Maternal Control of the Drosophila Embryonic Dorsal/Ventral Axis

描述（由申请人提供）：硫代碳水化合物如糖胺聚糖控制许多影响人体健康的不同过程，包括血液凝固、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。然而，对于其中的许多过程，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，这对我们利用这些分子的特性用于治疗目的的能力提出了障碍。果蝇胚胎的背腹侧极性是由嵌入蛋壳的硫酸碳水化合物决定的，该碳水化合物影响胚膜和蛋壳之间卵泡间隙的丝氨酸蛋白水解活性。原肠胚形成缺陷（GD）加工并激活Snake， Snake加工并激活Easter， Easter将Spaetzle前体切割成跨膜受体Toll的配体。在Pipe的控制下，Easter的激活仅发生在卵的腹侧，Pipe是脊椎动物糖胺聚糖硫酸酸化酶的同源物，在发育中的卵母细胞的腹侧卵泡细胞中表达。Pipe将硫酸盐转移到糖蛋白的碳水化合物侧链，这些糖蛋白被合并到蛋壳的卵黄膜层内，并构成一个腹侧线索，促进被激活的Snake处理复活节。这个项目的长期目标是确定硫酸酸化的腹侧线索如何控制空间受限的Easter加工，进而控制Toll的激活。中心假设是基于最近观察到的gfp标记的GD在卵泡周围空间的腹侧定位，即加工过的GD与硫酸酸化的腹侧线索结合，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Easter加工。提出的研究有三个具体目标：1)通过确定GD定位所需的GD加工程度，以及通过确定GD定位和加工所需的GD内部决定因素，阐明控制GD在卵黄周空间内腹侧定位的机制。2)通过鉴定其与Easter和Snake相互作用所需的GD结构域，以及确定GD促进Snake蛋白酶活性、Easter对裂解的敏感性或两种蛋白之间产生相互作用的程度，确定定位GD是如何介导Easter被Snake切割的。3)为了阐明包含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促进GD定位和Easter处理，通过分离管道修饰的关键蛋壳蛋白VML，通过质谱分析检测其相关碳水化合物，并确定以管道/腹侧线索依赖的方式与之相互作用的蛋白质。这些研究具有重要意义，因为在阐明这种典型发育途径的分子机制的过程中，它们将对丝氨酸蛋白酶和其他医学上重要的蛋白质受其与硫酸碳水化合物相互作用影响的方式产生新的见解。

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.