BIOSYNTHETIC PATHWAY FOR STREPTOCOCCAL GLYCOPROTEINS

描述（改编自申请人的摘要）：S。血竭 血小板聚集相关蛋白（PAAP）已被分离为 115 kDa的前体N-甲酰甲硫氨酰糖蛋白。而其他 在文献中已经提出了原核糖蛋白，这是 第一个来自革兰氏阳性生物体的蛋白质通过直接 实验证据表明存在共价结合的碳水化合物。 初步研究还表明，碳水化合物聚合物不是 以PAAP的壁相关形式存在。通过研究PAAP 生物合成和输出，S. sanguis提供了一个独特的模型系统， 了解原核糖蛋白的新生物合成途径。 申请人假设PAAP提供了一种机制， 将碳水化合物聚合物掺入S. sanguis。 作为一种协同转运蛋白，PAAP可以通过 膜与输出的蛋白质共价结合。 一旦 当PAAP被转运时，碳水化合物寡糖可以被切割， 从蛋白质骨架转移到其他细胞壁成分， 例如脂质或肽聚糖。为了验证这一假设， 该研究项目的目的是：1）识别和表征 含有PAAP寡糖的细胞壁组分，2）分离物 色葡萄血寡糖基转移酶负责 寡糖向PAAP蛋白骨架的细胞内转移， 3)分离负责去除的糖苷酶/转移酶， 将寡糖从输出的PAAP蛋白转移到另一个蛋白 在细胞壁中的位点，4）对分离的 与细胞内和细胞壁相关的蛋白质/酶 寡糖的转移，和5）筛选这些蛋白质/ 其他链球菌中的酶。 为了实现这些目标，免疫学， 将采用生物化学和分子生物学技术。 的 链球菌糖蛋白生物合成途径的阐明 可能会影响对其他生物合成途径的理解，例如 真核糖蛋白生物合成与原核细胞壁 多糖合成口腔链球菌的细胞壁丰富 在多糖中，如果这些结构被共同转运到 细胞表面作为PAAP糖蛋白的一部分， 糖基化途径可能提示改变糖基化途径功能的策略。 这些多糖。

DESCRIPTION (Adapted from applicant's abstract): The S. sanguis Platelet Aggregation-associated Protein (PAAP) has been isolated as a precursor N-formylmethionyl glycoprotein of 115 kDa. While other prokaryotic glycoproteins have been suggested in the literature, this is the first protein from a Gram-positive organism shown by direct experimental evidence to have covalently associated carbohydrate. Preliminary studies also suggest that the carbohydrate polymers are not present in the wall-associated form of PAAP. Therefore, by studying PAAP biosynthesis and export, S. sanguis provides a unique model system to understand the novel biosynthetic pathway of prokaryotic glycoproteins. The applicant hypothesizes that PAAP provides a mechanism for incorporation of carbohydrate polymers onto the cell wall of S. sanguis. Acting as a co-transporter, PAAP may translocate the carbohydrate through the membrane in covalent association with the exported protein. Once PAAP is transported, the carbohydrate oligosaccharides may be cleaved from the protein backbone and transferred to other cell wall components, such as lipid or peptidoglycan. To test this hypothesis, the specific aims of this research project are to: 1) identify and characterize the cell wall component which contains the PAAP oligosaccharides, 2) isolate the S. sanguis oligosaccharyltransferase responsible for the intracellular transfer of oligosaccharide to the PAAP protein backbone, 3) isolate the glycosidase/transferase(s) responsible for removal and transfer of oligosaccharides from the exported PAAP protein to another site in the cell wall, 4) biochemically characterize the isolated proteins/enzymes associated with intracellular and wall-associated transfer of the oligosaccharides, and 5) screen for these proteins/ enzymes in other streptococci. To accomplish these aims, immunologic, biochemical and molecular biological techniques will be employed. The elucidation of this biosynthetic pathway for streptococcal glycoproteins may impact the understanding of other biosynthetic pathways, such as eukaryotic glycoprotein biosynthesis and prokaryotic cell wall polysaccharide synthesis. The cell walls of oral streptococci are rich in polysaccharides, and, if these structures are cotransported to the cell surface as part of the PAAP glycoprotein, identification of the glycosylation pathway may suggest strategies to alter the function of these polysaccharides.