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Research project (§ 26 & § 27)
Duration : 2018-01-01 - 2020-12-31

Chlorite dismutases (Clds) are able to efficiently decompose chlorite (ClO2-) into harmless chloride (Cl-) and dioxygen (O2) with chlorite being the sole source of dioxygen. Thereby, a covalent oxygen-oxygen bond is formed, a unique biochemical reaction that in addition is only catalyzed by the water-splitting manganese complex of photosystem II of oxygenic phototrophic organisms. The mechanism of cleavage of chlorite is still under heavy debate. Computational studies suggest homolytic cleavage, thereby producing chlorine monoxide (ClO●) and Compound II [Por…Fe(IV)=O] followed by a rebound step and release of chloride and dioxygen, whereas biochemical studies on pentameric (clade 1) Clds suggest heterolytic cleavage of chlorite thereby forming Compound I [Por+●…Fe(IV)=O] and hypochlorite (HOCl/-OCl). However, there is no experimental proof for the generation of Compound I nor is it known why these oxidoreductases have their pH optimum around pH 5 and are inactivated at alkaline pH. In order to elucidate structure-function relationships in Cld and understand the molecular basis of chlorite degradation, we have selected the dimeric Cld from Cyanothece sp. PCC7425 (CCld) as model enzyme for several reasons. CCld can easily be produced in E. coli, allows the generation of crystals of optimum size at all relevant pH values to be probed by both X-ray and neutron crystallography and for the first time showed distinct spectral features of typical heme b Compound II, which was formed immediately after mixing the ferric enzyme with chlorite. Moreover, CCld exhibits further enzymatic features that contradict the proposed heterolytic but favour the homolytic cleavage mechanism of chlorite. Thus it is the aim of this project to fully clarify the molecular mechanism of chlorite cleavage and O2 formation by using a broad set of biochemical and biophysical methods: (i) the recombinant production and purification of the wild-type protein and selected single mutants; (ii) characterization of these iron-proteins by various spectroscopic (resonance Raman, UV-vis) and electrochemical techniques, (iii) analysis of all individual reaction steps of enzyme cycle by multi-mixing stopped-flow spectroscopy and freeze-quench electron paramagnetic resonance (EPR) spectroscopy; and (iv) elucidation of X-ray and neutron crystal structures in the pH range 5-10. The work will be performed in close cooperation with internationally well-known scientists from Austria (X-ray crystallography: Kristina Djinovic-Carugo), Italy (RR spectroscopy: Giulietta Smulevich), Belgium (EPR spectroscopy: Sabine Van Doorslaer) and USA (neutron crystallography: Leighton Coates). Understanding structure-function relationships of Cld will provide the basis for its application in chemical engineering and bioremediation.
Research project (§ 26 & § 27)
Duration : 2017-11-01 - 2021-08-31

PROJECT SUMMARY A number of Abs targeting oligomannose-type glycans on the HIV envelope spike (Env) have been described in recent years that exhibit broad neutralizing activity (bnAbs). However, eliciting such nAbs by immunization has not been very successful so far. A principal problem may be the host origin of the glycans, with immune tolerance mechanisms limiting the frequency or development of B cells capable of producing Abs with specificity for mammalian oligomannose. For example, Abs elicited by glycoconjugate immunogens presenting oligomannosides are generally unable to bind oligomannose on Env and even when Env-binding Abs have been obtained, such as with recombinant yeast, they appear to bind insufficiently avid to the virus and fail to exert meaningful neutralizing activity. Here, we propose to utilize bacterially derived oligosaccharide analogs of oligomannose to overcome these challenges. We focus in this application on a fairly conserved patch of high-mannose glycans at and surrounding Asn301 and Asn332 on HIV gp120. Prototypic for Abs targeting these oligomannose-type glycans is the PGT128 family of nAbs, which are potent and broadly active, suggesting that a vaccine component able to elicit similar nAbs could offer protection at even modest serum Ab concentrations. We not long ago discovered a bacterial oligosaccharide that closely resembles the D1 arm of mammalian oligomannose and subsequently made synthetic derivatives of it with a D3 arm-like extension. One of these derivatives, in the form of a neoglycoconjugate, is bound avidly by PGT128 family members and, notably, their predicted germline predecessor. More importantly, data from a pilot immunization with the lead conjugate in transgenic animals harboring an unarranged human Ab repertoire show elicitation of oligomannose-specific Abs with HIV cross-neutralizing activity. Here, we propose to expand on these encouraging preliminary studies. Specifically, we wish to elaborate on our conjugate design to heighten Ab responses and continue to utilize transgenic animals to identify an optimal adjuvant+conjugate combination. We also will dissect antibody responses at the serum and repertoire levels to determine similarities between the elicited responses and existing nAbs. Finally, we propose to test our strategy also in macaques to assess the extent to which it may work in outbred systems. In sum, this project will investigate whether glycan mimicry can serve to readily trigger the development of cross-reactive Abs to the highly vulnerable oligomannose patch on HIV Env. If so, this work could inform strategies for targeting other glyco-epitopes on HIV-1.
Research project (§ 26 & § 27)
Duration : 2018-01-01 - 2021-12-31

Das Austrian Biorefinery Center Tulln ist geplant als ein international führendes Zentrum in der angewandten Grundlagenforschung, basierend auf der internationalen Spitzenposition der beteiligten Institute in der Forschung und auf der Konzentration von Kompetenzen und Industrie-Kooperationen am Standort Tulln. Das BOKU ABC-T bündelt Grundlagen- und angewandte Forschung auf dem Gebiet der Bioraffinerie, der Chemie nachwachsender Rohstoffe, neuer Biomaterialien und Analytik von Bioraffinerieströmen am Technopol Tulln. In der vierjährigen ersten Projektphase werden in zehn Modulen mit zehn Formenpartnern grundlagenwissenschaftliche Forschungsfragen bearbeitet, wobei die praktische Relevanz immer durch die jeweilige Firmenkooperation sichergestellt ist.

Supervised Theses and Dissertations