A suite of tools for the study of tryptophan C-mannosylation

Tryptophan C-mannosylation is a co-translational modification unique to the metazoans and apicomplexan parasites. It is the only example of protein C-glycosylation reported to date and appears to play a role in protein folding, trafficking and/or stability. Our knowledge of the prevalence of this modification and its role in biology is limited, and hampered by a lack of tools for studying the modification. Here, we present a simple microbial system for the production of proteins with and without the modification, to facilitate characterisation of the effects of tryptophan mannosylation on protein stability and function. This system facilitates exploration of the C-mannosyltransferase substrate preference and catalytic machinery. The ability to readily produce proteins with this modification facilitated the generation and rigours characterisation of monoclonal antibodies capable of detecting C-mannosyl tryptophan. With these antibodies, and a robust expression system, we were able to identify the first small molecule capable of inhibiting C-mannosyltransferases: a susbtrate-mimicking peptide that is active in cells. Together, these tools provide a solid foundation for interrogating the role of C-mannosyl tryptophan in myriad biological systems.

色氨酸C-甘露糖基化（Tryptophan C-mannosylation）是后生动物（metazoans）与顶复门寄生虫（apicomplexan parasites）特有的共翻译修饰（co-translational modification）。该修饰是目前已报道的唯一一例蛋白质C-糖基化（protein C-glycosylation）现象，被认为在蛋白质折叠（protein folding）、转运（trafficking）及/或稳定性维持中发挥作用。目前学界对该修饰的发生频率（prevalence）及其生物学功能的认知仍较为有限，且受限于相关研究工具的匮乏。本文报道了一套简易的微生物表达系统（microbial system），可用于制备带有或不带有该修饰的蛋白质，以助力解析色氨酸C-甘露糖基化对蛋白质稳定性与功能的影响。该系统可用于探究C-甘露糖基转移酶（C-mannosyltransferase）的底物偏好性（substrate preference）及其催化机制（catalytic machinery）。借助该系统可便捷获取带有该修饰的蛋白质，进而开发并严谨表征可特异性识别C-甘露糖基化色氨酸的单克隆抗体（monoclonal antibodies）。依托上述抗体与稳定的表达系统，我们成功鉴定出首个可抑制C-甘露糖基转移酶的小分子物质（small molecule）：一种可在细胞内发挥活性的底物模拟肽（substrate-mimicking peptide）。综上，上述工具为研究色氨酸C-甘露糖基化在各类生物系统中的功能提供了坚实的研究基础。