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

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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