Structure, Biosynthesis & Function of Glycoproteins

项目摘要 这项研究计划的目的是为了从分子水平上了解 在新合成的酸的输送中起作用的磷甘油酰靶向系统 水解酶转化为溶酶体。这种细胞内蛋白质运输途径的遗传缺陷使 上升到严重的溶酶体储存疾病，说明了这一系统的重要性。关键的一步 在这个途径中是甘露糖残基在高甘露糖上的选择性磷酸化。 酸性水解酶的UDP-GlcNAc：溶酶体酶N-乙酰氨基葡萄糖-1-糖链 磷酸转移酶(Ptase)。这种顺式高尔基转移酶是一种III型跨膜型α2β2γ2 由两个基因编码的六聚体蛋白。我们确定了两个Notch Repeat模块 和α亚基的DNA甲基转移酶相关蛋白相互作用域 介导对酸性水解酶常见蛋白质决定簇的特异性识别。特定的 目标1旨在定义每个领域在这一过程中的作用。目标2侧重于 Ptase的N端胞质尾部在维持正常高尔基体定位中的作用。这一目标 源于我们的发现，三个ML III患者细胞质N-Tail的错义突变 α亚基的缺失导致突变的Ptase错误定位于内体/溶酶体系统。 我们的假设是，这些突变阻止了这种高尔基体酶的循环，原因是未能 被合并到COPI囊泡中，这是回收过程中的一个必要步骤。为了支持这一点， 我们的初步发现表明WT N-Tail直接与WT N-Tail结合，但不是突变多肽与 δ和ζ亚基。我们的目标是在分子水平上理解这些相互作用。 目的3是确定胞质N-尾的直接相互作用的一般性。 高尔基体型跨膜糖基转移酶大家族，含COPI亚基。 有强有力的证据表明，糖基转移酶从高尔基池晚期到早期或 内质网通过COPI囊泡，但转移酶如何包装到囊泡中的情况很差 明白了。这一目标有可能为这一进程建立一个新的范例。

Project Summary The objective of this research proposal is to obtain a molecular understanding of the phosphomannosyl targeting system which functions in the delivery of newly synthesized acid hydrolases to lysosomes. Genetic defects in this intracellular protein transport pathway give rise to severe lysosomal storage diseases, illustrating the importance of this system. A key step in this pathway is the selective phosphorylation of mannose residues on the high mannose glycans of the acid hydrolases by UDP-GlcNAc:lysosomal enzymes N-acetylgucosamine-1- phosphotransferase (Ptase). This cis-Golgi transferase is a type III transmembrane α2β2γ2 hexameric protein encoded by two genes. We established that the two Notch repeat modules and the DNA methyltransferase-associated protein (DMAP) interaction domain of the α subunit mediate the specific recognition of the common protein determinant of acid hydrolases. Specific Aim 1 is directed toward defining the role of each domain in this process. Aim 2 focuses on the role of the N-terminal cytoplasmic tail of Ptase in maintaining proper Golgi localization. This aim is derived from our finding that three ML III patient missense mutations in the cytoplasmic N-tail of the α-subunit result in mislocalization of the mutant Ptase to the endosome/lysosome system. Our hypothesis is that these mutations prevent recycling of this Golgi enzyme due to a failure to be incorporated into COPI vesicles, a required step for the recycling process. In support of this, our preliminary findings show direct binding of the WT N-tail, but not the mutant peptides, to the δ and ζ subunits of COPI. The goal is to understand these interactions at the molecular level. Aim 3 is to determine the generality of the direct interaction of the cytoplasmic N-tails of the large family of Golgi type II transmembrane glycosyltransferases with the COPI subunits. Strong evidence exists that the glycosyltransferases recycle from late to early Golgi cisternae or the ER via COPI vesicles, but how the transferases are packaged into the vesicles is poorly understood. This aim has the potential to establish a new paradigm for this process.