Maternal Control of the Drosophila Embryonic Dorsal/Ventral Axis

描述(申请人提供)：糖胺多聚糖等硫化碳水化合物控制许多不同的影响人类健康的过程，包括凝血、免疫和神经细胞迁移、病毒和微生物感染以及生长因子/受体信号转导。然而，对于这些过程中的许多过程，硫化碳水化合物如何在机制水平上调节其生物效应尚不清楚，这为我们利用这些分子的性质用于治疗目的提供了障碍。果蝇胚胎的背腹极性是由蛋壳中嵌入的一种硫酸盐碳水化合物决定的，这种碳水化合物影响胚膜和蛋壳之间卵周隙中丝氨酸的蛋白分解活性。原肠形成缺陷(GD)处理并激活Snake，Snake处理并激活Easter，Easter将Spaetzle前体裂解成跨膜受体Toll的配体。复活节的激活只发生在卵子的腹侧，在发育中的卵母细胞的腹侧卵泡细胞中表达的一种脊椎动物糖胺多聚糖硫酸盐酶的控制下。PIPE将硫酸盐转移到糖蛋白的碳水化合物侧链，这些糖蛋白结合到蛋壳的内卵黄膜层中，构成腹侧线索，促进激活的Snake处理复活节。这个项目的长期目标是确定硫酸盐化的腹侧线索如何控制复活节的空间受限处理，进而控制Toll的激活。这个中心假设基于最近的观察，即GFP标记的GD变得位于卵黄周空间的腹侧，即加工后的GD与硫酸盐化的腹侧线索结合，一旦集中在那里，就促进了Snake和Easter之间的相互作用，从而导致复活节加工。拟议的研究追求三个具体目标：1)通过确定GD定位所需的程度，并确定GD内定位和加工所需的决定因素，阐明控制GD在卵周间隙内腹侧定位的机制。2)通过确定与Easter和Snake相互作用所需的GD结构域，以及通过确定GD促进Snake和Easter的蛋白酶活性、Easter对切割的敏感性或这两种蛋白之间的生产性相互作用的程度，来确定定位的GD如何介导Snake对Easter的切割。3)阐明组成腹侧线索的管道修饰碳水化合物的结构，并确定这些分子如何通过分离管道修饰的关键蛋壳蛋白VML，通过质谱分析检查其相关碳水化合物，并确定以管道/腹侧线索依赖的方式与其相互作用的蛋白质，从而促进GD的定位和复活节加工。这项研究意义重大，因为在阐明这一聚合发育途径背后的分子机制的过程中，他们将对丝氨酸蛋白酶和其他医学上重要的蛋白质如何受到它们与硫酸盐碳水化合物相互作用的影响提供新的见解。

DESCRIPTION (provided by applicant): Sulfated carbohydrates such as glycosaminoglycans control many, diverse processes affecting human health including blood clotting, immune and neural cell migration, viral and microbial infection, and growth factor/receptor signaling. However, for many of these processes, how the sulfated carbohydrates mediate their biological effects at the mechanistic level is not understood, presenting a barrier to our ability to harness the properties of these molecules for therapeutic purposes. Drosophila embryo dorsal-ventral polarity is determined by a sulfated carbohydrate embedded in the eggshell that influences serine proteolytic activity in the perivitelline space between the embryonic membrane and the eggshell. Gastrulation Defective (GD) processes and activates Snake, Snake processes and activates Easter, and Easter cleaves the Spaetzle precursor into a ligand for the transmembrane receptor Toll. Activation of Easter occurs solely on the ventral side of the egg under the control of Pipe, a homologue of vertebrate glycosaminoglycan sulfating enzymes that is expressed in ventral follicle cells of the developing oocyte. Pipe transfers sulfate to the carbohydrate side chains of glycoproteins that become incorporated into the inner vitelline membrane layer of the eggshell and constitute a ventral cue that promotes processing of Easter by activated Snake. The long-term goal of this project is to determine how the sulfated ventral cue controls the spatially restricted processing of Easter, and in turn, the activation of Toll. Th central hypothesis, which is based on the recent observation that GFP-tagged GD becomes ventrally localized in the perivitelline space, is that processed GD binds to the sulfated ventral cue and once concentrated there, facilitates an interaction between Snake and Easter that results in Easter processing. The proposed investigations pursue three specific aims: 1) To elucidate the mechanism that controls the ventral localization of GD within the perivitelline space of the egg by determining the extent to which GD processing is required for its localization and by identifying determinants within GD that are required for localization and processing. 2) To determine how localized GD mediates the cleavage of Easter by Snake by identifying the GD domains that are required for its interactions with Easter and Snake, and by determining the extent to which GD promotes Snake protease activity, Easter susceptibility to cleavage, or productive interaction between the two proteins. 3) To elucidate the structure of the Pipe-modified carbohydrates that comprise the ventral cue and determine how these molecules facilitate GD localization and Easter processing by isolating VML, a key eggshell protein modified by Pipe, examining its associated carbohydrates by mass spectrometric analysis, and identifying proteins that interact with it in a Pipe/ventral cue-dependent manner. The proposed studies are significant because in the course of elucidating the molecular mechanism underlying this paradigmatic developmental pathway, they will yield new insights into the ways in which serine proteases and other medically important proteins are influenced by their interactions with sulfated carbohydrates.