Role of O-glycosylation in Animal Development

粘液型O-连接糖基化是一种广泛存在的、在进化上保守的蛋白质修饰，由一系列酶(果蝇中的pgants或哺乳动物中的pGalNActs)催化，将糖N-乙酰氨基半乳糖(GalNAc)转移到注定是膜结合或分泌的蛋白质中的丝氨酸和苏氨酸的羟基上。这种糖基化缺陷是导致人类疾病、家族性肿瘤、钙质沉着症和TN综合征的原因。此外，O-糖基化的改变与肿瘤的进展和转移有关。最近，全基因组相关研究已经确定了与高密度脂蛋白-胆固醇水平、甘油三酯水平、先天性心脏病、结肠癌和骨密度相关的基因中负责启动O-糖基化的酶的编码基因。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-葡聚糖在发育过程中的机制作用，以了解它们如何促进疾病的易感性和进展。我们小组以前的工作表明，O-连接的糖基化对于果蝇的生存是必不可少的。我们最近的研究证明了这种蛋白修饰在细胞外基质(ECM)蛋白分泌中的作用。具体地说，我们发现，在果蝇翅膀发育过程中，失去一个PGANT家族成员会改变ECM蛋白的分泌，从而影响基底膜的组成，破坏整合素介导的细胞黏附。同样，我们也证明了O-糖基化还调节哺乳动物器官发育过程中细胞外基质的组成，影响整合素和成纤维细胞生长因子信号，从而影响细胞增殖和发育中的唾液腺的生长。这些结果强调了O-糖基化在分泌和建立细胞微环境中的保守作用。今年发表的研究阐明了O-葡聚糖影响果蝇消化道分泌的机制。我们发现这个家族中的一个成员(PGANT4)通过糖基化分泌器官的一个基本成分(Tango1)来调节分泌，从而提供对Furin介导的蛋白分解的保护。Tango1是一种内质网/高尔基体跨膜蛋白，协调将大量货物包装成分泌囊泡。在没有PGANT4的情况下，Tango1被切割，导致分泌器官极化的丧失，分泌囊泡形成的丧失，以及排列和保护消化道的蛋白质的分泌中断。这些研究暗示了这种蛋白质修饰在高等真核生物肠道功能中的作用，因为这些基因在小鼠和人类的胃、小肠和结肠中大量表达。 我们还开发了一个实时成像系统，用于实时成像活体器官的分泌泡形成和极化分泌，以确定不同的PGANT家族成员是如何参与这些过程的。使用这个系统，我们定义了囊泡形成并最终与顶膜融合以将其内容物分泌到细胞外空间时发生的事件的顺序。我们还在确定特定肌动蛋白结构在融合和分泌事件中的作用。利用简单的基因中断，我们正在进一步研究这些植物如何通过在某些家庭成员缺失的器官中实时成像来中介对分泌和分泌器官结构的影响。 最后，我们正在继续与Tabak实验室合作，研究O-糖基化缺失对哺乳动物发育的其他方面的影响。具体地说，我们发现Galnt1的缺失通过影响胚胎心脏瓣膜的发育来影响小鼠的心脏功能。Galnt1基因缺陷的小鼠由于发育过程中细胞增殖增加而表现出瓣膜增大。伴随着细胞增殖增加的是某些ECM成分的增加以及BMP和MAPK信号通路的上调。这项研究为这种蛋白质修饰在心脏瓣膜发育中的作用提供了第一个证据，并可能代表一种治疗特发性瓣膜疾病的新模型。 总而言之，我们正在利用从果蝇中收集的信息，更好地关注在更复杂的哺乳动物系统中受O-糖基化影响的发育的关键方面。我们还使用活体器官内的实时成像来确定O-糖基化影响分泌的具体过程。我们希望，我们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的作用机制。

Mucin-type O-linked glycosylation is a widespread and evolutionarily conserved protein modification catalyzed by a family of enzymes (PGANTs in Drosophila or pGalNAcTs 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 recent 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 an ECM protein, thereby influencing basement membrane composition and disrupting integrin-mediated cell adhesion during Drosophila wing development. Likewise, we demonstrated that O-glycosylation also modulates the composition of the ECM during mammalian organ development, influencing integrin and FGF signaling, thereby affecting cell proliferation and growth of the developing salivary glands. These results highlight a conserved role for O-glycosylation in secretion and in the establishment of cellular microenvironments. Studies published this year elucidated the mechanism by which O-glycans influence secretion in the Drosophila digestive tract. We found that one member of this family (PGANT4) modulates secretion by glycosylating an essential component of the secretory apparatus (Tango1), conferring protection from furin-mediated proteolysis. Tango1 is an ER/Golgi transmembrane protein that coordinates packaging of large cargo into secretory vesicles. In the absence of PGANT4, Tango1 is cleaved, resulting in loss of secretory apparatus polarization, loss of secretory vesicle formation and disrupted secretion of proteins that line and protect the digestive tract. These studies have implications for the role of this protein modification in proper gut function in higher eukaryotes, as these genes are abundantly expressed in the stomach, small intestine and colon of mice and humans. We have also developed a system for real-time imaging of secretory vesicle formation and polarized secretion in a living organ, to define how various PGANT family members are involved in these processes. Using this system, we have defined the order of events that occur as vesicles are formed and eventually fuse with the apical membrane to secrete their contents into the extracellular space. We are also defining the role of specific actin structures in fusion and secretion events. Taking advantage of facile gene disruption, we are further investigating how the PGANTs are mediating effects on secretion and secretory apparatus structure through real-time imaging in organs where certain family members have been deleted. Finally, we are continuing to collaborate with the Tabak laboratory to investigate the effects of loss of O-glycosylation on other aspects of mammalian development. Specifically, we have found that loss of Galnt1 affects cardiac function in mice by influencing embryonic heart valve development. Galnt1-deficient mice displayed enlarged valves due to increased cell proliferation during development. Increased cell proliferation was accompanied by increases in certain ECM components and up-regulation of BMP and MAPK signaling pathways. This study provides the first evidence for the role of this protein modification in heart valve development and may represent a new model for idiopathic valve disease. In summary, we are using information gleaned from Drosophila to better focus on crucial aspects of development affected by O-glycosylation in more complex mammalian systems. We are also using real-time imaging within living organs to define the specific processes by which O-glycosylation influences secretion. 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.