Role of O-glycosylation in Animal Development

O-糖基化在动物发育中的作用

• 批准号: 8553342
• 负责人: KELLY G TEN HAGEN
• 金额: $ 133.79万
• 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/8553342
• 关键词: Acetylgalactosamine; Address; Affect; Animal Model; Animals; Basement membrane; Binding; Biological; Biological Models; Biological Process; Bone Density; Calcinosis; Cell Adhesion; Cell Proliferation; Cells; Colon; Colon Carcinoma; Communication; Complex; Congenital Heart Defects; Defect; Development; Disease; Disease Progression; Disease susceptibility; Drosophila genus; Drosophila melanogaster; Enzymes; Epithelial Cell Proliferation; Essential Genes; Eukaryota; Exclusion; Extracellular Matrix; Family; Family member; Fibroblast Growth Factor; Genes; Genetic; Gland; Glean; Glycobiology; Goals; Growth; High Density Lipoprotein Cholesterol; Human; Integrins; Laboratories; Laminin; Link; Location; MAP Kinase Gene; Mannose; Mediating; Membrane; Membrane Proteins; Mesoderm; Methods; Modification; Mucins; Multigene Family; Mus; Muscular Dystrophies; Mutation; Neoplasm Metastasis; Organ; Organogenesis; Phosphorylation; Play; Polysaccharides; Positioning Attribute; Post-Translational Protein Processing; Protein Glycosylation; Proteins; Proto-Oncogene Proteins c-akt; RNA Interference; Research; Role; Serine; Severity of illness; Signal Transduction; Site; Small Intestines; Staging; Stomach; Submandibular gland; Syndrome; System; Threonine; Tissues; Transferase Gene; Triglycerides; Work; alpha Dystroglycan; endoplasmic reticulum stress; gastrointestinal system; genome wide association study; gland development; glycosylation; glycosyltransferase; human disease; hydroxyl group; in vivo; interest; member; sugar; tumor progression

The overarching goal of the Developmental Glycobiology Unit is to determine the mechanisms by which protein O-glycosylation regulates basic biological processes to better understand the role of this modification in development and disease. Mucin-type O-linked glycosylation is a widespread and evolutionarily conserved protein modification catalyzed by a family of enzymes (PGANTs or GalNAcTs) 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 role of O-glycans during development in order to understand how they contribute to disease susceptibility and progression. Previous work from our group demonstrated that there are at least 9 functional transferase genes in Drosophila and that at least one is required for viability. These studies provided the first evidence that a member of this multigene family is required for development and viability in any eukaryote. Most recently, we have performed in vivo RNA interference (RNAi) to identify the remaining family members that are also essential for viability. We have discovered that 4 additional family members are required for viability. Specifically, RNAi to pgant4, pgant5, pgant7 or the putative glycosyltransferase CG30463 resulted in lethality. Additionally, these essential genes were required in specific tissues (mesoderm, digestive system and tracheal system), suggesting unique tissue-specific functions for each. Finally, we demonstrated that one of these newly defined essential genes is responsible for proper gut function. Loss of this glycosyltransferase resulted in reduced secretion of O-glycosylated proteins into the lumen of the gut and morphological alterations in the cells responsible for gut acidification. Mutations in this family member resulted in improper gut acidification. These studies have implications for the role of this protein modification in the proper gut function in higher eukaryotes, as these genes are abundantly expressed in the stomach, small intestine and colon of mice and humans. Following up on our Drosophila studies demonstrating a role for an O-glycosyltransferase in extracellular matrix (ECM) formation, we investigated whether the loss of a mammalian O-glycosyltransferase (Galnt1) has an effect on basement membrane formation and organogenesis using the murine submandibular gland (SMG) as a model system. The basement membrane of the developing SMG is a complex array of components that influence cell signaling, proliferation and differentiation; additionally, it is rich in O-glycosylated proteins. In these studies, we demonstrate that the loss of Galnt1 affects FGF-mediated cell proliferation during mammalian SMG organogenesis by influencing the secretion of basement membrane proteins. Mice deficient for the enzyme Galnt1 (that adds sugars to proteins during early stages of SMG development) resulted in intracellular accumulation of major BM components along with increased endoplasmic reticulum (ER) stress. Along with changes in BM composition, Galnt1 deficient glands displayed decreased FGF signaling, reduced AKT and MAPK phosphorylation, and reduced epithelial cell proliferation. Exogenous addition of BM component laminin to Galnt1 deficient glands rescued FGF signaling and the growth defects in a β1-integrin-dependent manner. Our work demonstrated that O-glycosylation influences the composition of the secreted ECM during mammalian organ development, with resultant effects on cell signaling, proliferation and organ growth. These results highlight a conserved role for O-glycosylation in the establishment of cellular microenvironments and have implications for the role of this protein modification in both development and disease. Finally, we investigated the interplay between two different forms of glycosylation that are present on the same protein (alpha-dystroglycan). Specifically, we examined how the presence of O-mannosylation at specific sites on alpha- dystroglycan influences the subsequent addition of O-GalNAc by the GalNAcTs. By using a combination of enzymatic and mass spectroscopic methods, we found that the presence and specific location of O-mannose can result in either the regional exclusion or changes in the specific position of GalNAc addition. Our study demonstrates that one form of glycosylation can influence the presence and/or position of another form of glycosylation, suggesting that changes in both types of glycosylation may contribute to disease severity, as is commonly seen in muscular dystrophies. 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. Our hope is that the cumulative results of the studies described above will elucidate the mechanisms by which this conserved protein modification operates in both normal development and in disease susceptibility.

发育糖生物学单元的首要目标是确定蛋白质 O-糖基化调节基本生物过程的机制，以更好地了解这种修饰在发育和疾病中的作用。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，由酶家族（PGANT 或 GalNAcT）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，从而是膜结合的或分泌的。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，果蝇中至少有 9 个功能性转移酶基因，并且至少有一个是生存所必需的。这些研究提供了第一个证据，证明任何真核生物的发育和生存都需要这个多基因家族的成员。最近，我们进行了体内 RNA 干扰 (RNAi)，以确定对生存也至关重要的其余家族成员。 我们发现还需要 4 名额外的家庭成员才能生存。 具体来说，对 pgant4、pgant5、pgant7 或假定的糖基转移酶 CG30463 的 RNAi 导致致死。 此外，这些必需基因是特定组织（中胚层、消化系统和气管系统）所必需的，这表明每种组织都具有独特的组织特异性功能。 最后，我们证明这些新定义的必需基因之一负责正常的肠道功能。这种糖基转移酶的缺失导致 O-糖基化蛋白分泌到肠腔中的减少以及负责肠道酸化的细胞的形态变化。该家族成员的突变导致肠道酸化不当。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。 我们的果蝇研究证明了 O-糖基转移酶在细胞外基质 (ECM) 形成中的作用，随后我们利用小鼠颌下腺研究了哺乳动物 O-糖基转移酶 (Galnt1) 的缺失是否对基底膜形成和器官发生有影响（SMG）作为模型系统。发育中的 SMG 的基底膜是一系列复杂的成分，影响细胞信号传导、增殖和分化；此外，它还富含O-糖基化蛋白质。在这些研究中，我们证明 Galnt1 的缺失通过影响基底膜蛋白的分泌，影响哺乳动物 SMG 器官发生过程中 FGF 介导的细胞增殖。 缺乏 Galnt1 酶（在 SMG 发育的早期阶段将糖添加到蛋白质中）的小鼠会导致主要 BM 成分在细胞内积累，同时内质网 (ER) 应激增加。 随着 BM 组成的变化，Galnt1 缺陷腺体表现出 FGF 信号传导减少、AKT 和 MAPK 磷酸化减少以及上皮细胞增殖减少。 向 Galnt1 缺陷腺体中外源添加 BM 成分层粘连蛋白，以 β1 整合素依赖性方式挽救了 FGF 信号传导和生长缺陷。我们的工作表明，O-糖基化会影响哺乳动物器官发育过程中分泌的 ECM 的组成，从而对细胞信号传导、增殖和器官生长产生影响。 这些结果强调了 O-糖基化在细胞微环境建立中的保守作用，并对这种蛋白质修饰在发育和疾病中的作用具有影响。 最后，我们研究了同一蛋白质（α-肌营养不良聚糖）上存在的两种不同形式的糖基化之间的相互作用。 具体来说，我们研究了α-肌营养不良聚糖上特定位点O-甘露糖基化的存在如何影响GalNAcT随后添加O-GalNAc。 通过结合使用酶法和质谱方法，我们发现O-甘露糖的存在和特定位置可以导致区域排除或GalNAc添加的特定位置的变化。 我们的研究表明，一种形式的糖基化可以影响另一种形式的糖基化的存在和/或位置，这表明两种类型的糖基化的变化可能会导致疾病的严重程度，正如肌营养不良症中常见的那样。 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。我们希望上述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。