Glycobiology of Giardia

描述（由申请人提供）：蓝氏贾第鞭毛虫通过粪口途径传播，是美国和发展中国家腹泻的重要寄生虫原因。贾第虫的感染和诊断阶段是四核囊肿，其具有部分由独特的GalNAc均聚物的原纤维组成的壁。在之前的资助期间，我们已经发现了许多关于贾第虫蛋白质糖基化（目标1）和囊壁形成（目标2）的发现。 这些发现提出了两个相关的假设，将在拟议的实验中进行。首先，贾第虫中的蛋白质糖基化比人类宿主或模式真核生物如酵母菌中的蛋白质糖基化简单得多。因此，目标1中的贾第虫聚糖的研究将提供关于蛋白质糖基化的多样性和进化的见解，以及提供贾第虫糖蛋白的更好描述。第二个假设是贾第虫的囊壁由少量蛋白质组成，这些蛋白质是结合GalNAc均聚物的原纤维的凝集素。目标2中的研究表征了参与贾第虫囊壁合成和降解的酶，这将有助于深入了解这种寄生虫的发病机制，并提供一种不同于含有多种糖聚合物和约100种蛋白质的真菌的替代壁模型。 在实现目标1方面取得的进展包括证明贾第虫产生2-糖N-聚糖前体，而不是后生动物和真菌产生的14-糖前体。转移N-聚糖的贾第虫寡糖基转移酶（OST）具有单个亚基而不是8个亚基。在真核生物中存在对N-连接聚糖的位点的选择，具有蛋白质折叠的N-聚糖依赖性质量控制（QC），但在贾第虫中不存在这样的选择JDP-GlcNAc是唯一转运到贾第虫的ER腔中的核苷酸-糖。针对特定目标1的拟议实验将表征贾第虫的单亚基OST，并表征我们预测的贾第虫独特的GPI锚。目标2的进展包括使用麦胚凝集素，它与贾第虫N-聚糖结合，以显着富集分泌和膜蛋白。胱氨酸特异性糖蛋白包括GalNAc均聚物合酶的候选者。分离的GalNAc均聚物不含蛋白质，基于卷曲原纤维的互锁环保持其中空球形。贾第虫囊壁蛋白1的富含亮氨酸的重复结构域是结合GalNAc均聚物的凝集素。针对特定目标1的拟议实验将进一步探索贾第虫囊壁的卷曲原纤维和凝集素模型，分离独特GalNAc均聚物的合酶，并表征在脱囊期间分解GalNAc聚合物的糖水解酶。

DESCRIPTION (provided by applicant): Giardia lamblia, which is spread by the fecal-oral route, is an important parasitic cause of diarrhea in the US and the developing world. The infectious and diagnostic stage of Giardia is the quadranucleate cyst, which has a wall composed in part by fibrils of a unique GalNAc homopolymer. During the previous funding period, we have made many discoveries concerning glycosylation of Giardia proteins (Aim 1) and the formation of the cyst wall (Aim 2). These discoveries have suggested two related hypotheses that will be pursued in the proposed experiments. First, protein glycosylation in Giardia is much simpler than that of the human host or of model eukaryotes such as Saccharomyces. Therefore studies of Giardia glycans in Aim 1 will provide insights concerning the diversity and evolution of protein glycosylation, as well as providing a better description of Giardia glycoproteins. The second hypothesis is that the cyst wall of Giardia is composed of a small number of proteins, which are lectins that bind fibrils of the GalNAc homopolymer. Studies in Aim 2 that characterize enzymes involved in synthesis and degradation of the Giardia cyst wall will lead to insights into the mechanism of pathogenesis of this parasite and provide an alternative wall model that is distinct from those of fungi that contain multiple sugar polymers and ~100 proteins. Progress towards Aim 1 included demonstration that Giardia makes a 2-sugar N-glycan precursor rather than the 14-sugar precursor made by metazoans and fungi. The Giardia oligosaccharyl transferase (OST) that transfers N-glycans has a single subunit rather than 8 subunits. There is selection for sites of N- linked glycans in eukaryotes with N-glycan-dependent quality control (QC) of protein folding, but there is no such selection in Giardia.UDP-GlcNAc is the only nucleotide-sugar transported into the ER lumen of Giardia. Proposed experiments towards Specific Aim 1 will characterize the single-subunit OST of Giardia and characterize what we predict is a unique GPI anchor of Giardia. Progress toward Aim 2 included use of wheat germ agglutinin, which binds to the Giardia N-glycans, to dramatically enrich secreted and membrane proteins. Cyst-specific glycoproteins included a candidate for the GalNAc homopolymer synthase. The GalNAc homopolymer, which was isolated free of protein, maintains its hollow spherical shape based upon interlocking loops of curled fibrils. The Leu-rich repeat domain of the Giardia cyst wall protein 1 is a lectin that binds the GalNAc homopolymer. Proposed experiments towards Specific Aim 1 will explore further a curled fibril and lectin model of the Giardia cyst wall, isolate the synthase for the unique GalNAc homopolymer, and characterize the glycohydrolase that breaks down the GalNAc polymer during excystation.