Role of O-glycosylation in Animal Development

O-糖基化在动物发育中的作用

• 批准号: 9155525
• 负责人: KELLY G TEN HAGEN
• 金额: $ 250.83万
• 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/9155525
• 关键词: ADAMTS1 gene; Acetylgalactosamine; Actins; Address; Affect; Animal Model; Animals; Basement membrane; Binding; Biological; Bone Density; Calcinosis; Cardiac; Cell Adhesion; Cell Proliferation; Cleaved cell; Collaborations; Colon; Colon Carcinoma; Communication; Complex; Congenital Heart Defects; Defect; Development; Disease; Disease Progression; Disease susceptibility; Drosophila genus; Drosophila melanogaster; EFRAC; Embryo; Enzymes; Eukaryota; Event; Extracellular Matrix; Extracellular Matrix Proteins; Extracellular Space; F-Actin; 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; MAP Kinase Gene; Mammals; Mediating; Membrane; Modeling; Modification; Mucins; Mus; Myosin ATPase; Neoplasm Metastasis; Organ; Peptide Hydrolases; Play; Polysaccharides; Post-Translational Protein Processing; Process; Protein Glycosylation; Protein Secretion; Proteins; Proteoglycan; Proteolysis; Pulmonary Valve Stenosis; Real-Time Systems; Research; Role; Salivary Glands; Secretory Component; Secretory Vesicles; Serine; Signal Transduction; Small Intestines; Stomach; Structure; Syndrome; System; Threonine; Time; Triglycerides; Vesicle; Wing; Work; aortic valve; apical membrane; cell growth; genome wide association study; glycosylation; human disease; hydroxyl group; in vivo; interest; member; organ growth; stem; sugar; tumor progression; versican

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.

粘蛋白型 O 连接糖基化是一种广泛且进化上保守的蛋白质修饰，由一系列酶（果蝇中的 PGANT 或哺乳动物中的 pGalNAcTs）催化，将糖 N-乙酰半乳糖胺 (GalNAc) 转移到蛋白质中丝氨酸和苏氨酸的羟基上注定是膜结合或分泌的。这种类型的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、先天性心脏缺陷、结肠癌和骨矿物质密度相关的酶中的 O-糖基化。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-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。我们小组之前的工作表明，O-连接糖基化对于果蝇的生存能力至关重要。 我们的研究证明了这种蛋白质修饰在细胞外基质 (ECM) 蛋白质分泌中的作用。 具体来说，我们发现失去一个 PGANT 家族成员会改变 ECM 蛋白的分泌，从而影响基底膜组成并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 O-糖基化还在哺乳动物器官发育过程中调节 ECM 的组成，影响整合素和 FGF 信号传导，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-糖基化在分泌和细胞微环境建立中的保守作用。 我们小组最近的研究阐明了 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) 通过糖基化分泌装置的重要组成部分 (Tango1) 来调节分泌，从而提供免受弗林蛋白酶介导的蛋白水解作用的保护。 Tango1 是一种内质网/高尔基体跨膜蛋白，可协调将大量货物包装到分泌囊泡中。 在缺乏 PGANT4 的情况下，Tango1 被裂解，导致分泌器极化丧失、分泌小泡形成丧失以及衬里和保护消化道的蛋白质分泌中断。 这些研究对这种蛋白质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。 此外，我们还开发了一种实时成像活体器官中分泌囊泡形成和极化分泌的系统，以确定 O-糖基化如何参与这些过程。 使用该系统，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顺序。通过 3D 延时成像，我们发现 F-肌动蛋白募集到融合囊泡发生在融合孔形成之后但肌球蛋白募集之前。 此外，我们还展示了分支肌动蛋白成核剂 Arp2/3 和 WASp 在调节分泌的特定方面的重要作用。 利用体内容易基因破坏的优势，我们正在进一步研究 PGANT 如何通过对某些家族成员已被删除的器官进行实时成像来介导对分泌和分泌器结构的影响。 最后，我们与 Tabak 实验室合作，研究 O-糖基化缺失对哺乳动物发育其他方面的影响。具体来说，我们发现 Galnt1 的缺失会影响小鼠的心脏功能，导致主动脉瓣和肺动脉瓣狭窄、反流、射血分数改变和心脏扩张。 导致功能受损的主要缺陷源于瓣膜发育过程中细胞增殖的增加。 我们发现 Galnt1 的缺失导致胚胎第 11.5 天 (E11.5) 发育瓣膜中 O-聚糖的缺失。 此外，O-聚糖的损失伴随着蛋白酶 ADAMTS1 和 ADAMTS5 的损失，以及蛋白多糖多功能蛋白聚糖裂解的减少以及 BMP 和 MAPK 信号传导的增加。 早在 E11.5 就观察到发育瓣膜内细胞增殖增加。 综上所述，我们的研究表明，Galnt 家族成员的缺失可以通过影响发育过程中细胞外基质的形成/重塑来影响器官发育和功能，从而对细胞增殖产生影响。此外，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，并可能代表特发性瓣膜疾病的新模型。 总之，我们正在利用从果蝇收集的信息来更好地关注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。我们还在活体器官内使用实时成像来确定 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发挥作用的机制。