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

• 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
• 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

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.

B3 GLCT（b3-葡糖基转移酶）将b3连接的葡萄糖添加到凝血酶敏感蛋白1型重复序列上的O-岩藻糖上 （TSR），形成二糖葡萄糖1 - 3岩藻糖。通过蛋白质O-岩藻糖基转移酶添加O-岩藻糖 2（POFUT 2）与位于TSR：C1 XX（S/T）C2内的拟定共有序列中的丝氨酸/苏氨酸。 数据库检索与这一共识揭示了49个潜在的POFUT 2目标（从而预测B3 GLCT 目标）。这些目标中近一半是ADAMTS或类似ADAMTS的超级家族的成员， 其中许多已知在重塑细胞外基质中发挥重要的生物学作用。消除 小鼠中的Pofut 2导致早期胚胎死亡，这与O-岩藻糖基化的基本功能一致， 这些蛋白质的一部分或全部。相反，B3 GLCT突变导致Peters+综合征（PPS，OMIM #261540），一种罕见的常染色体隐性遗传病，其特征是结构畸形，包括Peters 眼睛异常、身材矮小、短指、发育迟缓和特征性颅面 异常其他几种异常在患者中常见，包括心脏缺陷， 唇/腭、泌尿生殖系统、耳朵和中枢神经系统。在小鼠中消除B3 glct导致几种类似的 表型，包括颅面和长骨生长缺陷，这表明突变体将是一个优秀的， 体内模型研究B3 GLCT功能。我们最近的出版物表明，POFUT 2和B3 GLCT都是 对TSR折叠的质量控制具有重要意义。使用RNAi介导的敲低，我们证明了这种损失 POFUT 2的敲低导致所有分析的靶的分泌缺陷，而B3 GLCT的敲低仅是必需的 用于分泌某些靶标。这些结果为表型的差异提供了一个潜在的解释 Pofut 2基因敲除小鼠和PPS患者之间的差异。这些观察结果共同导致了我们的中心假设， B3 GLCT介导的葡萄糖添加是有效折叠TSR-蛋白亚类所必需的， 在PPS中观察到的异常是由于少数敏感靶的分泌受损。这里我们将 在三个目标中检验这个假设。目的1研究PPS患者中确定的突变如何影响B3 GLCT 活性和稳定性，使用基于细胞的和生物化学测定。目标2检查预测POFUT 2的因素 靶点需要B3 GLCT进行分泌以及原因。我们将测试B3 GLCT是否是分泌 POFUT 2目标与PPS相关，并检查一些TSR是否需要B3 GLCT进行折叠，而其他TSR则需要 没有最后，目的3研究B3 glct的缺失是否损害体内POFUT 2靶的分泌，以及是否 B3 glct敲除对TSR数目不同的靶具有不同的影响。此外，这一目标测试 对蛋白质分泌的影响是否是细胞类型特异性的，以及观察到的骨生长异常是否 在B3 glct突变体中，由功能蛋白减少或未解析未折叠蛋白产生 由于错误折叠的目标的积累而引起的反应。结合起来，这些目标将提供分子 机制来解释葡萄糖如何参与TSR折叠的新质量控制途径。