Ligand-Directed Chemistry on Glycoside Hydrolases – A Proof of Concept Study

Selective covalent labelling of enzymes using small molecule probes has advanced the scopes of protein profiling. The covalent bond formation to a specific target is the key step of activity-based protein profiling (ABPP), a method which has become an indispensable tool for measuring enzyme activity in complex matrices. With respect to carbohydrate processing enzymes, strategies for ABPP so far involve labelling the active site of the enzyme, which results in permanent loss of activity. Here, we report in a proof of concept study the use of ligand-directed chemistry (LDC) for labelling glycoside hydrolases near – but not in – the active site. During the labelling process, the competitive inhibitor is cleaved from the probe, departs the active site and the enzyme maintains its catalytic activity. To this end, we designed a building block synthetic concept for small molecule probes containing iminosugar-based reversible inhibitors for labelling of two model β-glucosidases. The results indicate that the LDC approach can be adaptable for covalent proximity labelling of glycoside hydrolases.T. M. W. thanks the FWF (Wien, Austria) for financial support (project number P30372-B21). Authors from TU Graz acknowledge support from NAWI Graz.Peer reviewe

Tools for functional dissection of site-specific <i>O</i>-GlcNAcylation

Protein O-GlcNAcylation is an abundant post-translational modification of intracellular proteins with the monosaccharide N-acetylglucosamine covalently tethered to serines and threonines. Modification of proteins with O-GlcNAc is required for metazoan embryo development and maintains cellular homeostasis through effects on transcription, signalling and stress response. While disruption of O-GlcNAc homeostasis can have detrimental impact on cell physiology and cause various diseases, little is known about the functions of individual O-GlcNAc sites. Most of the sites are modified sub-stoichiometrically which is a major challenge to the dissection of O-GlcNAc function. Here we discuss the application, advantages and limitations of the currently available tools and technologies utilised to dissect the function of O-GlcNAc on individual proteins and sites in vitro and in vivo. Additionally, we provide a perspective on future developments required to decipher the protein- and site-specific roles of this essential sugar modification

Enzym-Substrat Interaktionen in bioaktiven Materialien

Bioaktive Materialen sind allgegenw\ue4rtig und ihre Interaktionen mit den unterschiedlichsten Enzymen bestimmen eine Vielzahl biologischer Prozesse. Spezifische Enzym-Substrat Interaktionen wurden in Rahmen dieser Arbeit untersucht, um ihre Tauglichkeit f\ufcr die Detektion von Wundinfektion und Infektionsprevention zu testen. Es wurden Enzymsubtrate f\ufcr die Detektion von Lysozym und Myeloperoxidase hergestellt, zwei Infektionsbiomarker, die vom Immunsystem in einer fr\ufchen Phase einer beginnenden Infektion produziert werden und in die Wundfl\ufcssigkeit abgegeben werden. In diesem konnte mit Hilfe der hergestellen Substrate eine Infektion schon in einer fr\ufchen Phase durch Farb\ue4nderung signalisiert werden. Diese funktionalen Enzymsubstrate k\uf6nnen in Diagnosesysteme eingebaut werden und dort durch schnelle Infektionsdetektion eine rechtzeitge Wundbehandlung einleiten. Aufgrund der vermehrten Probleme mit bakteriellen Resistenzen bei Infektionbehandlungen liegt weltweit Augenmerk in der Entwicklung neuer antimikrobieller Behandlungsmethoden. In dieser Arbeit wurde ein alternatives antimikrobielles System entwickelt, dass kontinuierlich Wasserstoffperoxid (H2O2) \ufcber 24 h produzieren kann. Das System beruht auf dem H2O2 produzierendem Enzym Cellobiose Dehydrogenase, welches auf Chitosan immobilisiert wurde und das Wachstum representativer Bakterien vollst\ue4ndig inhibieren konnte. Dabei wurde H2O2 in Konzentrationen produziert, die nicht sch\ue4dlich f\ufcr intaktes Gewebe ist, was bei herk\uf6mmlichen H2O2-Behandlungsmethoden bei l\ue4ngerer Behandlungsdauer der Fall ist. Im weiteren Verlauf der Arbeit wurde eine neue Methode zu Produktion antimikrobieller Chitooligosaccharide entwickelt, basierend auf Cellobiohydralasen, dessen F\ue4higkeit zur Hydrolyse von Chitosan nicht bekannt war. Durch unzureichende Produktion und Analyse von bisher beschriebenen Chitooligosacchariden ist wenig \ufcber Auswirkung struktureller Merkmale auf ihre biologisch Aktivit\ue4t bekannt, weshalb ein Hauptaugenmerk dieser Arbeit in der genauen Analyse der Reaktionsprodukte lag.Bioactive materials are ubiquitous in nature and their interactions with specific enzymes drive a variety of biological processes, which can be utilized for various biomedical applications. In this work, various enzymes were studied regarding their suitability for the detection wound infection and infection prevention. Substrates were synthesized for the detection of the immune system-derived wound infection biomarkers lysozyme and myeloperoxidase. These enzymes show highly elevated activities in wound fluids from infected wounds already before an infection is evident. Wound fluids were investigated using the enzyme-responsive substrates and occurring infections could be visually detected within short-time of incubation. The functional substrates are possible candidates for the incorporation in point-of-care-diagnostics to allow early infection detection, which enables a timely initiation of treatment. Another issue in wound treatment is the emergence of bacterial resistances, which complicates wound treatment and strengthens the need for new antimicrobial strategies. An antimicrobial system was developed based on cellobiose dehydrogenase immobilized on chitosan, which led to the continuous production of hydrogen peroxide (H2O2) over 24 h and completely inhibited the growth of representative bacteria. A concentration of H2O2 was produced that is not harmful for intact skin, which was the main obstacle of former treatments. In course of this thesis, an alternative route for the production of antimicrobial chito-oligosaccharides was developed based on the hydrolysis of chitosan by cellobiohydrolases, enzymes with so far unknown activity towards chitosan. Insufficient production and analysis limited the knowledge about structure-function relationships and thus special emphasis was put to the thorough product analysis.eingereicht von DI Gregor TeglAbweichender Titel laut cbersetzung der Verfasserin/des VerfassersZusammenfassung in deutscher SpracheDissertation Universit\ue4t f\ufcr Bodenkultur Wien 201

Enzym-Substrat Interaktionen in bioaktiven Materialien

Bioaktive Materialen sind allgegenwärtig und ihre Interaktionen mit den unterschiedlichsten Enzymen bestimmen eine Vielzahl biologischer Prozesse. Spezifische Enzym-Substrat Interaktionen wurden in Rahmen dieser Arbeit untersucht, um ihre Tauglichkeit für die Detektion von Wundinfektion und Infektionsprevention zu testen. Es wurden Enzymsubtrate für die Detektion von Lysozym und Myeloperoxidase hergestellt, zwei Infektionsbiomarker, die vom Immunsystem in einer frühen Phase einer beginnenden Infektion produziert werden und in die Wundflüssigkeit abgegeben werden. In diesem konnte mit Hilfe der hergestellen Substrate eine Infektion schon in einer frühen Phase durch Farbänderung signalisiert werden. Diese funktionalen Enzymsubstrate können in Diagnosesysteme eingebaut werden und dort durch schnelle Infektionsdetektion eine rechtzeitge Wundbehandlung einleiten. Aufgrund der vermehrten Probleme mit bakteriellen Resistenzen bei Infektionbehandlungen liegt weltweit Augenmerk in der Entwicklung neuer antimikrobieller Behandlungsmethoden. In dieser Arbeit wurde ein alternatives antimikrobielles System entwickelt, dass kontinuierlich Wasserstoffperoxid (H2O2) über 24 h produzieren kann. Das System beruht auf dem H2O2 produzierendem Enzym Cellobiose Dehydrogenase, welches auf Chitosan immobilisiert wurde und das Wachstum representativer Bakterien vollständig inhibieren konnte. Dabei wurde H2O2 in Konzentrationen produziert, die nicht schädlich für intaktes Gewebe ist, was bei herkömmlichen H2O2-Behandlungsmethoden bei längerer Behandlungsdauer der Fall ist. Im weiteren Verlauf der Arbeit wurde eine neue Methode zu Produktion antimikrobieller Chitooligosaccharide entwickelt, basierend auf Cellobiohydralasen, dessen Fähigkeit zur Hydrolyse von Chitosan nicht bekannt war. Durch unzureichende Produktion und Analyse von bisher beschriebenen Chitooligosacchariden ist wenig über Auswirkung struktureller Merkmale auf ihre biologisch Aktivität bekannt, weshalb ein Hauptaugenmerk dieser Arbeit in der genauen Analyse der Reaktionsprodukte lag.Bioactive materials are ubiquitous in nature and their interactions with specific enzymes drive a variety of biological processes, which can be utilized for various biomedical applications. In this work, various enzymes were studied regarding their suitability for the detection wound infection and infection prevention. Substrates were synthesized for the detection of the immune system-derived wound infection biomarkers lysozyme and myeloperoxidase. These enzymes show highly elevated activities in wound fluids from infected wounds already before an infection is evident. Wound fluids were investigated using the enzyme-responsive substrates and occurring infections could be visually detected within short-time of incubation. The functional substrates are possible candidates for the incorporation in point-of-care-diagnostics to allow early infection detection, which enables a timely initiation of treatment. Another issue in wound treatment is the emergence of bacterial resistances, which complicates wound treatment and strengthens the need for new antimicrobial strategies. An antimicrobial system was developed based on cellobiose dehydrogenase immobilized on chitosan, which led to the continuous production of hydrogen peroxide (H2O2) over 24 h and completely inhibited the growth of representative bacteria. A concentration of H2O2 was produced that is not harmful for intact skin, which was the main obstacle of former treatments. In course of this thesis, an alternative route for the production of antimicrobial chito-oligosaccharides was developed based on the hydrolysis of chitosan by cellobiohydrolases, enzymes with so far unknown activity towards chitosan. Insufficient production and analysis limited the knowledge about structure-function relationships and thus special emphasis was put to the thorough product analysis.eingereicht von DI Gregor TeglAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZusammenfassung in deutscher SpracheDissertation Universität für Bodenkultur Wien 201

Leloir glycosyltransferases of natural product <i>C</i>-glycosylation: structure, mechanism and specificity

A prominent attribute of chemical structure in microbial and plant natural products is aromatic C-glycosylation. In plants, various flavonoid natural products have a β-C-d-glucosyl moiety attached to their core structure. Natural product C-glycosides have attracted significant attention for their own unique bioactivity as well as for representing non-hydrolysable analogs of the canonical O-glycosides. The biosynthesis of natural product C-glycosides is accomplished by sugar nucleotide-dependent (Leloir) glycosyltransferases. Here, we provide an overview on the C-glycosyltransferases of microbial, plant and insect origin that have been biochemically characterized. Despite sharing basic evolutionary relationships, as evidenced by their common membership to glycosyltransferase family GT-1 and conserved GT-B structural fold, the known C-glycosyltransferases are diverse in the structural features that govern their reactivity, selectivity and specificity. Bifunctional glycosyltransferases can form C- and O-glycosides dependent on the structure of the aglycon acceptor. Recent crystal structures of plant C-glycosyltransferases and di-C-glycosyltransferases complement earlier structural studies of bacterial enzymes and provide important molecular insight into the enzymatic discrimination between C- and O-glycosylation. Studies of enzyme structure and mechanism converge on the view of a single displacement (SN2)-like mechanism of enzymatic C-glycosyl transfer, largely analogous to O-glycosyl transfer. The distinction between reactions at the O- or C-acceptor atom is achieved through the precise positioning of the acceptor relative to the donor substrate in the binding pocket. Nonetheless, C-glycosyltransferases may differ in the catalytic strategy applied to induce nucleophilic reactivity at the acceptor carbon. Evidence from the mutagenesis of C-glycosyltransferases may become useful in engineering these enzymes for tailored reactivity.</jats:p

Ineffectiveness of hemoadsorption in large animals with abdominal sepsis: a randomized controlled porcine study

Abstract Objectives The use of hemoadsorption (HA) has become popular in the treatment of vasoplegic states associated with massive cytokine release, including septic shock. However, this approach does not seem to be based on robust evidence, and it does not follow international guidelines. To understand the pathophysiological rationale and timing of HA, we conducted a large animal septic shock experiment. Design Prospective randomized large-animal peritoneal septic shock experiment. Setting Laboratory investigation. Subjects Twenty-six anesthetized, mechanically ventilated, and instrumented pigs randomly assigned into (1) sham-operated group with HA (SHAM, n = 5); (2) sepsis animals without HA (SEPSIS, n = 5); (3) sepsis group with HA at norepinephrine initiation (EARLY, n = 8); and (4) sepsis group with HA initiated at norepinephrine rate reaching 0.5 μg/kg/min (LATE, n = 8). Interventions Peritoneal sepsis was induced by cultivated autologous feces inoculation. A CytoSorb cartridge (200 g) with a blood flow rate of 200 mL/min and heparin anticoagulation was used to perform HA. The animals received sedation and intensive organ support up to 48 h or until they experienced cardiovascular collapse. Measurements and main results Systemic hemodynamics, multiple-organ functions, and immune-inflammatory response were measured at predefined periods. The HA treatment was not associated with any measurable benefit in terms of systemic hemodynamics and organ support. The systemic inflammatory markers were unaffected by any of the treatment timings. In contrast, the HA resulted in higher vasopressor load and decreased 36-h survival (5 animals in SHAM (100%), 4 (80%) in SEPSIS, 4 (57%) in EARLY, and 2 (25%) in LATE; p = 0.041). The HA exposure in healthy animals was associated with hemodynamic deterioration, systemic inflammatory response, and cytopenia. Conclusions In this large-animal-controlled fulminant sepsis study, the HA was unable to counteract the disease progression in the early or advanced septic shock phase. However, findings from the HA-exposed sham animals suggest potential safety concerns

Discovery of β-<i>N</i>-acetylglucosaminidases from screening metagenomic libraries and their use as thioglycoligase mutants

Screening of a human gut metagenomic library yielded a set of novel beta-N-acetyl-glucosaminidases. Mutation of active site residues yielded a thioglycoligase that efficiently S-GlcNAcylates protein targets as well as various thiosugar acceptors.</jats:p