Structural basis of the interplay between the carbohydrate recognition and protein ubiquitination systems in the glycoprotein degradation in cells

为了更好地理解糖基化和泛素化控制的细胞内糖蛋白降解的潜在机制的分子基础，我们使用了融合糖生物学和结构生物学方法的方法。利用糖库研究细胞内凝集素的糖结合特性我们利用多维HPLC方法构建了一个高甘露糖型糖库，并将其应用于前沿亲和色谱（frontal affinity chromatography，FAC）分析了多种细胞内凝集素的糖结合特异性，这些凝集素参与了细胞内糖蛋白的质量控制。FAC数据显示，内质网（ER）分子伴侣、货物受体、泛素连接酶和去糖基化酶的糖结合结构域特异性识别N-聚糖加工途径中部分修剪的高甘露糖型寡糖上展示的不同糖表位，从而决定糖蛋白命运 关于我们 s在细胞中。以结构生物学方法阐明糖蛋白降解系统的分子机制。稳定同位素辅助NMR数据的基础上，我们阐明了分子识别的结构基础的糖结合域的泛素连接酶SCFFbs 1，泛素化错误折叠的糖蛋白逆行转移到细胞质，和泛素结合域的去糖基化酶肽：N聚糖酶，这有利于蛋白酶体降解的糖蛋白。此外，我们确定了泛素连接酶和去泛素化酶的泛素识别模式，通过使用泛素链库。此外，我们的NMR数据揭示了泛素连接酶复合物的激活机制后，共价连接Nedd 8。这些结果提供了深入了解糖蛋白降解过程中糖识别和蛋白泛素化系统之间相互作用的分子基础。少

For a better understanding the molecular basis for the underlying mechanisms of the intracellular glycoprotein degradation governed by glycosylation and ubiquitination, we used a fusional approach integrating the methodologies of glycobiology and structural biology.1. Elucidation of sugar-binding properties of the intracellular lectins by using a sugar library.We constructed a high-mannose-type sugar library using the multi-dimensional HPLC method and applied them to frontal affinity chromatography (FAC) analyses of sugar-binding specificities of a variety of intracellular lectins involved in the quality control of glycoproteins in cells. The FAC data revealed that the sugar-binding domains of the endoplasmic reticulum (ER) chaperones, cargo receptors, ubiquitin ligases, and deglycosylation enzyme specifically recognize distinct glycotopes displayed on the partially trimmed high-mannose-type oligosaccharides in the N-glycan processing pathway and thereby determine the glycoprotein-fate … More s in cells.2. Elucidation of the molecular mechanisms of the glycoprotein degradation system by structural biology approaches. On the basis of the stable-isotope-assisted NMR data, we elucidated the structural basis of the molecular recognition by the sugar-binding domain of the ubiquitin ligase SCFFbs1, which ubiquitinates malfolded glycoproteins retrogradely transferred from the ER to the cytosol, and by the ubiquitin-binding domain of the deglycosylation enzyme peptide:Nglycanase, which facilitates the proteasomal degradation of glycoproteins. Furthermore, we determined the ubiquitin recognition modes of ubiquitin ligases and de-ubiquitinating enzymes by using a library of ubiquitin chains. In addition, our NMR data revealed the mechanisms of the activation of the ubiquitin-ligase complexes upon covalent attachment of Nedd8. These results provide insights into the molecular basis of the interplay between the carbohydrate recognition and protein ubiquitination systems in the glycoprotein degradation in cells. Less