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

用于结构研究的细菌聚唾液酸转移酶的定向进化及其在治疗性蛋白质的酶促聚唾液酸化中的应用

• 批准号: 243357277
• 负责人: Dr. Lars Baumann
• 金额: --
• 依托单位: Arbeitsgruppe Biochemie und Bioorganische Chemie
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/243357277?language=en
• 关键词: Directed; evolution; bacterial; polysialyltransferases; structural

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.

唾液酸由大约50个九碳糖组成，在更广泛的意义上可以被视为神经酸的衍生物。其中最重要的成员之一是n -乙酰基神经酸（Neu5Ac），这种构建块的一种特殊形式是聚唾液酸，这是一种长链均聚物，几乎完全与神经元标记蛋白NCAM相连。它的表达暂时局限于产前发育阶段，并在周围神经损伤后在成人中重新激活，以加速再生。人们普遍认为，PSA抑制细胞间接触的形成，而细胞间接触是中枢神经系统发育中神经元可塑性的必要前提。有趣的是，一些神经侵入性病原体（如脑膜炎奈瑟菌）通过形成含有psa的胶囊结构来完美地适应宿主，这些结构有助于伪装自己并防止免疫系统的攻击。PSA是在多阶段生物合成过程中酶促产生的，最后通过所谓的多唾液基转移酶（PSTs）转移到目标分子上。尽管进行了深入的研究，但到目前为止，无论是细菌还是脊椎动物的pst的三维结构都没有得到。结构细节将提供令人兴奋的机制洞察这些迷人的，渐进的工作酶，并进一步为定制抗微生物药物对抗危及生命的传染病铺平道路。此外，先驱研究表明，PSA与蛋白质疗法（如胰岛素、天冬酰胺酶）的化学偶联增加了它们在循环中的稳定性和保质期。由于PSA是无毒的，可生物降解的，只具有低免疫原性的潜力，它提供了一个有意义的替代常见的蛋白质聚乙二醇化。因此，聚唾液基转移酶作为快速、清洁、安全生产psa蛋白偶联物的体外催化剂具有广阔的应用前景。为了提高细菌多唾液转移酶的稳定性和活性，它们应该受到定向进化方法的影响，以确定有益的突变。为此，建议开发一种新的高通量分析方法，以快速可靠地筛选有希望的候选物，这些候选物将在随后的大规模表达和纯化中用于核磁共振和x射线的结构研究。在项目的后期，我们将对这些突变体进行应用和优化，用于体外对具有临床意义的蛋白质如SDF-1、不同凝血因子、丝氨酸型蛋白酶抑制剂等进行psayation。生成的PSA-蛋白偶联物将在体外测试PSA链长、多分散性和整体偶联稳定性。将已获得的结果与报道的纯化学PSA修饰数据进行比较分析，将揭示酶促PSA化的优势和劣势，并有助于估计缀合物的药代动力学性质。