Structure-function Analysis of the SP85/PsB Spore Coat Protein in Dictyostelium

细胞壁对许多真核微生物、植物和动物的生命至关重要。包括纤维素在内的多糖是许多细胞壁的主要成分。对动物、衣单胞菌和涡虫富含蛋白质的细胞壁的研究表明，细胞-基质和基质-基质相互作用是由涉及离散蛋白质结构域的高度特异性接触介导的。该项目的目标是研究盘基骨菌的孢子外壳，以便从长远来看，蛋白质在富含纤维素的细胞壁组装中所起的关键作用。像许多细胞壁一样，孢子外壳由一个富含纤维素的中心区域组成，两侧被富含蛋白质的层包围。其中一种主要的被毛蛋白SP85或PsB，是协调被毛中纤维素-蛋白质相互作用的主要嫌疑人。SP85位于靠近质膜的被膜内层，在体外可以同时结合纤维素和另一种被膜蛋白SP65。根据SP85敲除菌株和个体结构域过度表达菌株的表型，SP85被认为有助于纤维素合成的时间（早期功能）和外层的组织，包括蛋白质的结合和纤维素的限制（晚期功能）。其中一种结构是n端和富含半胱氨酸的c1结构域的融合，它阻断了85%的细胞中纤维素合成所需的检查点，并破坏了15%的细胞中突破阻滞的外层。c1结构域唯一地结合纤维素和SP65。为了确定SP85与纤维素和SP65结合对体内功能的作用，计划进行以下实验：1)在生长细胞中表达的全长SP85基因（SP85从生长细胞中分泌出来，远离其他外壳蛋白）将在c1结构域随机突变，以特异性阻断纤维素或SP65的结合；2)绘制c1结构域和附近黏蛋白样结构域的二硫键和糖基化模式，并用于监测正常和突变sp85的结构；3)将目标1中定义的失活突变引入孢子前细胞中表达的SP85和NC1中，并整合到被皮中。表型分析将评估早期和晚期依赖sp85的功能是如何受到影响的。这些结果有望提供有关SP65如何调节SP85在检查点抑制中的作用的信息，SP65在桥接内层和外层蛋白质中的假设适配器作用，以及纤维素结合是否对SP85的定位和/或在系结和组织纤维素微原纤维中很重要。如果时间允许，另外一组旨在测试阻断sp65结合的突变特异性的实验也将进行。这些方法将测试由体外研究确定的特定结构域活性的体内相关性，因此避免了部分长度表达构建体的单一使用所固有的潜在伪影。据预测，纤维素-SP85- sp65三聚体是一个外壳组装的核心模块，可以在未来的研究中扩展，以了解SP85和其他外壳蛋白中其他地方的离散蛋白质结构域如何组织壁组装。更广泛的影响：SP85富含半胱氨酸结构域的结构-功能关系被定义，预计将推广到在植物和真核微生物中发现的其他富含纤维素的细胞壁的相关结构域，具有经济和健康意义。最终，这些研究很可能为纤维素合酶如何被调节、纤维素如何结晶、纤维素原纤维如何被限制在外蛋白层内以及细胞壁层如何形成建立有用的先例。该项目将继续吸引研究生、本科生和高中生，包括妇女和代表性不足的少数民族，作为研究受训者参与执行与PI在其学术环境中的教育承诺相一致的目标。将与美国和国际上的核心实验室和其他研究实验室进行重要的互动。这些活动将为蛋白质组学、糖生物学和细胞壁生物学的总体关键质量做出贡献，并有望为整个大学社区提供持续的资源。所有发现将通过会议和出版物及时与科学界分享。

Cell walls are essential for the life of many microbial eukaryotes, plants and animals. Polysaccharides including cellulose are a major component of many walls. Studies of protein-rich walls in animals, Chlamydomonas and Volvox have shown that cell-matrix and matrix-matrix interactions are mediated by highly specific contacts involving discrete protein domains. The goal of this project is to study the spore coat of Dictyostelium in order to understand, in the long term, the critical roles played by proteins in the assembly of cellulose-rich walls. Like many cell walls, the spore coat consists of a central cellulose-rich region surrounded on either side by protein-rich layers. One of the major coat proteins, SP85 or PsB, is a prime suspect for coordinating cellulose- protein interactions in the coat. SP85 is located in the inner layer of the coat near the plasma membrane, and can simultaneously bind cellulose and another coat protein, SP65, in vitro . Based on the phenotypes of SP85 knockout strains and strains in which individual domains have been overexpressed, SP85 is thought to contribute to both the timing of cellulose synthesis (early function) and the organization of the outer layer including incorporation of proteins and confinement of cellulose (late function). One construct, a fusion of the N-terminal and cysteine-rich C1-domains, blocks a checkpoint required for cellulose synthesis in 85% of the cells, and disrupts the outer layer in the 15% that break through the block. The C1-domain uniquely binds both cellulose and SP65.To determine the role of binding of SP85 to cellulose and SP65 for function in vivo, the following experiments are planned: 1), the gene for full-length SP85 expressed in growing cells, from which it is secreted away from other coat proteins, will be subjected to random mutagenesis in its C1-domain to specifically block either cellulose-or SP65-binding; 2), the disulfide-bonding and glycosylation patterns of the C1-domain and nearby mucin-like domains will be mapped and used to monitor the structures of normal and mutant SP85s; and 3), the inactivating mutations defined in aim 1 will be introduced into SP85 and NC1 expressed in prespore cells and incorporated into the coat. Phenotype analyses will assess how the early and later SP85-dependent functions are affected. The results are expected to provide information about how SP65 regulates SP85 action in checkpoint inhibition, the hypothesized adaptor role for SP65 in bridging inner-and outer-layer proteins, and whether cellulose-binding is important for localizing SP85 and/or in tethering and organizing cellulose microfibrils. An additional set of experiments, aimed at testing the specificity of mutations that block SP65-binding, will also be carried out if time permits. These approaches will test the in vivo relevance of specific domain activities defined by in vitro studies, therefore avoiding potential artifacts intrinsic to the sole use of partial-length expression constructs. The cellulose-SP85-SP65 trimer is predicted to be a coat assembly core module which can be extended in future studies to understand how discrete protein domains elsewhere in SP85 and in other coat proteins function to organize wall assembly.Broader impacts: The structure-function relationships to be defined for the cysteine-rich domains of SP85 are expected to be generalizable to related domains of other cellulose-rich walls found in plants and eukaryotic microbes of economic and health significance. Ultimately, these studies are likely to establish useful precedents for how cellulose synthase is regulated, how cellulose is crystallized, how cellulose fibrils are constrained within the outer protein layer, and how cell wall layers are formed. The project will continue to engage the participation of graduate,undergraduate and high school students, including women and underrepresented minorities,as research trainees in execution of the aims consistent with the educational commitment of the PI in his academic setting. There will be significant interaction with core laboratories and other research laboratories in both the USA and internationally. These activities will contribute to the overall critical mass of effort in proteomics, glycobiology and cell wall biology, and is expected to constitute a continuing resource for the overall university community. All findings will be shared with the scientific community in timely fashion via meetings and publications.