文昌鱼肽聚糖识别蛋白BbtPGRP3的结构和功能研究

发布时间：2018-07-21 16:55

【摘要】：固有免疫,也叫做非特异性免疫或者天然免疫,在脊椎动物中是抵御病原菌入侵的第一道防线,在无脊椎动物和植物中则是唯一的防御系统。天然免疫系统可以通过数目有限的一系列由胚系编码的模式识别受体(pattern-recognition receptors, PRRs)来识别病原微生物产生的种类相对比较固定的代谢产物,也就是病原相关分子模式(pathogen-associated molecular patterns, PAMPs).病原相关分子模式中非常具有代表性的一类就是作为大多数细菌细胞壁的重要组成成分的肽聚糖(Peptidoglycans, PGNs)。而肽聚糖识别蛋白(Peptidoglycan Recognition Proteins, PGRPs),就是一类可以专一性识别和/或水解肽聚糖的模式识别受体。从除线虫以外的低等无脊椎动物到高等哺乳动物都存在结构上比较保守的肽聚糖识别蛋白,但它们的数量和功能有很大差异。文昌鱼通常被视作脊椎动物祖先的模式生物,由于经历了一个被称作“免疫大爆发”的过程,从而具有了种类非常丰富的天然免疫分子。文昌鱼中目前已经鉴定出来17-18种肽聚糖识别蛋白,通过生物信息学对它们进行分析,这些肽聚糖识别蛋白均含有酰胺酶活性所必须的关键性氨基酸残基,可能作为效应分子行使功能。目前已经被鉴定出来的青岛文昌鱼肽聚糖识别蛋白中,有3个被分别命名为BbtPGRP1-3的蛋白具有非常独特结构域组成,它们都包含一个N端几丁质结合结构域和一个C端肽聚糖识别结构域。而几丁质结合结构域通常广泛存在于各种动物和植物当中,通过多糖结合能力行使抗真菌和／或抗细菌功能。我们选取了青岛文昌鱼的BbtPGRP3蛋白作为研究靶标,解析了它的全长蛋白2.7 A的晶体结构,包括一个N端的类橡胶素(hevein-like)的几丁质结合结构域和一个C端的具有催化活性的肽聚糖识别结构域。同时我们通过定点突变结合酶活实验,确定C端的肽聚糖识别结构域同时具有对DAP类型和Lys类型肽聚糖的酶活,并且对DAP类型肽聚糖的活性更高。而N端的几丁质结合结构域在几丁质结合能力之外,还可以通过多糖结合能力使全长BbtPGRP3相比单独C端肽聚糖识别结构域对Lys类型肽聚糖的活性提高5倍,从而使得BbtPGRP3有一个更为广谱的底物识别特性。最后,我们推测文昌鱼的进化可能是一个基因水平转移结合结构域重排的“模块化进化”过程。
[Abstract]:Innate immunity, also known as nonspecific or innate immunity, is the first line of defense against pathogens in vertebrates and the only defense system in invertebrates and plants. The innate immune system can identify relatively fixed metabolites produced by pathogenic microorganisms by a limited number of pattern-recognition receptor receptors (PRRs) encoded by embryoids, that is, pathogen-associated molecular patternsassociated patterns (PAMPs). Peptidoglycans (PGNs), an important component of the cell wall of most bacteria, is one of the most representative of pathogen-associated molecular patterns. Peptidoglycan recognition proteins (PGRPs) are a kind of pattern recognition receptors that can specifically recognize and / or hydrolyze peptidoglycan. From low invertebrates except nematodes to higher mammals, there are conserved peptidoglycan recognition proteins, but their numbers and functions are very different. Amphioxus is often regarded as a model animal of vertebrate ancestors, with a very rich variety of innate immune molecules due to a process known as "immune explosion". At present, 17-18 peptidoglycan recognition proteins have been identified in amphioxus and analyzed by bioinformatics. These peptidoglycan recognition proteins all contain the key amino acid residues necessary for the activity of actamases. It may function as an effector molecule. Among the peptidoglycan recognition proteins of Qingdao amphioxus, three proteins named BbtPGRP1-3 have very unique domain composition. They contain a N-terminal chitin binding domain and a C-terminal peptidoglycan recognition domain. Chitin binding domains are commonly found in various animals and plants and function as antifungal and / or antimicrobial agents through polysaccharide binding ability. The BbtPGRP3 protein of Qingdao amphioxus was selected as the research target, and the crystal structure of its full-length protein 2.7 A was analyzed. It includes a chitin binding domain of N-terminal rubberoid (hevein-like) and a C-terminal peptidoglycan recognition domain with catalytic activity. At the same time, we confirmed that C-terminal peptidoglycan recognition domain had enzyme activity for DAP type and Lys type peptidoglycan, and had higher activity for DAP type peptidoglycan by site-directed mutation combined with enzyme activity experiment. In addition to the chitin binding ability, the N-terminal chitin binding domain can enhance the activity of the full-length BbtPGRP3 to Lys type peptidoglycan by 5 times compared with the single C-terminal peptidoglycan recognition domain. Thus, BbtPGRP3 has a wider spectrum of substrate recognition characteristics. Finally, we speculate that the evolution of amphioxus may be a "modular evolution" process of gene horizontal transfer and domain rearrangement.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q51

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