嗜盐古菌S-层蛋白O-糖基化及脂修饰相关基因的鉴定及功能研究

Best known for their capacities as extremophiles, Archaea express proteins that enable them to succeed in habitats, which are hostile to many other organisms. Indeed, archaeal proteins are able to remain properly folded and functional in the face of extremes of salinity, temperature, and other adverse physical conditions. Posttranslational modifications may help archaeal proteins overcome the challenges presented by their surroundings. In Archea, the cell wall consists solely of S-layers, which are formed by glycoprotein lattices that function as a shape determining and maintaining framework. S-layer proteins have been widely used as reporter proteins for the study of a variety of posttranslational modifications in Archaea, including both N-/O-glycosylation and lipid modification. Presently, little is known of the steps involved in archaeal O-glycosylation and lipid modification or the relation of such steps to the respective eucaryal or bacterial processes. In this proposed study, at first we plan to identify haloarchael homologues of eucaryal and bacterial O-glycosylation and lipid modification genes through bioinformatic tools. Furthermore, by using gene knock-out/complementation, biochemistry and mass spectrometry, the specific function of the candidate genes will be characterized. The roles of O-glycosylation and lipid modification in maturation, trafficking, and assemly of S-layer proteins and their role in halophilic adaption of this organism will be studied. This study will continue to expand our understanding of the diversity, the biogenesis and the roles of O-glycosylation and lipid modification experienced by S-layer glycoproteins. Such information could provide insight into the strategies adopted by Archaea in the face of the extreme environments in which they can exist. One immediate benefit of relating posttranslational protein modifications to protein structure, stability, and function will be the utilization of enzymes from extremophilic Archaea tailored for a broad spectrum of biotechnological and industrial applications.

古菌可以在高盐、高温等极端环境下生存，这与其蛋白质在极端环境下仍可正确折叠及发挥功能密切相关，而蛋白质的翻译后修饰可能在其中起重要的作用。S-层蛋白是古菌细胞表面最常见的结构之一，对维持菌体形态具有重要作用。嗜盐古菌的S-层蛋白含有N-/O-糖链及C-末端的脂修饰，但目前对S-层糖蛋白的O-糖基化及脂修饰的合成与功能仍完全未知。本项目拟通过生物信息学筛选参与嗜盐古菌S-层蛋白O-糖基化及脂修饰的基因，结合基因敲除/互补、生化分析、质谱等技术鉴定基因功能，并探讨O-糖基化及脂修饰对S-层蛋白成熟、转运、组装及其对菌体适应高盐环境的影响。该项目的研究将完善对古菌蛋白质翻译后修饰途径多样性的认识，并对了解古菌适应极端生存环境的机制及利用蛋白质工程改造蛋白具有指导意义。

蛋白质的糖基化修饰是一种重要的翻译后修饰，在生命三域中普遍存在。在真核生物和细菌中，对糖基化修饰的机制和功能已有较为详尽的研究，而在古菌中的研究则比较滞后。对古菌蛋白质糖基化修饰的研究仅在少数几种模式古菌中有开展，并且主要集中于N-糖基化修饰的研究，对古菌O-糖基化修饰的机制则基本不了解。 在本研究中，我们以嗜盐古菌西班牙盐盒菌(Haloarcula hispanica)为研究对象，从糖蛋白、糖链结构、糖链合成相关基因以及糖基化修饰的生物学功能等方面进行了研究。我们首先鉴定了西班牙盐盒菌的表层蛋白（S-层蛋白），发现该菌的S-层由两个同源的蛋白组成，并证明这两种S-层蛋白均被N-连接的6-sulfo-QuiNβ-(1, 6)-[Glcα-(1, 2)-]Gal和O-连接的Glcα-(1, 4)-Gal糖链修饰。. 此外，在西班牙盐盒菌中，我们以多萜醇磷酸甘露糖-蛋白质O-甘露糖基转移酶(PMT)检索得到了两个基因agl22和aogA，并构建了单基因的敲除株。在aogA基因敲除株中，多种多萜醇磷酸连接的己糖（Dol-P-Hex）积累，并且S-层蛋白不能被N-和O-糖链所修饰；生物信息学分析表明，AogA具有PMT的典型拓扑结构和保守区。根据这些结果推测，AogA可能是负责向S-层蛋白转移O-糖链中第一个半乳糖的蛋白质O-半乳糖基转移酶（PGT），而N-糖基化修饰受阻可能是由于缺失O-糖基化修饰所致。此外，该突变株在高盐环境中的生长能力严重下降，S-层结构增厚，细胞形态的维持及细胞分裂也受到了严重影响。. 在agl22基因敲除株中，多萜醇磷酸连接的N-糖链前体仍可合成，但S-层糖蛋白不能被N-糖链所修饰；与寡糖基转移酶AglB的序列比对发现Agl22含有保守的DXD、DXXK，但不具有WWDYG保守区。这些结果表明，Agl22可能是一种催化N-糖基化修饰的新型寡糖基转移酶（OTase）。另外，突变株在高盐环境下的生存能力和S-层结构未发生明显变化，表明N-糖基化修饰对西班牙盐盒菌的生存并非必需的，但可能具有抑制细菌或真菌生长的生态功能。