Role of O-glycosylation in Animal Development

粘蛋白型0-连接的糖基化是一种广泛的和进化上保守的蛋白质修饰，其由酶家族（果蝇中的PGANTs或哺乳动物中的ppGalNAcTs）催化，所述酶家族将糖N-乙酰半乳糖胺（GalNAc）转移到蛋白质中的丝氨酸和苏氨酸的羟基，所述丝氨酸和苏氨酸注定是膜结合的或分泌的。这种类型的糖基化缺陷是人类疾病家族性肿瘤性钙质沉着症和Tn综合征的原因。此外，O-糖基化的变化与肿瘤进展和转移相关。最近，全基因组关联研究已经确定了编码酶的基因，这些酶负责启动与HDL-胆固醇水平，甘油三酯水平，先天性心脏病，结肠癌和骨矿物质密度相关的O-糖基化。从这些研究中，很明显，这种保守的蛋白质修饰具有多种生物学作用。我们研究小组的重点是阐明O-聚糖在发育过程中的机制作用，以了解它们如何促进疾病易感性和进展。我们小组以前的工作表明，O-连接的糖基化对果蝇的生存能力是必不可少的。 我们的研究已经证明了这种蛋白质修饰在细胞外基质（ECM）蛋白分泌中的作用。 具体来说，我们发现，一个PGANT家族成员的损失改变ECM蛋白的分泌，破坏果蝇发育过程中整合素介导的细胞粘附，并影响哺乳动物器官发育过程中整合素和FGF信号传导。 从机制上讲，我们已经表明，保守的货物受体的O-糖基化调节其稳定性和形成分泌囊泡的能力。这些结果突出了O-糖基化在分泌和细胞微环境建立中的保守作用。 本课题组最近的研究主要集中在进一步研究O-糖基化在分泌和分泌囊泡形成中的作用。 此外，我们有兴趣研究如何调节大型、高度O-糖基化的蛋白质（例如粘蛋白）的合成、包装和分泌。 因此，我们已经开发了一个强大的成像平台，使用果蝇唾液腺，使我们能够可视化含粘蛋白的分泌囊泡的初始形成，它们的成熟，因为它们经历同型融合和调节分泌激素刺激后。果蝇遗传学与体内和离体成像的强大组合允许人们快速询问分泌的许多方面中的因素的作用，包括囊泡生物发生、囊泡运动、与质膜融合、颗粒货物的释放和货物一旦在管腔中膨胀。 考虑到参与分泌的许多基因和机制在物种间是保守的，该系统可以提供关于参与哺乳动物分泌的许多组分的功能的详细机制信息。 我们最近发现一个pgant家族成员在分泌囊泡形成中的作用。我们确定了一个新的成员的pgant家庭进行组织特异性差异剪接内的唾液腺的果蝇。 该剪接事件产生2种亚型，其在该酶的亚结构域内不同，并差异调节分泌颗粒形态。 有趣的是，该亚结构域的剪接赋予独特的底物特异性，允许货物粘蛋白的完全糖基化。 在没有一个剪接变体的情况下，货物粘蛋白没有完全糖基化，分泌颗粒呈现不规则的碎片样外观。 我们的研究提供了第一个证据，pgant家族成员的选择性剪接可以改变底物特异性，粘蛋白货物的糖基化状态可以影响分泌颗粒形态。 我们也在检查粘膜的功能，粘膜是我们身体各种器官系统的内部上皮细胞的衬里。我们消化道的粘膜屏障是抵御病原体和损害的第一道防线。 这种屏障的破坏与疾病有关，如克罗恩病、结肠炎和结肠癌。 在我们的研究中，我们表明，在果蝇的粘膜屏障的基因消融导致上皮细胞表达的IL-6样细胞因子Upd 3，导致分化的细胞，形成祖细胞龛。 生态位的破坏导致祖细胞的异常增殖。 生态位破坏可以通过过度表达upd 3来重现，并通过删除upd 3来拯救，突出了这种细胞因子的关键作用。 此外，生态位的完整性和细胞增殖可以通过过度表达保守的货物受体Tango 1或补充外源性粘蛋白来拯救。 我们的研究阐明了由受损的粘膜屏障激活的旁分泌信号传导事件，并为粘蛋白模拟物和其他治疗口腔粘膜和消化道疾病的策略提供了一种新的体内筛选平台。 总之，我们正在使用从果蝇收集的信息，以更好地专注于在更复杂的哺乳动物系统中受O-糖基化影响的发育和器官功能的关键方面。我们的希望是，我们的研究的累积结果将阐明这种保守的蛋白质修饰在正常发育和疾病易感性中起作用的机制。

Mucin-type O-linked glycosylation is a widespread and evolutionarily conserved protein modification catalyzed by a family of enzymes (PGANTs in Drosophila or ppGalNAcTs in mammals) that transfer the sugar N-acetylgalactosamine (GalNAc) to the hydroxyl group of serines and threonines in proteins that are destined to be membrane-bound or secreted. Defects in this type of glycosylation are responsible for the human diseases familial tumoral calcinosis and Tn syndrome. Additionally, changes in O-glycosylation have been associated with tumor progression and metastasis. More recently, genome-wide association studies have identified the genes encoding the enzymes that are responsible for initiating O-glycosylation among those associated with HDL-cholesterol levels, triglyceride levels, congenital heart defects, colon cancer and bone mineral density. From these studies, it is apparent that this conserved protein modification has a multitude of biological roles. The focus of our research group is to elucidate the mechanistic roles of O-glycans during development in order to understand how they contribute to disease susceptibility and progression. Previous work from our group demonstrated that O-linked glycosylation is essential for viability in Drosophila. Our studies have demonstrated roles for this protein modification in the secretion of extracellular matrix (ECM) proteins. Specifically, we found that loss of one PGANT family member alters secretion of ECM proteins, disrupting integrin-mediated cell adhesion during Drosophila development and influencing integrin and FGF signaling during mammalian organ development. Mechanistically, we have shown that O-glycosylation of a conserved cargo receptor modulates its stability and ability to form secretory vesicles. These results highlight a conserved role for O-glycosylation in secretion and in the establishment of cellular microenvironments. Recent studies by our group have focused on further investigation of the role of O-glycosylation in secretion and secretory vesicle formation. Additionally, we are interested in investigating how the synthesis, packaging and secretion of large, highly O-glycosylated proteins, such as mucins, are regulated. We have therefore developed a powerful imaging platform using Drosophila salivary glands, allowing us to visualize the initial formation of mucin-containing secretory vesicles, their maturation as they undergo homotypic fusion and regulated secretion after hormone stimulation. The powerful combination of Drosophila genetics with in vivo and ex vivo imaging allows one to rapidly interrogate the role of factors in many aspects of secretion, including vesicle biogenesis, vesicle movement, fusion with the plasma membrane, release of granule cargo and expansion of cargo once in the lumen. Given that many genes and mechanisms involved in secretion are conserved across species, this system can provide detailed mechanistic information regarding the function of many components involved in mammalian secretion. We have recently discovered a role for one member of the pgant family in secretory vesicle formation. We identified a novel member of the pgant family that undergoes tissue-specific differential splicing within the salivary gland of Drosophila. This splicing event generates 2 isoforms that differ within a subdomain of this enzyme and differentially modulate secretory granule morphology. Interestingly, splicing of this subdomain confers unique substrate specificity, allowing the complete glycosylation of a cargo mucin. In the absence of one splice variant, the cargo mucin is not fully glycosylated and secretory granules take on an irregular, shard-like appearance. Our study provides the first evidence that alternative splicing of a member of the pgant family can alter substrate specificity and that the glycosylation status of mucin cargo can influence secretory granule morphology. We are also examining the function of the mucous membrane that lines the internal epithelia of various organ systems of our bodies. The mucous barrier of our digestive tract is the first line of defense against pathogens and damage. Disruptions in this barrier are associated with diseases, such as Crohns disease, colitis and colon cancer. In our research, we show that genetic ablation of the mucosal barrier in Drosophila causes epithelial expression of the IL-6-like cytokine Upd3, leading to differentiation of cells that form the progenitor cell niche. Disruption of the niche leads to abnormal proliferation of progenitor cells. Niche disruption could be recapitulated by overexpressing upd3 and rescued by deleting upd3, highlighting a crucial role for this cytokine. Additionally, niche integrity and cell proliferation could be rescued by overexpression of the conserved cargo receptor Tango1 or by supplementation with exogenous mucins. Our studies elucidate the paracrine signaling events activated by a compromised mucosal barrier and provide a novel in vivo screening platform for mucin mimetics and other strategies to treat diseases of the oral mucosa and digestive tract. In summary, we are using information gleaned from Drosophila to better focus on crucial aspects of development and organ function affected by O-glycosylation in more complex mammalian systems. Our hope is that the cumulative results of our research will elucidate the mechanisms by which this conserved protein modification operates in both normal development and disease susceptibility.