Auf dem Weg zu maßgeschneiderten Oligosacchariden - Chemo-Enzymatische Synthese und physiologische Funktionen von neuartigen Kohlenhydratstrukturen

It is known that eukaryotic cells have an envelope of carbohydrates – the glycocalyx – which plays a crucial role in cell-cell and cell-ligand interaction processes. Additionally, bacterial glycosylation patterns are in the spotlight. Carbohydrate structures influence inflammation, tomor formation and progression, infections and many other biological processes. Glycobiology is a science on the crossroads of chemistry and biology. Synthesis and analysis of carbohydrate structures demand the application of methods from both fields of science and beyond. Carbohydrates are on the one hand chemically similar, on the other hand its structure determination demands basic chemical methods like nuclear magnetic resonance (NMR) and mass spectroscopy (MS) or even their combination. Glycosyl transferases are carbohydrate-active enzymes and are of outstanding relevance in glycobiology. They are involved in the synthesis as well as the degradation and carbohydrate recognition. In this thesis novel oligosaccharides were synthesized by the consecutive action of two fructosyltransferases. The fructosyltransferases SacB from Bacillus megaterium transfers a fructosyl moiety to different acceptors (galactose, mannose, fucose and xylose). The fructosyltransferase Suc1 from Aspergillus niger synthesizes defined tri- and tetrasaccharides. These oligofructosides were examined concerning their immuno-stimulatory properties by a co-cultivation with human colorectal epithelial carcinoma cells (Caco-2). A test regarding the secretion of 25 different cytokines yielded a significantly high level of interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1). The fructosyltransferase SacB from Bacillus megaterium was examined by a intensive mutagenesis study. By enzyme engineering, 27 variants were created on the basis of sequence alignments with fructosyltransferases from other organisms and the solved structure of the fructosyltransferase from Bacillus megaterium (in cooperation with Rebekka Biedendieck and Martin Gamer, Department of Microbiology, TU Braunschweig and Christian Strube, Helmholtz-Centre for Infection Research). The biochemical data of these variants proved their influence on the polysaccharide formation. The variants of the amino acids Y247, N252, K315 and K373 are not located in the active site, but have a clear impact on the poly- vs. oligosaccharide formation. In spite of their altered product spectrum, their kinetic data are similar to the wild-type SacB. On the basis of these examinations, the polymer vs. Oligosaccharide formation mechanism of glycosyltransferases became clearer. Furthermore, a sucrose isomerase from Protaminobacter rubrum was examined. Substrate engineering of this enzyme led to the synthesis of an isomaltulose analogue consisting of a galactose unit instead of glucose. The linkage was determined as alpha-(1,6) analogous to isomaltulose. Finally, chemically functionalized carbohydrates were synthesized and presented on the surface of eukaryotic cells by glycoengineering techniques. In this position they are specific targets on the surface of human larynx carcinoma cells (HEp-2) for further chemical modification. This was performed by the incorporation of azido-functionalized N-acetyl-glucosamine and alkyne-functionalized N-acetyl neuraminic acid. By “click chemistry” with alkynylated rhodamine or azido fluorescein, respectively, these modified carbohydrates were detected.Seit langem ist bekannt, dass eukaryotische Zellen eine Hülle aus Kohlenhydraten (Glycocalyx) besitzen, die eine entscheidende Rolle bei Zell-Zell und Zell-Ligand Interaktionsprozessen spielt. Mehr und mehr treten aber auch bakterielle Glycosylierungen in den Vordergrund. Zuckerstrukturen beeinflussen Entzündungen, Tumorentstehung und -entwicklung, Infektionen und viele andere biologische Prozesse. Die Glycobiologie ist eine Wissenschaft an der Kreuzung zwischen Biologie und Chemie. Aufbau und Analyse von Kohlenhydraten erfordern den Einsatz von Methoden aus beiden Wissenschaftsgebieten und darüber hinaus. Zuckerstrukturen sind einerseits chemisch sehr ähnlich, ihre Strukturaufklärung erfordert aber gängige chemische Methoden wie nuclear magnetic resonance (NMR), mass spectrometry (MS) oder deren Kombinationen. Glycosyltransferasen sind Enzyme, die auf Zucker Aktivität zeigen und sind in der Glycobiologie von besonderer Bedeutung. Sie sind sowohl am Aufbau als auch an Abbau von Zuckerstrukturen sowie der Zuckererkennung beteiligt. In dieser Arbeit wurden durch den prozessiven Einsatz zweier Fructosyltransferasen neuartige Oligosaccharide synthetisiert. Die Fructosyltransferase SacB aus Bacillus megaterium überträgt Fruktose auf verschiedene Akzeptoren (Galactose, Mannose, Fucose und Xylose). Die Fructosyltransferase Suc1 aus Aspergillus niger synthetisiert Tri- und Tetrafructoside von definierter Länge. Diese Oligofructoside wurden auf ihre immmunstimulierende Funktion hin mittels einer Co-Kultivierung mit humanen Darmepithelzellen (Caco-2) untersucht. Ein Test auf Sekretion von 25 unterschiedlichen Cytokinen ergab einen erhöhten Level von Interleukin 8 (IL-8) und monocyte chemoattractant protein 1 (MCP-1). Die Fructosytransferase SacB aus Bacillus megaterium wurde einer intensiven Mutagenesestudie unterzogen. Mittels Enzym-Engineering wurden 27 verschiedene Varianten aufgrund von Sequenzvergleichen mit anderen Fructosyltransferasen sowie der erzeugten Struktur der SacB aus Bacillus megaterium hergestellt (Kooperation mit Rebekka Biedendieck und Martin Gamer, Institut für Mikrobiologie, TU Braunschweig und Christian Strube, Helmholtz-Zentrum für Infektionsforschung). Die biochemische Untersuchung der Varianten zeigte deutlich ihren Einfluss auf die Polysaccharidbildung. Die Varianten der Aminosäuren Y247, N252, K315 und K373 liegen nicht im aktiven Zentrum, beeinflussen aber deutlich die Polysaccharidbildung. Trotz der Variation ihres Polymer-Produktspektrums sind ihre kinetischen Daten dem Wildtyp sehr ähnlich. Der Polymer- gegenüber Oligosaccharid-Bildungsmechanismus von Glykosyltransferasen wurde durch diese Untersuchungen klarer. Weiterhin wurde eine Saccharose-Isomerase aus Protaminobacter rubrum untersucht. Substrat-Engineering dieses Enzyms führte zur Synthese eines Isomaltulose-Analogons, das Galaktose anstelle der Glucose-Einheit besitzt. Die Verknüpfungsart wurde durch Spektroskopie als alpha-(1,6), also analog der Isomaltulose, bestimmt. Als letzter Punkt dieser Arbeit wurden chemisch funktionalisierte Kohlenhydrate erzeugt, die mittels Glyko-Engineering auf Zelloberflächen von eukaryotischen Zellen präsentiert werden sollen. Dort sollen sie als spezifisch funktionalisierbare Angriffspunkte dienen, um die Zelloberflächen von humanen Kehlkopfepithelzellen (HEp-2) gezielt zu modifizieren. Dies gelang durch Einbau von Azid-funktionalisiertem N-acetyl-glucosamin sowie Alkin-funktionalisierter N-acetyl-neuraminsäure. Durch „Click Chemie“ mit Alkin-Rhodamin bzw. Azido-Fluoreszein wurden diese Gruppen auf den Zelloberflächen detektiert

Drug-Induced Exposure of Schistosoma mansoni Antigens SmCD59a and SmKK7

BACKGROUND: Schistosomiasis is a serious health problem especially in developing countries and affects more than 243 million people. Only few anthelmintic drugs are available up to now. A major obstacle for drug treatment is the different developmental stages and the varying host compartments during worm development. Anthelmintic drugs have been tested mainly on adult schistosomes or freshly transformed cercariae. Knowledge concerning the larval stages is lacking. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we used in vitro-grown schistosomula (aged between 2 to 14 days) to investigate drug effects of the three anthelmintics praziquantel, artemether, and oxamniquine. Further, we analyzed the antibody accessibility of two exemplary schistosome antigens SmCD59a and SmKK7, before and after drug treatment. Our results demonstrated that praziquantel applied at a concentration of 1 μM inhibited development of all life stages. Application of 10 μM praziquantel led to dramatic morphological changes of all schistosomula. Artemether at 1 and 10 μM had differential effects depending on whether it was applied to 2-day as compared to 7- and 14-day schistosomula. While 2-day schistosomula were not killed but inhibited from further development, severe morphological damage was seen in 7- and 14-day schistosomula. Oxamniquine (1 and 10 μM) led to severe morphological impairment in all life stages. Analyzing the accessibility of the antigens SmCD59a and SmKK7 before drug treatment showed no antibody binding on living intact schistosomula. However, when schistosomula were treated with anthelmintics, both antigens became exposed on the larvae. Oxamniquine turned out to be most effective in promoting antibody binding to all schistosomula stages. CONCLUSION: This study has revealed marked differences in anthelmintic drug effects against larvae. Drug treatment increases surface antigen presentation and renders larvae accessible to antibody attack

NCX1 represents an ionic Na+ sensing mechanism in macrophages

Inflammation and infection can trigger local tissue Na(+)accumulation. This Na+-rich environment boosts proinflammatory activation of monocyte/macrophage-like cells (M phi s) and their antimicrobial activity. Enhanced Na+-driven M phi function requires the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5), which augments nitric oxide (NO) production and contributes to increased autophagy. However, the mechanism of Na(+)sensing in M phi s remained unclear. High extracellular Na(+)levels (high salt [HS]) trigger a substantial Na(+)influx and Ca(2+)loss. Here, we show that the Na+/Ca(2+)exchanger 1 (NCX1, also known as solute carrier family 8 member A1 [SLC8A1]) plays a critical role in HS-triggered Na(+)influx, concomitant Ca(2+)efflux, and subsequent augmented NFAT5 accumulation. Moreover, interfering with NCX1 activity impairs HS-boosted inflammatory signaling, infection-triggered autolysosome formation, and subsequent antibacterial activity. Taken together, this demonstrates that NCX1 is able to sense Na(+)and is required for amplifying inflammatory and antimicrobial M phi responses upon HS exposure. Manipulating NCX1 offers a new strategy to regulate M phi function

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements