Role of O-glycosylation in Animal Development

粘蛋白型o -连接糖基化是一种广泛的、进化上保守的蛋白质修饰，由一系列酶（果蝇中的pants或哺乳动物中的pGalNAcTs）催化，这些酶将糖n-乙酰半乳糖胺（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。这种糖基化的缺陷是人类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，全基因组关联研究已经确定了编码酶的基因，这些酶负责启动与高密度脂蛋白胆固醇水平、甘油三酯水平、先天性心脏缺陷、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的易感性和进展。我们小组之前的工作表明，o链糖基化对果蝇的生存能力至关重要。我们最近的研究已经证明了这种蛋白修饰在细胞外基质（ECM）蛋白分泌中的作用。具体来说，我们发现一个pant家族成员的缺失会改变ECM蛋白的分泌，从而影响基底膜的组成，破坏果蝇翅膀发育过程中整合素介导的细胞粘附。同样，我们证明了o -糖基化也可以调节哺乳动物器官发育过程中ECM的组成，影响整合素和FGF信号，从而影响发育中的唾液腺的细胞增殖和生长。这些结果强调了o -糖基化在分泌和细胞微环境建立中的保守作用。今年发表的研究阐明了o -聚糖影响果蝇消化道分泌的机制。我们发现该家族的一个成员（PGANT4）通过糖基化分泌器官的一个重要组成部分（Tango1）来调节分泌，从而保护免受furin介导的蛋白质水解。Tango1是一种内质网/高尔基跨膜蛋白，协调大货物进入分泌囊泡的包装。在缺乏pant4的情况下，Tango1被切割，导致分泌器极化丧失，分泌囊泡形成丧失，排列和保护消化道的蛋白质分泌中断。这些研究对这种蛋白修饰在高等真核生物正常肠道功能中的作用具有重要意义，因为这些基因在小鼠和人类的胃、小肠和结肠中大量表达。

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.