ANALYSIS OF NUCLEAR-PORE PROTEIN P62 GLYCOSYLATION
ANALYSIS OF NUCLEAR-PORE PROTEIN P62 GLYCOSYLATION
复制标题
DOI：
10.1021/bi00005a025
复制
发表时间：
1995-02-07
期刊：
影响因子：
2.9
通讯作者：
HANOVER, JA
中科院分区：
文献类型：
--
作者：
LUBAS, WA;SMITH, M;HANOVER, JA
核孔的糖蛋白组分对于核转运是必需的，并且通过糖基化和磷酸化修饰。这些翻译后修饰的功能和控制知之甚少。使用重组p62作为底物，在体外检查主要大鼠核孔糖蛋白p62的糖基化。大鼠p62在大肠杆菌中表达并纯化至接近同质。使用部分纯化的哺乳动物转移酶的动力学分析表明，重组蛋白是一个很好的底物（Km = 0.30 μ M）的转移GIcNAc从UDP-GlcNAc（Km = 1.8 μ M）。通过体外翻译产物的缺失分析和[C-14] GlcNAc标记的蛋白水解片段的免疫沉淀相结合，对大鼠p62的O-连接GlcNAc糖基化位点进行定位。大鼠p62的氨基末端糖基化较差，在Lys（22）和Lys（97）之间没有O-连接的GlcNAc位点;羧基末端在Ser（471）处有一个已知的糖基化位点。大鼠p62中的大多数糖基化位点可能发生在从Ser（270）到Thr（294）的中心Ser/Thr富集区中的六个聚集的Ser残基上。衍生自该区域的合成肽是O-GlcNAc加成的良好底物（K-m = 30 μ M）和p62糖基化的有效竞争性抑制剂（K-i = 15 μ M）。有人提出，这个富含Ser/Thr的结构域作为氨基端β-折叠片层和羧基端α-螺旋结构域之间的接头区域起作用。该连接区的O-糖基化和磷酸化可以提供在核孔组装和拆卸期间改变p62构象的动态手段。
Glycoprotein components of the nuclear pore are essential for nuclear transport and are modified by both glycosylation and phosphorylation. The function and control of these post-translational modifications are poorly understood. Glycosylation of the major rat nuclear pore glycoprotein, p62, was examined in vitro using recombinant p62 as a substrate. Rat p62 was expressed in Escherichia coli and purified to near homogeneity. Kinetic analysis using a partially purified mammalian transferase suggests that the recombinant protein is an excellent substrate (K-m = 0.30 mu M) for the transfer of GlcNAc from UDP-GlcNAc (K-m = 1.8 mu M). Localization of the sites of O-linked GlcNAc glycosylation of rat p62 was performed by a combination of deletion analysis of in vitro translation products and by immunoprecipitation of [C-14]GlcNAc-labeled proteolytic fragments. The amino terminus of rat p62 is poorly glycosylated with no O-linked GlcNAc sites between Lys(22) and Lys(97); the carboxyl terminus has one known glycosylation site at Ser(471). The majority of the glycosylation sites in rat p62 are likely to occur on the six clustered Ser residues in the central Ser/Thr-rich region from Ser(270) to Thr(294). A synthetic peptide derived from this region is a good substrate for O-GlcNAc addition (K-m = 30 mu M) and a potent competitive inhibitor of p62 glycosylation (K-i = 15 mu M). It is proposed that this Ser/Thr-rich domain functions as a linker region between the amino-terminal beta-pleated sheet and the carboxyl terminal alpha-helical domains. O-Glycosylation and phosphorylation of this linker region could provide a dynamic means of altering the conformation of p62 during nuclear pore assembly and disassembly.