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

• 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
• 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

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O-葡萄糖转移酶2和3(POGLUT2和POGLUT3)将O-连接的葡萄糖添加到丝氨酸上 表皮生长因子样(EGF)重复序列中的残基包含假定的共同序列 C3xNTxGS(Y/F)xC4，其中C3和C4是EGF重复序列中第三和第四个保守的半胱氨酸。数据库 研究发现，纤维蛋白(FBN)和潜伏的转化生长因子β结合蛋白(LTBP)具有大量的表皮生长因子 按照共识重复，而其他蛋白质只有一到两个。FBN1和FBN2是主要的 细胞外基质中10-12 nm微纤维中的结构蛋白，它们在那里充当 弹性组织中的弹性蛋白，为肺等组织的功能提供必要的弹性和回弹力。微纤丝 还结合了许多其他分子，包括LTBP，调节组织的动态平衡。FBN1基因突变 导致马凡综合征(MFS)，可导致主动脉瘤/夹层，还可导致肺、骨生长、 还有眼睛缺陷。在小鼠中消除Fbn1、Ltbp1或Ltbp4会导致围产期死亡，概括起来有几个 在MFS患者中所见的表型。目前尚不清楚表皮生长因子O-糖基化对血管内皮生长因子的影响 FBN或LTBP函数。使用我们的糖蛋白组质谱方法，我们证明了47个中的27个 从人皮肤成纤维细胞培养物中免疫纯化的FBN1中的EGF重复序列被O-葡萄糖修饰。 在HEK293T细胞中表达的重组FBN2、LTPB1和LTBP4中的O-葡萄糖水平相似。 POGLUT2和POGLUT2显著减少FBN1 N末端片段(EGF1-26)的分泌 POGLUT3在HEK293T细胞中被敲除，表明这些酶的修饰都是定位的 在内质网上，是有效折叠和分泌这些底物所必需的。这个 我们最近的观察强调了这种O-葡萄糖修饰的重要性，我们的大多数 Poglut2/Poglut3双基因敲除(DKO)小鼠围产期死亡。幸存者有类似于FBN和 包括肺缺如、体积小、并指畸形等。基于这些观察结果，我们假设 FBN和LTPBs的O-糖基化对于功能微纤维网络是必不可少的。我们预测 O-糖基化的丧失将减少这些蛋白质的分泌和/或破坏与微原纤维的结合 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 有三个目标。目标1利用细胞为基础研究O-葡萄糖丢失如何影响POGLUT2/3底物蛋白 分泌物分析，微纤维超微结构分析，以及FBN1与配体相互作用的分析。AIM 2测试 O-糖基化是否需要POGLUT2/3共识中的保守氨基酸，以及 这些保守残基中的MFS突变是否通过失去O-葡萄糖来调节其对FBN1的影响。目标 3研究了POGLUT2/3的缺失是否损害了小鼠肺发育过程中的微原纤维网络 模型、组织学方法和遗传相互作用研究。结合起来，这些目标将提供分子 解释O-葡萄糖如何影响微原纤维网络的结构和功能的机制。