Role of O-glycosylation in Animal Development

O-糖基化在动物发育中的作用

• 批准号: 8344134
• 负责人: KELLY G TEN HAGEN
• 金额: $ 127.97万
• 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/8344134
• 关键词: Actins; Address; Affect; Animal Model; Animals; Apoptosis; Architecture; Basement membrane; Binding Proteins; Biological; Biological Models; Carbohydrates; Cell Adhesion; Cell Culture Techniques; Cell Line; Cell Proliferation; Cell physiology; Cells; Cellular Morphology; Cellular Structures; Colon; Communication; Complex; Cytokinesis; Defect; Development; Disease; Disease susceptibility; Drosophila genus; Drosophila melanogaster; Enzymes; Epithelial Cell Proliferation; Epithelial Cells; Essential Genes; Eukaryota; Event; Extracellular Matrix; Extracellular Matrix Proteins; Family; Family Sizes; Family member; Fibroblast Growth Factor; Fishes; Genes; Genetic; Gland; Glean; Goals; Golgi Apparatus; Growth; Human; Insecta; Integrin Binding; Integrin-mediated Cell Adhesion Pathway; Integrins; Intracellular Transport; Laboratories; Laminin; Link; MAP Kinase Gene; Mammals; Mediating; Membrane Proteins; Modification; Mucins; Multigene Family; Mus; Mutation; Organ; Organism; Organogenesis; Phosphorylation; Play; Polypeptide N-acetylgalactosaminyltransferase; Post-Translational Protein Processing; Protein Glycosylation; Protein Secretion; Proteins; Proto-Oncogene Proteins c-akt; RNA Interference; Role; Signal Transduction; Small Intestines; Staging; Stomach; Structure; Submandibular gland; System; Tissues; Transferase; Transferase Gene; Wing; Work; endoplasmic reticulum stress; fly; follow-up; fungus; gland development; glycosylation; glycosyltransferase; in vivo; interest; member; migration; mutant; neuronal cell body; polypeptide; sugar

Cells of the body are decorated with a variety of carbohydrates (sugars) that serve many diverse functions. These sugars not only act as a protective barrier on the outside of the cell, but are also involved in cell adhesion, migration, communication and signaling events in many organisms. Our group studies one type of sugar addition to proteins, known as mucin-type O-linked glycosylation, which is initiated by the polypeptide GalNAc transferase (ppGalNAcT or PGANT) enzyme family. This sugar addition is seen in most eukaryotic organisms including mammals, fish, insects, worms and some types of fungi. The conservation of this protein modification across species suggests that it plays crucial roles during many aspects of development. It is known that there are as many as 20 family members encoding functional ppGalNAcTs in mammals. Given the size of the family and the complexity it generates, we have taken advantage of a simpler model system (Drosophila melanogaster) to investigate the biological role of glycosylation during development. 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. More 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 and are essential in specific tissues. Moreover, one of these newly defined essential genes is responsible for proper gut function. Loss of this glycosyltransferase results in reduced secretion of O-glycosylated proteins into the lumen of the gut and affects the structure of the cells responsible for proper gut acidifcation. Mutations in this family member result in improper gut acidification. 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 also performed RNAi to each pgant in fly cell culture to examine the effects of each gene on specific cellular processes (cell adhesion, division, viability, apoptosis, morphological changes, intracellular transport, subcellular alterations). Using this approach, we obtained evidence for the role of pgants in the proper formation and structure of the secretory apparatus. RNAi to either pgant3 or pgant6 resulted in altered Golgi organization. Disruption of the normal Golgi structure in both cases was accompanied by a reduction in secretion, indicating a functional consequence of the loss of each transferase. Additionally, RNAi to pgant3 also resulted in alteration of the normal actin cytoskeletal architecture, changes in cell morphology and loss of cell adhesion. Other effects observed included multi-nucleated cells seen after RNAi to pgant2 or pgant35A in both cell lines, suggesting a role for these genes in the completion of cytokinesis. These studies provide a new platform for interrogating the cellular effects of mucin-type O-linked glycosylation and evidence for unique subcellular roles of the pgants in secretory apparatus structure and function. By examining the consequences of mutations in pgant family members in the fly, we found that mutations in pgant3 alter integrin-mediated epithelial cell adhesion in the Drosophila wing blade. We discovered that the loss of pgant3 resulted in the improper secretion and localization of the extracellular matrix (ECM) protein Tiggrin. Tiggrin is an integrin-binding protein that is normally O-glycosylated in wild type wing discs. Loss of Tiggrin within the basement membrane region in pgant3 mutants resulted in disruption of integrin-mediated cell adhesion and defects in wing formation. These studies provided the first example of the effects of O-glycosylation on protein secretion, establishment of the basement membrane and modulation of integrin-mediated cell adhesion in vivo. We followed up on these results to ask 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 demonstrates 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. 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.

身体的细胞被各种碳水化合物（糖）装饰，具有多种不同的功能。这些糖不仅充当细胞外部的保护屏障，而且还参与许多生物体中的细胞粘附、迁移、通讯和信号传导事件。我们的小组研究了一种类型的蛋白质糖添加，称为粘蛋白型 O 连接糖基化，它是由多肽 GalNAc 转移酶（ppGalNAcT 或 PGANT）酶家族启动的。这种糖添加存在于大多数真核生物中，包括哺乳动物、鱼类、昆虫、蠕虫和某些类型的真菌。这种蛋白质修饰在物种间的保守性表明它在发育的许多方面发挥着至关重要的作用。据了解，哺乳动物中有多达20个编码功能性ppGalNAcT的家族成员。考虑到该家族的规模及其产生​​的复杂性，我们利用一个更简单的模型系统（黑腹果蝇）来研究糖基化在发育过程中的生物学作用。 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，生存需要 4 个额外的家庭成员，并且在特定组织中至关重要。 此外，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会导致 O-糖基化蛋白向肠腔的分泌减少，并影响负责适当肠道酸化的细胞的结构。 该家族成员的突变会导致肠道酸化不当。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。 我们还对果蝇细胞培养物中的每个基因进行RNAi，以检查每个基因对特定细胞过程（细胞粘附、分裂、活力、凋亡、形态变化、细胞内转运、亚细胞改变）的影响。使用这种方法，我们获得了 pgants 在分泌器官的正确形成和结构中的作用的证据。对 pgant3 或 pgant6 的 RNAi 导致高尔基体组织发生改变。在这两种情况下，正常高尔基体结构的破坏都伴随着分泌的减少，表明每种转移酶损失的功能后果。 此外，对 pgant3 的 RNAi 还导致正常肌动蛋白细胞骨架结构的改变、细胞形态的变化和细胞粘附的丧失。 观察到的其他效应包括在两种细胞系中对 pgant2 或 pgant35A 进行 RNAi 后观察到的多核细胞，表明这些基因在完成胞质分裂中的作用。这些研究为探究粘蛋白型O-连接糖基化的细胞效应提供了一个新的平台，并为pgants在分泌装置结构和功能中独特的亚细胞作用提供了证据。 通过检查果蝇中 pgant 家族成员突变的后果，我们发现 pgant3 的突变改变了果蝇翼片中整合素介导的上皮细胞粘附。我们发现pgant3的缺失导致细胞外基质（ECM）蛋白Tiggrin的分泌和定位不当。 Tiggrin 是一种整合素结合蛋白，在野生型翼盘中通常是 O-糖基化的。 pgant3 突变体基底膜区域内 Tiggrin 的缺失导致整合素介导的细胞粘附破坏和翼形成缺陷。这些研究提供了第一个关于 O-糖基化对蛋白质分泌、基底膜的建立和体内整合素介导的细胞粘附调节的影响的例子。 我们以小鼠下颌下腺 (SMG) 作为模型系统，对这些结果进行了追踪，询问哺乳动物 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。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。 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。