Establishing strategies for sortase-catalyzed multi-peptide assemblies to profile T-cell selectivity

建立分选酶催化的多肽组装策略来分析 T 细胞选择性

• 批准号: 283011643
• 负责人: Professor Dr. Christian Freund
• 金额: --
• 依托单位: Interfakultäres Institut für Biochemie (IFIB)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/283011643?language=en
• 关键词: Establishing; strategies; sortase; catalyzed; multi

The bacterial transpeptidase sortase A has become a versatile tool for protein chemistry by catalyzing chemoselective ligation reactions of peptides, proteins and synthetic probes. During catalysis sortase cleaves a so-called sorting motif consisting of the amino acid sequence LPxTG or LPxTA at the threonine residue under concomitant formation of a sortase-bound thioester. The enzyme is deacylated by ligating the thioester to the glycine residue of a second peptide that serves as nucleophile. While sortase A of S. aureus is widely used in biochemistry and chemical biology the assembly of multiple peptides is limited by the fact that each sorting motif and nucleophile is recognized by sortase, thereby leading to fragment shuffling and polymerization as a manifesting side reaction when fragment numbers increase. We plan to address these problems by establishing switchable ligation sites. Sorting motif and/or N-Gly nucleophile will be installed in either active on-states, allowing sortase-mediated ligation, or inactive off-state. Simple chemical conversions will allow switching between states and enable multi-fragment assemblies. These developments will go hand-in-hand with directed evolution of sortase selectivity and activity aimed to (a) engineer sortase mutants specific for ligation site switching approaches as well as (b) to generate new sortases with orthogonal substrate selectivity. We plan to use these new tools for establishing tailor-made probes for immunology, in particular for the investigation of T-cell activation. Antigen peptide tetramers equipped with biophysical probes will be assembled by the methods described above. Avidity effects of the assembled antigen tetramers will be exploited for subsequent orthogonal ligation with MHCII molecules and T-cells. These tools should be useful to study T-cell activation and for tracking rare T-cell populations in infection or autoimmune disease models.

细菌转肽酶分选酶A通过催化肽、蛋白质和合成探针的化学选择性连接反应而成为蛋白质化学的通用工具。在催化过程中，分选酶在伴随形成分选酶结合的硫酯的情况下在苏氨酸残基处切割由氨基酸序列LPxTG或LPxTA组成的所谓分选基序。通过将硫酯连接到充当亲核试剂的第二肽的甘氨酸残基上，使酶脱酰。而S.金黄色葡萄球菌广泛用于生物化学和化学生物学。多个肽的组装受到以下事实的限制：每个分选基序和亲核体被分选酶识别，从而导致片段改组和聚合，这是当片段数目增加时的明显副反应。我们计划通过建立可转换的连接位点来解决这些问题。分选基序和/或N-Gly亲核体将以允许分选酶介导的连接的活性开启状态或非活性关闭状态安装。简单的化学转换将允许在状态之间切换并实现多片段组装。这些发展将与分选酶选择性和活性的定向进化齐头并进，目的是（a）工程改造对连接位点转换方法特异性的分选酶突变体以及（B）产生具有正交底物选择性的新分选酶。我们计划使用这些新工具来建立用于免疫学的定制探针，特别是用于T细胞活化的研究。配备有生物物理探针的抗原肽四聚体将通过上述方法组装。组装的抗原四聚体的亲合力效应将用于随后与MHCII分子和T细胞的正交连接。这些工具对于研究T细胞活化和追踪感染或自身免疫性疾病模型中的罕见T细胞群应该是有用的。