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。本课题组最近的研究阐明了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。 此外，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O-糖基化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。通过3D时间推移成像，我们发现F-肌动蛋白募集到融合小泡发生在融合孔形成之后，但在肌球蛋白募集之前。此外，我们还展示了分支肌动蛋白核因子Arp2/3和Wasp在调节分泌的特定方面所起的重要作用。利用体内简单的基因破坏，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，与Tabak实验室合作，我们正在研究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的缺失会影响小鼠的心功能，导致主动脉和肺动脉瓣狭窄、返流、射血分数改变和心脏扩张。导致功能受损的主要缺陷来自瓣膜发育过程中细胞增殖的增加。我们发现Galnt1的缺失导致了胚胎11.5天(E11.5)发育瓣膜中O-糖链的丢失。此外，O-多糖的丢失伴随着ADAMTS1和ADAMTS5蛋白酶的丢失，同时蛋白多糖的切割减少，BMP和MAPK信号转导增加。在发育中的瓣膜内，早在E11.5就观察到细胞增殖增加。综上所述，我们的研究表明，Galnt家族成员的丧失可以通过影响发育过程中细胞外基质的形成/重塑，从而影响器官的发育和功能，从而影响细胞增殖。此外，这项研究为这种蛋白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 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. Recent studies by our group 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. Additionally, we have developed a system for real-time imaging of secretory vesicle formation and polarized secretion in a living organ, to define how O-glycosylation is 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. Through 3D time-lapse imaging, we show that F-actin recruitment to fused vesicles occurs after fusion pore formation but before myosin recruitment. Moreover, we show essential roles for the branched actin nucleators, Arp2/3 and WASp in specific aspects of regulated secretion. Taking advantage of facile gene disruption in vivo, 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, in collaboration with the Tabak laboratory, we are investigating 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, resulting in aortic and pulmonary valve stenosis, regurgitation, altered ejection fraction and cardiac dilation. The primary defect resulting in compromised function stems from increased cell proliferation during valve development. We found that loss of Galnt1 resulted in the loss of O-glycans in developing valves at embryonic day 11.5 (E11.5). Moreover, the loss of O-glycans was accompanied by loss of the proteases ADAMTS1 and ADAMTS5, along with decreased cleavage of the proteoglycan versican and increased BMP and MAPK signaling. Increased cell proliferation within the developing valves was observed as early as E11.5. Taken together, our study demonstrates that loss of a member of the Galnt family can influence organ development and function by affecting the formation/remodeling of the extracellular matrix during development, with resultant effects on cell proliferation. Furthermore, 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.