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

B3GLCT(b3-葡萄糖基转移酶)在凝血酶反应蛋白1型重复序列上将b3连接的葡萄糖添加到O-岩藻糖上 (TSRs)，形成二糖葡萄糖b1-3岩藻糖。O-岩藻糖由蛋白O-岩藻糖基转移酶加成 2(POFUT2)到一个丝氨酸/苏氨酸，位于TSR：C1XX(S/T)C2内的一个建议的共识序列中。 根据这一共识进行的数据库搜索显示了49个潜在的POFUT2目标(从而预测了B3GLCT 目标)在人类身上。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 在小鼠中，Pofut2导致早期胚胎死亡，这与O-岩藻糖基化在小鼠体内的一个重要功能一致 这些蛋白质中的一部分或全部。相反，B3GLCT的突变导致彼得斯综合征(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，包括彼得斯 眼畸形、矮小、短指、发育迟缓和特征性的颅面 异常现象。其他几种异常在患者中也很常见，包括心脏缺陷、裂隙 唇腭部、泌尿生殖系统、耳朵和中枢神经系统。在小鼠中消除B3glct会导致几个类似的结果 表型，包括颅面和长骨生长缺陷，表明突变将是一个优秀的 活体模型研究B3GLCT功能。我们最近的出版物表明，POFUT2和B3GLCT都是 对TSR折叠的质量控制具有重要意义。使用RNAi介导的基因敲除，我们证明了这种损失 POFUT2基因的缺失会导致所有被分析靶标的分泌缺陷，而B3GLCT基因的敲除只是必需的 用于分泌某些靶标。这些结果为表型差异提供了一个潜在的解释。 在Pofut2基因缺失小鼠和PPS患者之间。这些观察结果共同得出了我们的中心假说， B3GLCT介导的葡萄糖加成是TSR蛋白质子集有效折叠所必需的，并且 PPS中出现的异常是由于少数敏感靶点的分泌受损所致。在这里，我们将 从三个方面来检验这一假设。目的1检查PPS患者中已发现的突变如何影响B3GLCT 基于细胞和生化分析的活性和稳定性。目标2检查预测的POFUT2 靶标需要B3GLCT来分泌，以及为什么。我们将测试B3GLCT是否需要分泌 与PPS相关的POFUT2目标，并检查一些TSR是否需要B3GLCT进行折叠，而其他TSR是否需要 不。最后，目标3调查B3glct的缺失是否会损害体内POFUT2靶标的分泌，以及是否 B3glct基因敲除对TSR数量不同的目标有不同的影响。此外，这个目标还测试了 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 在B3glct中，突变是由于功能蛋白的减少或未分解的未折叠蛋白所致 由于错误折叠的目标积累而产生的反应。结合起来，这些目标将提供分子 解释葡萄糖如何参与TSR折叠的新质量控制途径的机制。

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.