Glucosidase II (Glc'ase II) is a glycan-processing enzyme that trims two ␣1,3-linked Glc residues in succession from the glycoprotein oligosaccharide Glc2Man9GlcNAc2 to give Glc1Man9GlcNAc2 and Man9GlcNAc2 in the endoplasmic reticulum (ER). Monoglucosylated glycans, such as Glc1-Man9GlcNAc2, generated by this process play a key role in glycoprotein quality control in the ER, because they are primary ligands for the lectin chaperones calnexin (CNX) and calreticulin (CRT). A precise analysis of the substrate specificity of Glc'ase II is expected to further our understanding of the molecular basis to glycoprotein quality control, because Glc'ase II potentially competes with CNX/CRT for the same glycans, Glc1Man7-9GlcNAc2. In this study, a quantitative analysis of the specificity of Glc'ase II using a series of structurally defined synthetic glycans was carried out. In the presence of CRT, Glc'ase II-mediated trimming from Glc2Man9GlcNAc2 stopped at Glc1Man9GlcNAc2, supporting the notion that the glycan structure delivered to the CNX/CRT cycle is Glc1Man9GlcNAc2. Unexpectedly, our experiments showed that Glc1Man8(B)GlcNAc2 had nearly the same reactivity as Glc1Man9GlcNAc2, which was markedly greater than that of its positional isomer Glc1Man8(C)GlcNAc2. An analysis with glycoprotein-like probes revealed the stepwise formation of Glc1Man9GlcNAc2 and Man9GlcNAc2 from Glc2-Man9GlcNAc2, even in the presence of CRT. It was also shown that Glc1Man8(B)GlcNAc2 had even greater reactivity than Glc1Man9GlcNAc2 at the glycoprotein level. Moreover, inhibitory activities by nonglucosylated glycans suggested that Glc'ase II recognized the C arm (Man␣1,2Man␣1,6Man-) of high mannose-type glycans.

The folding of glycoproteins is primarily mediated by a quality control system in the ER, in which UDP-Glc:glycoprotein glucosyltransferase (UGGT) serves as a "folding sensor". In this system, client glycoproteins are delivered to UGGT after the trimming of their innermost glucose residue by glucosidase II, which releases them from the lectin chaperones calnexin (CNX) and calreticulin (CRT). UGGT is inactive against folded proteins, allowing them to proceed to the Golgi apparatus for further processing to complex- or hybrid-type glycoforms. On the other hand, this enzyme efficiently glucosylates incompletely folded glycoproteins to monoglucosylated structures, providing them with an opportunity to interact with CNX/CRT. In order to clarify the mode of this enzyme's substrate recognition, we conducted a structure-activity relationship study using a series of synthetic probes. The inhibitory activities of various glycans suggest that UGGT has a strong affinity for the core pentasaccharide (Man3GlcNAc2) of high-mannose-type glycans. Our comparison of the reactivity of acceptors that have been modified by various aglycons supports the hypothesis that UGGT recognizes the hydrophobic region of client glycoproteins. Moreover, we discovered fluorescently labeled substrates that will be valuable for highly sensitive detection of UGGT activity.

Intracellular environments are highly crowded due to the presence of various biomacromolecules. In this study, we estimated the property of the endoplasmic reticulum glucosidase II (G-II) under macromolecular crowding conditions. A crowded milieu that contains bovine serum albumin greatly enhanced the second trimming step (cleavage 2), which deglucosylates Glc1Man9GlcNAc2, but not the first trimming step (cleavage 1), which removes the terminal glucose residue from Glc2Man9GlcNAc2. A similar effect was obtained with ribonuclease A and high molecular weight polyethylene glycol 20,000. An analysis of CD spectra suggested that G-II enhanced its cleavage 2 activity through conformational change. We also investigated the effects of molecular crowding on other N-linked glycan-processing enzymes, UDP-Glc:glycoprotein glucosyltransferase and 1,2-alpha-mannosidase. Our results indicate that the kinetics of glycan processing under crowded conditions may be quite different from those measured in dilute buffers.