Glycobiology of Giardia

描述（由申请人提供）：兰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。贾第鞭毛虫的感染和诊断阶段是四核囊肿，其壁部分由一种独特的GalNAc均聚物的原纤维组成。在之前的资助期间，我们在贾第鞭毛虫蛋白的糖基化（Aim 1）和囊壁的形成（Aim 2）方面取得了许多发现。这些发现提出了两个相关的假设，这些假设将在拟议的实验中进行。首先，贾第鞭毛虫体内的蛋白质糖基化比人类宿主或酵母菌等模式真核生物要简单得多。因此，Aim 1中贾第鞭毛虫多糖的研究将提供有关蛋白质糖基化的多样性和进化的见解，并提供贾第鞭毛虫糖蛋白的更好描述。第二种假设是贾第鞭毛虫的囊壁由少量蛋白质组成，这些蛋白质是结合GalNAc均聚物原纤维的凝集素。Aim 2中对贾第鞭毛虫囊壁合成和降解酶的研究将有助于深入了解这种寄生虫的发病机制，并提供不同于含有多种糖聚合物和约100种蛋白质的真菌的另一种囊壁模型。目标1的进展包括证明贾第鞭毛虫产生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 -聚糖的贾第鞭草寡糖转移酶（OST）只有一个亚基而不是8个亚基。真核生物中存在N-聚糖依赖性蛋白折叠质量控制（QC）对N-聚糖位点的选择，但贾第鞭毛虫没有这种选择。UDP-GlcNAc是唯一被转运到贾第鞭毛虫内质网腔的核苷酸糖。针对Specific Aim 1的拟议实验将表征贾第鞭毛虫的单亚基OST，并表征我们预测的贾第鞭毛虫独特的GPI锚点。Aim 2的进展包括使用小麦胚芽凝集素，它与贾第鞭毛虫n -聚糖结合，显著丰富分泌蛋白和膜蛋白。囊肿特异性糖蛋白包括GalNAc均聚合酶的候选蛋白。GalNAc均聚物，被分离出来不含蛋白质，基于卷曲原纤维的互锁环保持其中空的球形。贾第鞭毛虫囊壁蛋白1的富leu重复结构域是一种结合GalNAc均聚物的凝集素。针对Specific Aim 1的实验将进一步探索贾第鞭毛虫囊壁的卷曲纤维和凝集素模型，分离独特的GalNAc均聚物的合酶，并表征在脱落过程中分解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. PUBLIC HEALTH RELEVANCE: Giardia lamblia, which is spread by poor hygiene, is an important cause of diarrhea in the US, Europe, and developing countries. Studies of the sugars that cover the parasite and that make its protective cyst wall are important to understand how Giardia causes disease and may in the long term lead to better diagnostics, vaccines, or therapeutics.