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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：
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
项目类别：
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

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）增加了一个b3连接的葡萄糖到O-聚焦的血栓反应蛋白1型重复