Directed evolution of bacterial polysialyltransferases for structural studies and their application for enzymatic polysialylation of therapeutic proteins

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Sialic acids comprise a family of around 50 nine-carbon sugars, which can be regarded as derivatives of the neuramic acid in a broader sense. One of the most important members is N-acetyl neuramic acid (Neu5Ac) and a special form of this building block is the polysialic acid, a long-chain homopolymer which is found tethered almost exclusively to the neuronal marker protein NCAM. Its expression is temporally restricted to the prenatal development phase and is reactivated in adults after injury of peripheral nerves in order to accelerate regeneration. It is well accepted that PSA suppresses the formation of cell-cell contacts - an essential prerequisite for neuronal plasticity in the developing central nervous system. Interestingly, some neuroinvasive pathogens (e.g. Neisseria meningitidis) perfectly adapted to their hosts by forming PSA-containing capsule structures which help to camouflage themselves and prevent immune system attacks.PSA is produced enzymatically in a multistage biosynthesis and finally transferred onto the target molecule by so-called polysialyltransferases (PSTs). Despite intensive research no three-dimensional structures of PSTs are available so far, neither bacterial nor from their vertebrate counterparts. Structural details would offer exciting mechanistic insights into these fascinating, processive working enzymes, and furthermore pave the way for tailored antimicrobial drugs fighting life-threatening infectious diseases.Moreover, pioneer studies demonstrate that chemical conjugation of PSA to protein therapeutics (e.g. insulin, asparaginase) increases their stability and shelf-life in the circulation. Since PSA is non-toxic, biodegradable and possess only low immunogenic potential, it provides a meaningful alternative to common protein PEGylations. Therefore, polysialyltransferases offer the great opportunity for broad application as in vitro catalysts for fast, clean and safe production of PSA-protein conjugates.In order to improve stability and activity of bacterial polysialtransferases, they shall be subjected to directed evolution approaches in order to identify beneficial mutations. For this purpose the development of a novel high-throughput assay is proposed to screen fast and reliable for promising candidates, which are intended to be expressed and purified afterwards in larger scale for structural studies using NMR and X-Ray. Later on in the project, these mutants shall be applied and optimized for in vitro PSAylation of proteins with clinical relevance such as SDF-1, different coagulation factors and serpine-type protease inhibitors. The generated PSA-protein conjugates will be tested in terms of PSA chain length, polydispersity and overall conjugate stability in vitro. Comparative analysis of achieved results along with reported data of pure chemical PSA modifications will reveal strengths and weaknesses of enzymatic PSAylation and help to estimate pharmacokinetic properties of the conjugates.