Role of O-glycosylation in Animal Development

O-糖基化在动物发育中的作用

• 批准号: 8929684
• 负责人: KELLY G TEN HAGEN
• 金额: $ 147.96万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8929684
• 关键词: Acetylgalactosamine; Actins; Address; Affect; Animal Model; Animals; Basement membrane; Binding; Biological; Bone Density; Calcinosis; Cardiac; Cell Adhesion; Cell Proliferation; Cleaved cell; Colon; Colon Carcinoma; Communication; Complex; Congenital Heart Defects; Defect; Development; Disease; Disease Progression; Disease susceptibility; Drosophila genus; Drosophila melanogaster; Embryonic Heart; Enzymes; Eukaryota; Event; Extracellular Matrix; Extracellular Matrix Proteins; Extracellular Space; Family; Family member; Fibroblast Growth Factor; Gastrointestinal tract structure; Genes; Genetic; Glean; Goals; Golgi Apparatus; Heart Valves; High Density Lipoprotein Cholesterol; Human; Image; Integral Membrane Protein; Integrin-mediated Cell Adhesion Pathway; Integrins; Laboratories; Life; Link; MAPK Signaling Pathway Pathway; Mammals; Mediating; Membrane; Modeling; Modification; Mucins; Mus; Neoplasm Metastasis; Organ; Play; Polysaccharides; Post-Translational Protein Processing; Process; Protein Glycosylation; Protein Secretion; Proteins; Proteolysis; Publishing; Real-Time Systems; Research; Role; Salivary Glands; Secretory Component; Secretory Vesicles; Serine; Signal Transduction; Small Intestines; Stomach; Structure; Syndrome; System; Threonine; Time; Triglycerides; Up-Regulation; Vesicle; Wing; Work; apical membrane; cell growth; genome wide association study; glycosylation; human disease; hydroxyl group; interest; sugar; tumor progression

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.

粘蛋白型O连接糖基化是一种广泛而进化的蛋白质修饰，由酶（果蝇中的PGANTS或PGALNACTS中的PGANTS）催化，可将糖N-乙酰乳糖苷（galnac）转移到糖N-乙酰乳糖酰胺（galNAC）中，将蛋白蛋白蛋白蛋白蛋白透射蛋白固定在内，以供素化。或分泌。这种类型的糖基化缺陷导致人类疾病家族性肿瘤钙化和TN综合征。另外，O-糖基化的变化与肿瘤进展和转移有关。最近，全基因组关联研究已经确定了编码与HDL-胆固醇水平，甘油三酸酯水平，先天性心脏缺陷，结肠癌和骨矿物质密度相关的酶的编码基因。从这些研究中可以明显看出，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-Glycans在开发过程中的机械作用，以了解它们如何促进疾病的敏感性和进展。我们小组的先前工作表明，O连接的糖基化对于果蝇的生存能力至关重要。 我们最近的研究表明，这种蛋白质修饰在细胞外基质（ECM）蛋白的分泌中的作用。 具体而言，我们发现一个PGANT家族成员的丧失会改变ECM蛋白的分泌，从而影响基底膜组成并破坏果蝇翼发育过程中整联蛋白介导的细胞粘附。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，从而影响整联蛋白和FGF信号传导，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中的保守作用。 今年发表的研究阐明了O-Glycans在果蝇消化道中影响分泌的机制。 我们发现该家族的一个成员（PGANT4）通过糖基化分泌仪（Tango1）的基本组成部分来调节分泌，从而允许蛋白酶介导的蛋白水解保护。 Tango1是一种ER/Golgi跨膜蛋白，可将大型货物包装到分泌囊泡中。 在没有pgant4的情况下，探戈1被切割，从而导致分泌仪的极化丧失，分泌囊泡形成的丧失以及破坏蛋白质的分泌，蛋白质分泌并保护消化道。 这些研究对这种蛋白质修饰在适当的肠道功能中的作用具有影响，因为这些基因在小鼠和人类的胃，小肠和结肠中大量表达。 我们还开发了一种用于分泌囊泡形成和活器官两极分化分泌的实时成像的系统，以定义各种PGANT家族成员如何参与这些过程。 使用此系统，我们定义了形成囊泡时发生的事件的顺序，并最终与顶膜融合，将其内容分泌到细胞外空间中。 我们还定义了特定肌动蛋白结构在融合和分泌事件中的作用。 利用便利的基因破坏，我们正在进一步研究PGANTS如何通过删除某些家庭成员的器官的实时成像来介导对分泌和分泌设备结构的影响。 最后，我们将继续与TABAK实验室合作，研究O-糖基化丧失对哺乳动物发育其他方面的影响。具体而言，我们发现GALNT1的损失通过影响胚胎心脏瓣膜发育而影响小鼠的心脏功能。 由于细胞在发育过程中的增殖增加，缺乏GALNT1缺乏的小鼠显示出瓣膜增大。细胞增殖的增加伴随着某些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，并且可能代表了特发性瓣膜疾病的新模型。 总而言之，我们正在使用从果蝇收集的信息，以更好地专注于更复杂的哺乳动物系统中O-糖基化影响的发展的关键方面。我们还使用活器官中的实时成像来定义O-糖基化影响分泌的特定过程。 我们的希望是，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起作用的机制。