Role of Beta3-Glucosyltransferase in a non-canonical quality control pathway

Beta3-葡萄糖基转移酶在非规范质量控制途径中的作用

基本信息

批准号：
10221012
负责人：
BERNADETTE C HOLDENER
金额：
$ 56.19万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-17 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10221012
关键词：
ADAMTS Affect Alleles Animals Binding Biochemical Biological Biological Assay Blood coagulation Bone Growth Brain Cell Culture Techniques Cell Line Cell Lineage Cells Characteristics Child Clinical Congenital Abnormality Consensus Consensus Sequence Craniofacial Abnormalities Cultured Cells Cysteine-Rich Domain Data Defect Dependence Developmental Delay Disorders Disaccharides Disease Ear Embryo Endoplasmic Reticulum Enzymes Extracellular Matrix Eye Face Family Fucose Genes Genitourinary system Glucose Glucosyltransferase Heart Human Impairment In Vitro Irido-corneo-trabecular dysgenesis Kidney Knock-out Knockout Mice Krause-Kivlin syndrome Limb structure Link Mediating Mesenchyme Modification Molecular Mus Mutation Myelogenous Neonatal Null Lymphocytes Online Mendelian Inheritance In Man Pathway interactions Patients Phenotype Play Polysaccharides Process Property Protein Secretion Proteins Publications Quality Control RNA Interference Rapid screening Reagent Role Secretor blood group alpha-2-fucosyltransferase Serine Set protein Skeleton Structural defect Testing Threonine Thrombospondins Tissues base cell motility cell type cleft lip and palate craniofacial bone developmental disease endoplasmic reticulum stress glycosylation in vivo in vivo Model knock-down long bone member mouse model mutant novel protein folding response searchable database tool

项目摘要

B3GLCT (b3-glucosyltransferase) adds a b3-linked glucose to O-fucose on Thrombospondin type 1 Repeats (TSRs), forming the disaccharide Glucoseb1-3Fucose. The O-fucose is added by Protein O-fucosyltransferase 2 (POFUT2) to a Serine/Threonine located in a proposed consensus sequence within the TSR: C1XX(S/T)C2. Database searches with this consensus reveal 49 potential POFUT2 targets (and thus predicted B3GLCT targets) in humans. Nearly half of these targets are members of the ADAMTS or ADAMTS-like super-families, many of which are known to play essential biological roles in remodeling extracellular matrix. Elimination of Pofut2 in mice results in early embryonic lethality, consistent with an essential function for O-fucosylation for some or all of these proteins. In contrast, mutations in B3GLCT result in Peters plus syndrome (PPS, OMIM #261540), a rare autosomal recessive disorder characterized by structural malformations including Peters anomaly of the eye, short stature, brachydactyly, developmental delay, and characteristic craniofacial abnormalities. Several other abnormalities are commonly seen in patients including defects in heart, cleft lip/palate, genitourinary system, ear, and CNS. Elimination of B3glct in mice results in several similar phenotypes, including craniofacial and long bone growth defects, suggesting the mutants will be an excellent in vivo model to study B3GLCT function. Our recent publication suggests that both POFUT2 and B3GLCT are important for the quality control of TSR folding. Using RNAi-mediated knockdown, we demonstrated that loss of POFUT2 causes a secretion defect for all targets analyzed, while knockdown of B3GLCT is only necessary for secretion of some targets. These results provide a potential explanation for the difference in phenotype between Pofut2 null mice and PPS patients. Together these observations have led to our central hypothesis, that B3GLCT-mediated addition of glucose is required for efficient folding of a subset of TSR-proteins, and that the anomalies seen in PPS results from impaired secretion of a small number of sensitive targets. Here we will test this hypothesis in three Aims. Aim 1 examines how identified mutations in PPS patients affect B3GLCT activity and stability using cell-based and biochemical assays. Aim 2 examines which predicted POFUT2 targets require B3GLCT for secretion and why. We will test whether B3GLCT is required for secretion of POFUT2 targets relevant to PPS, and examine whether some TSRs require B3GLCT for folding and others do not. Finally, Aim 3 investigates whether loss of B3glct impairs secretion of POFUT2 targets in vivo and whether the B3glct knockout has different affects on targets that vary in number of TSRs. In addition, this aim tests whether effects on protein secretion are cell-type specific and whether the bone growth abnormalities observed in B3glct mutants result from reduction of functional protein or alternatively to unresolved unfolded protein response due to the accumulation of misfolded targets. Combined, these aims will provide molecular mechanisms to explain how glucose participates in a novel quality control pathway for TSR folding.

B3GLCT（B3-葡萄糖基转移酶）在血小板传播1型重复序列上添加B3连接的葡萄糖（TSR），形成二糖葡萄糖B1-3fucose。通过蛋白O-凝血素转移酶添加O-凝血液 2（pofut2）到位于TSR：C1XX（S/T）C2内提议的共有序列中的丝氨酸/苏氨酸。通过此共识进行数据库搜索揭示了49个潜在的POFUT2目标（因此预测了B3GLCT 目标）在人类中。这些目标中有将近一半是Adamts或Adamts样的超级家庭的成员，其中许多人在重塑细胞外基质中起着重要的生物学作用。消除小鼠中的pofut2导致早期胚胎致死性，与O型糖基化的基本功能一致这些蛋白质中的一些或全部。相反，B3GLCT的突变导致Peters Plus综合征（PPS，OMIM ＃261540），一种以结构性畸形为特征的罕见常染色体隐性疾病眼睛的异常，身材矮小，腕足，发育延迟和特征性颅面异常。患者通常会出现其他几种异常，包括心脏缺陷，裂。唇/pa，泌尿生殖系统，耳朵和CNS。消除小鼠的B3GLCT导致了几种相似的表型，包括颅面和长骨生长缺陷，表明这些突变体将是一种极好的研究B3GLCT功能的体内模型。我们最近的出版物表明POFUT2和B3GLCT都是对于TSR折叠的质量控制很重要。使用RNAi介导的敲低，我们证明了损失 POFUT2的of ta造成分析所有目标的分泌缺陷，而B3GLCT的敲低是必要的为了分泌一些目标。这些结果为表型差异提供了潜在的解释在POFUT2无效小鼠和PPS患者之间。这些观察结果一起导致了我们的中心假设， B3GLCT介导的葡萄糖的添加是有效折叠TSR蛋白需要的，并且 PPS中看到的异常是由于少数敏感靶标的分泌受损而导致的。我们会在这里在三个目标中检验该假设。 AIM 1检查PPS患者中发现的突变如何影响B3GLCT 使用基于细胞和生化测定的活性和稳定性。 AIM 2检查了预测POFUT2的哪些目标需要B3GLCT进行分泌以及原因。我们将测试是否需要B3GLCT才能分泌 POFUT2靶向与PPS相关的目标，并检查某些TSR是否需要B3GLCT进行折叠，而其他TSR是否需要不是。最后，AIM 3调查了B3GLCT的损失是否会损害POFUT2靶标在体内的分泌以及是否会损害体内的分泌物 B3GLCT敲除对TSR数量变化的靶标有不同的影响。此外，此目标测试对蛋白质分泌的影响是否是细胞类型的特异性，以及是否观察到骨骼生长异常在B3GLCT突变体中，功能蛋白的降低或未解决的展开蛋白由于堆积错误的靶标而导致的响应。这些目标结合在一起，将提供分子解释葡萄糖如何参与TSR折叠的新型质量控制途径的机制。