Role of POGLUT2 and POGLUT3 in regulating microfibril structure and function

POGLUT2和POGLUT3在调节微纤维结构和功能中的作用

基本信息

批准号：
10636927
负责人：
BERNADETTE C HOLDENER
金额：
$ 68.74万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10636927
关键词：
Address Affect Affinity Alanine Amino Acids Aorta Binding Binding Proteins Binding Sites Biological Assay Birth Bone Growth Calcium Calcium Binding Cell Differentiation process Cells Cessation of life Consensus Consensus Sequence Cysteine Data Databases Defect Deposition Dermal Development Disease Dissection Elastic Tissue Elasticity Elastin Electron Microscopy Endoplasmic Reticulum Ensure Enzymes Epidermal Growth Factor Extracellular Matrix Eye FBN1 Fibroblasts Genetic Glucose Glucosyltransferases Heart Histologic Homeostasis Human Impairment In Vitro Knock-out Knockout Mice Label Ligand Binding Ligands Link Location Lung Marfan Syndrome Mediating Methods Microfibrils Modification Molecular Mus Mutate Mutation N-terminal Patients Peptide Hydrolases Perinatal Perinatal mortality demographics Phenotype Play Protein Secretion Proteins Recombinants Role Serine Signal Transduction Site Structural Protein Structural defect Structure Survivors Syndactyly Tensile Strength Testing Tissues Transforming Growth Factor beta autosome fibrillin fibrillin-2 glycoproteomics glycosylation in vitro Assay in vivo lung development mouse model mutant notch protein novel protein function pulmonary function response scaffold trafficking

项目摘要

Protein O-glucosyltransferases 2 and 3 (POGLUT2 and POGLUT3) add an O-linked glucose to a serine residue in Epidermal Growth factor-like (EGF) repeats containing the putative consensus sequence C3xNTxGS(Y/F)xC4, where C3 and C4 are the third and fourth conserved cysteine in the EGF repeat. Database searches reveal that the fibrillins (FBNs) and Latent TGFβ Binding Proteins (LTBPs) have numerous EGF repeats with the consensus, while other proteins only have one or two. FBN1 and FBN2 are the major structural proteins in the 10-12 nm microfibrils in extracellular matrix, where they serve as the scaffold for elastin in elastic tissues, providing elasticity and recoil essential for function of tissues such as lung. Microfibrils also bind a number of other molecules, including LTBPs, that regulate tissue homeostasis. Mutations in FBN1 cause Marfan Syndrome (MFS) that can result in aortic aneurism/dissection and also cause lung, bone-growth, and eye defects. Elimination of Fbn1, Ltbp1, or Ltbp4 in mice results in perinatal lethality, recapitulating several of the phenotypes seen in MFS patients. Nothing is known about the impact of EGF O-glucosylation on FBN or LTBP function. Using our glycoproteomic mass spectral methods, we demonstrated that 27 of the 47 EGF repeats in FBN1 immunopurified from human dermal fibroblast cultures are modified with O-glucose. Similar levels of O-glucose were found in recombinant FBN2, LTPB1, and LTBP4 expressed in HEK293T cells. Secretion of an N-terminal fragment (EGF1-26) of FBN1 was significantly reduced when POGLUT2 and POGLUT3 were knocked out in HEK293T cells, suggesting that modification by these enzymes, both localized in the endoplasmic reticulum, are required for efficient folding and secretion of these substrates. The importance of this O-glucose modification is underscored by our recent observation that the majority of our Poglut2/Poglut3 double knockout (DKO) mice die perinatally. Survivors have abnormalities similar to Fbn and Ltbp KOs including lung defects, small size, and syndactyly. Based on these observations, we hypothesize that O-glucosylation of FBNs and LTPBs is essential for a functional microfibril network. We predict that loss of O-glucosylation will reduce secretion of these proteins and/or disrupt binding to microfibril associated proteins, leading to structural and/or signaling defects in tissues. We will test this hypothesis in three Aims. Aim 1 addresses how loss of O-glucose affects POGLUT2/3 substrate proteins using cell-based secretion assays, ultrastructural analysis of microfibrils, and analysis of FBN1-ligand interactions. Aim 2 tests whether conserved amino acids in the putative POGLUT2/3 consensus are required for O-glucosylation, and whether MFS mutations in these conserved residues mediate their effects on FBN1 by loss of O-glucose. Aim 3 examines whether loss of POGLUT2/3 impairs the microfibril network in developing lungs using mouse models, histological approaches, and genetic interaction studies. Combined, these aims will provide molecular mechanisms to explain how O-glucose affects the structure and function of the microfibril network.

蛋白质 O-葡萄糖基转移酶 2 和 3（POGLUT2 和 POGLUT3）将 O-连接葡萄糖添加到丝氨酸上含有推定共有序列的表皮生长因子样 (EGF) 重复序列中的残基 C3xNTxGS(Y/F)xC4，其中C3和C4是EGF重复序列中的第三个和第四个保守的半胱氨酸。搜索显示原纤维蛋白 (FBN) 和潜在 TGFβ 结合蛋白 (LTBP) 具有大量 EGF 重复一致，而其他蛋白质只有一两个FBN1和FBN2为主。细胞外基质中 10-12 nm 微纤维中的结构蛋白，它们充当支架弹性组织中的弹性蛋白，提供肺等组织功能所必需的弹性和回缩力。还结合许多其他分子，包括调节 FBN1 组织稳态的 LTBP。引起马凡氏综合症 (MFS)，可导致主动脉瘤/夹层，还会导致肺部、骨骼生长、消除小鼠中的 Fbn1、Ltbp1 或 Ltbp4 会导致围产期死亡，概括了几种情况。关于 EGF O-葡萄糖基化对 MFS 患者表型的影响尚不清楚。使用我们的糖蛋白组质谱方法，我们证明了 47 个中的 27 个。从人真皮成纤维细胞培养物中免疫纯化的 FBN1 中的 EGF 重复序列用 O-葡萄糖进行修饰。在 HEK293T 细胞中表达的重组 FBN2、LTPB1 和 LTBP4 中发现了相似水平的 O-葡萄糖。当 POGLUT2 和 FBN1 N 末端片段 (EGF1-26) 的分泌显着减少 POGLUT3 在 HEK293T 细胞中被敲除，表明这些酶的修饰均位于局部在内质网中，这些底物的有效折叠和分泌是必需的。我们最近的观察强调了这种 O-葡萄糖修饰的重要性，即我们的大多数 Poglut2/Poglut3 双敲除 (DKO) 小鼠在围产期死亡，幸存者具有与 Fbn 和 Fbn 类似的异常。根据这些观察，我们捕获了 Ltbp KO，包括肺缺陷、小尺寸和并指。我们预测 FBN 和 LTPB 的 O-葡萄糖基化对于功能性微纤维网络至关重要。 O-葡萄糖基化的丧失将减少这些蛋白质的分泌和/或破坏与微纤维的结合相关蛋白质，导致组织中的结构和/或信号缺陷，我们将检验这一假设。三个目标中的目标 1 解决了 O-葡萄糖的损失如何影响基于细胞的 POGLUT2/3 底物蛋白。分泌测定、微纤维超微结构分析以及 FBN1-配体相互作用分析 Aim 2 测试。 O-葡萄糖基化是否需要假定的 POGLUT2/3 共有序列中的保守氨基酸，以及这些保守残基中的 MFS 突变是否通过 O-葡萄糖的缺失介导其对 FBN1 的影响。 3 使用小鼠检查 POGLUT2/3 的缺失是否会损害发育中肺部的微纤维网络结合起来，这些目标将提供分子模型、组织学方法和遗传相互作用研究。解释 O-葡萄糖如何影响微纤维网络的结构和功能的机制。