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: 10427381
• 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 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10427381
• 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.

B3GLCT（b3-葡萄糖基转移酶）将 b3 连接的葡萄糖添加到血小板反应蛋白 1 型重复序列上的 O-岩藻糖上 (TSR)，形成二糖 Glucoseb1-3Fucose。 O-岩藻糖由蛋白质 O-岩藻糖基转移酶添加 2 (POFUT2) 到位于 TSR 内提议的共有序列中的丝氨酸/苏氨酸：C1XX(S/T)C2。 以此共识进行的数据库搜索揭示了 49 个潜在的 POFUT2 靶标（因此预测了 B3GLCT 目标）在人类中。这些目标中近一半是 ADAMTS 或类 ADAMTS 超家族的成员， 已知其中许多在重塑细胞外基质中发挥重要的生物学作用。消除 Pofut2 在小鼠中导致早期胚胎致死，这与 O-岩藻糖基化的基本功能一致 这些蛋白质的一些或全部。相反，B3GLCT 突变会导致 Peters plus 综合征（PPS、OMIM #261540)，一种罕见的常染色体隐性遗传疾病，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畸形 异常。患者中常见的其他几种异常包括心脏缺陷、裂隙 唇/腭、泌尿生殖系统、耳朵和中枢神经系统。消除小鼠体内的 B3glct 会导致几种类似的结果 表型，包括颅面和长骨生长缺陷，表明突变体将是一个优秀的 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 的敲低会导致所有分析目标的分泌缺陷，而仅需要敲除 B3GLCT 用于分泌某些靶标。这些结果为表型差异提供了潜在的解释 Pofut2 缺失小鼠和 PPS 患者之间的比较。这些观察结果共同得出了我们的中心假设， B3GLCT 介导的葡萄糖添加是 TSR 蛋白子集的有效折叠所必需的，并且 PPS 中出现的异常现象是由于少数敏感目标的分泌受损所致。在这里我们将 在三个目标中检验这个假设。目标 1 检查 PPS 患者中已识别的突变如何影响 B3GLCT 使用基于细胞和生化测定的活性和稳定性。目标 2 检查预测 POFUT2 的内容 目标需要 B3GLCT 才能分泌以及原因。我们将测试B3GLCT是否需要分泌 POFUT2 目标与 PPS 相关，并检查某些 TSR 是否需要 B3GLCT 进行折叠，而其他 TSR 是否需要 B3GLCT 进行折叠 不是。最后，Aim 3 研究 B3glct 的缺失是否会损害体内 POFUT2 靶点的分泌，以及是否 B3glct 敲除对 TSR 数量不同的靶标有不同的影响。此外，这个目标测试 对蛋白质分泌的影响是否具有细胞类型特异性以及是否观察到骨生长异常 B3glct 突变体是由于功能蛋白的减少或未分解的未折叠蛋白的减少造成的 由于错误折叠目标的积累而产生的响应。结合起来，这些目标将提供分子 解释葡萄糖如何参与 TSR 折叠的新型质量控制途径的机制。