GIARDIA ENCYSTMENT: CONTROL OF GA1NAC SYNTHESIS

贾第鞭毛虫是美国和世界上许多其他国家的一种主要肠道疾病。生活史简单而直接，需要附着在寄主肠道微绒毛边缘的营养滋养体和通过粪口途径从寄主传给寄主的包囊。在胆汁诱导的包囊形成过程中，贾第虫滋养体形成一种保护性的囊壁细丝，富含囊壁特异糖N-乙酰半乳糖胺(GalNAc)，作为一种新型的[GalNAcβ1产生3-GalNAc]n均聚物。GalNAc在非包囊滋养体中未检测到，它是由葡萄糖在包囊过程中通过五种可诱导的非沉淀酶的活性合成的：氨基葡萄糖6-磷酸异构酶、GPI、氨基葡萄糖6-磷酸N-乙酰基酶(GlcNPA)、磷酸乙酰氨基葡萄糖变位酶(PAG1cNM)、UDP-N-乙酰氨基葡萄糖焦磷酸化酶(UDP-GlcNAcPP)和UDP-N-乙酰氨基葡萄糖4‘-表异构酶(UDP-GlcNAcE)。我们实验室的目标是深入研究酶的调节，控制贾第鞭毛虫病的分子生物学，以及可能开发的新的化疗药物，这些药物可以靶向这一途径，从而防止包囊的形成，从而帮助控制贾第鞭毛虫病。到目前为止，已对GPI、UDP-GlcNAcPP和CWS进行了纯化(或部分纯化)和鉴定，并对GPI和UDP-GlcNAcPP进行了克隆和测序。我们还表明，GPI是转录调控的，而UDP-GlcNAcPP是结构性的，但对GPI的合成产物氨基葡萄糖6-PO4合成GalNAc具有单向激活作用。在对这一途径的调控进行更深入的研究之前，有必要更多地了解这三种尚未纯化或表征的酶。因此，我们计划使用分子技术与酶分析相结合的方法来1)确定GlcNPA、PAGlcNM和UDP-GlcNAcE的活性是在转录水平(如GPI的情况下)还是在转录后水平(如贾第虫的UDP-N-乙酰氨基葡萄糖焦磷酸化酶)诱导的，以及2)通过纯化(或表达)这些酶并从酶动力学的角度表征它们以及可能的激活剂和抑制剂，来确定GlcNPA、PAG1cNM和UDP-GlcNAcE是否是GalNAc合成途径中的调节酶。

Giardia causes is a major intestinal illness in the United States as well as many other countries worldwide. The life cycle is simple and direct requiring a vegetative trophozoite which attaches to the microvillus brush border of the host's intestine and a cyst which passes from host to host by the fecal-oral route. During cyst formation (encystment) induced by bile, Giardia trophozoites form a protective cyst wall filament rich in the cyst wall specific sugar N-acetylgalactosamine (GalNAC, as a novel [GalNAC beta1 yields 3 GalNAc]n homopolymer). GalNAc, undetected in non-encysting trophozoites, is synthesized from glucose during encystment by the activity of five inducible, nonsedimentable enzymes: Glucosamine 6-phosphate isomerase, GPI, glucosamine 6-phosphate N-acetylase (GlcNPA), phosphoacetylglucosamine mutase (PAG1cNM), UDP-N- acetylglucosamine pyrophosphorylase (UDP-GlcNAcPP), and UDP-N- acetylglucosamine 4' epimerase (UDP-GlcNAcE). The goal of our laboratories is an in-depth study of enzyme regulation for, the molecular biology for the control of, and the possible development of new chemotherapeutic agents that can target this pathway thus acting to prevent the formation of cysts and thus aiding in the control of giardiasis. To date GPI, UDP-GlcNAcPP and CWS have been purified (or partially purified) and characterized, and GPI and UDP-GlcNAcPP have been cloned and sequenced. We have shown also that GPI is transcriptionally regulated while UDP-GlcNAcPP is constitutive but unidirectionally activated toward GalNAc synthesis by glucosamine 6-PO4 the anabolic product of GPI. Before more in depth studies of the regulation of this pathway can be undertaken, it is essential to understand more about the three enzymes which have not yet been purified or characterized. Thus, we plan to use molecular techniques coupled with enzyme analyses to 1) determine whether GlcNPA, PAGlcNM, and UDP-GlcNAcE activities are induced at a transcriptional level (as is the case with GPI) or at a post- transcriptional level (as is the case with Giardia's UDP-N- acetylglucosamine pyrophosphorylase), and 2) determine if GlcNPA, PAG1cNM, and UDP-GlcNAcE are regulatory enzymes in the GalNAc synthetic pathway by purifying (or expressing) these enzymes and characterizing them with respect to enzyme kinetics, and possible activators and inhibitors.