPO) and lactoperoxidase (LPO) are rapidly and irreversibly inactivated by chlorite in the micromolar concentration range. Chlorite acts as efficient one-electron donor for Compound I and Compound II of MPO and LPO and reacts with the corresponding ferric resting states in a biphasic manner. The first (rapid) phase is shown to correspond to the formation of a MPO-chlorite high-spin complex, whereas during the second (slower) phase degradation of the prosthetic group was observed. Cyanide, chloride and hydrogen peroxide can block or delay heme bleaching. In contrast to HRP, the MPO/chlorite system does not mediate chlorination of target molecules. Irreversible inactivation is shown to include heme degradation, iron release and decrease in thermal stability. Differences between mammalian peroxidases and HRP are discussed with respect to differences in active site architecture and heme modification.

The influence of glycosaminoglycans on IL-8-mediated functions of neutrophils.

Glycosaminoglycans (GAGs) of the extracellular matrix (ECM) contribute to the regulation of physiological processes by binding various immune-competent proteins. Due to their large structural diversity, the analysis of the binding properties and their functional consequences is challenging. The cytokine interleukin-8 (IL-8) is involved in the recruitment of neutrophils to inflammatory sites. Here, we investigated the interaction of heparin hexasaccharides and recombinant human IL-8, consisting of 77 amino acids using fluorescence and NMR spectroscopy. A dissociation constant of 2.0±0.4 µM was determined for the heparin-IL-8 complex, which is slightly higher than what has been found for chondroitin-6-sulfate (K(D)=1.4±0.4 µM) [Pichert, A.; Samsonov, S. A.; Theisgen, S.; Thomas, L.; Baumann, L.; Schiller, J.; Beck-Sickinger, A. G.; Huster, D.; Pisabarro, M. T. Glycobiology2012, 22, 134-145], suggesting an important role of the sulfate group at position 6 of the second ring in the disaccharide unit of the GAGs in this interaction. In addition, the influence of long-chain hyaluronan, chondroitin sulfate, and heparin on IL-8-induced chemotaxis and oxidative activity of neutrophils was examined. Only the incubation of heparin with IL-8 affected the IL-8-mediated chemotaxis of neutrophils. However, all investigated GAGs enhanced the IL-8-induced formation of reactive oxygen species in neutrophils, which is an entirely new finding. This work provides a representative example of how protein functions can be regulated by different GAGs of the ECM.

Functional aspects of the interaction between interleukin-8 and sulfated glycosaminoglycans.

During the immune response, the cytokine interleukin 8 (IL-8, CXCL8) functions as a strong chemoattractant for polymorphonuclear leukocytes helping to direct these cells to infected/injured sites. This review focuses on the interaction of IL-8 with sulfated glycosaminoglycans expressed on cell surfaces and the extracellular matrix. This interaction contributes to the recruitment of polymorphonuclear cells from blood, penetration of these cells through the vessel wall, and their directed migration to inflammatory sites. Regulatory aspects of the interplay between IL-8 and heparan sulfate, the most abundant glycosaminoglycan, are highlighted. In this field, the large natural heterogeneity of glycosaminoglycans represents a great challenge that impedes the modeling of IL-8 functions. The interaction of IL-8 with newly developed artificial sulfated hyaluronan derivatives is also considered as these artificial substrates are an important tool for development of new materials in regenerative medicine.