Endoplasmic Reticulum Glucosidase II Is Composed of a Catalytic Subunit, Conserved from Yeast to Mammals, and a Tightly Bound Noncatalytic HDEL-containing Subunit (original) (raw)
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Endoplasmic Reticulum Glucosidase II Is Inhibited by Its End Products
Biochemistry, 2008
The calnexin/calreticulin cycle is a quality control system responsible for promoting the folding of newly synthesized glycoproteins entering the endoplasmic reticulum (ER). The association of calnexin and calreticulin with the glycoproteins is regulated by ER glucosidase II, which hydrolyzes Glc 2 Man X GlcNAc 2 glycans to Glc 1 Man X GlcNAc 2 and further to Glc 0 Man X GlcNAc 2 (X represents any number between 5 and 9). To gain new insights into the reaction mechanism of glucosidase II, we developed a kinetic model that describes the interactions between glucosidase II, calnexin/calreticulin, and the glycans. Our model accurately reconstructed the hydrolysis of glycans with nine mannose residues and glycans with seven mannose residues, as measured by Totani et al. [Totani, K., Ihara, Y., Matsuo, I., and Ito, Y. (2006) J. Biol. Chem. 281, 31502-31508]. Intriguingly, our model predicted that glucosidase II was inhibited by its nonglucosylated end products, where the inhibitory effect of Glc 0 Man 7 GlcNAc 2 was much stronger than that of Glc 0 Man 9 GlcNAc 2 . These predictions were confirmed experimentally. Moreover, our model suggested that glycans with a different number of mannose residues can be equivalent substrates of glucosidase II, in contrast to what had been previously thought. We discuss the possibility that nonglucosylated glycans, existing in the ER, might regulate the entry of newly synthesized glycoproteins into the calnexin/calreticulin cycle. Our model also shows that glucosidase II does not interact with monoglucosylated glycans while they are bound to calnexin or calreticulin. * Corresponding authors. S.B.-N.
Glucosidase I, a transmembrane endoplasmic reticular glycoprotein with a luminal catalytic domain
The Journal of biological chemistry, 1991
We have analyzed the functional domain structure of rat mammary glucosidase I, an enzyme involved in N-linked glycoprotein processing, using biochemical and immunological approaches. The enzyme contains a high mannose type sugar chain that can be cleaved by endo-beta-N-acetyl-D-glucosaminidase H without significantly affecting the catalytic activity. Based on trypsin digestion pattern and the data on membrane topography, glucosidase I constitutes a single polypeptide chain of 85 kDa with two contiguous domains: a membrane-bound domain that anchors the protein to the endoplasmic reticulum and a luminal domain. A catalytically active 39-kDa domain could be released from membranes by limited proteolysis of saponin-permeabilized membranes with trypsin. This domain appeared to contain the active site of the enzyme and had the ability to bind to glucosidase I-specific affinity gel. Phase partitioning with Triton X-114 indicated the amphiphilic nature of the native enzyme, consistent with ...
UDP-GlC:glycoprotein glucosyltransferase-glucosidase II, the ying-yang of the ER quality control
Seminars in Cell & Developmental Biology, 2010
The N-glycan-dependent quality control of glycoprotein folding prevents endoplasmic to Golgi exit of folding intermediates, irreparably misfolded glycoproteins and incompletely assembled multimeric complexes. It also enhances folding efficiency by preventing aggregation and facilitating formation of proper disulfide bonds. The control mechanism essentially involves four components, resident lectin-chaperones that recognize monoglucosylated polymannose glycans, a lectinassociated oxidoreductase acting on monoglucosylated glycoproteins, a glucosyltransferase that creates monoglucosytlated epitopes in protein-linked glycans and a glucosidase that removes the glucose units added by the glucosyltransferase. This last enzyme is the only mechanism component sensing glycoprotein conformations as it creates monoglucosylated glycans exclusively in not properly folded species or in not completely assembled complexes. The glucosidase is a dimeric heterodimer composed of a catalytic subunit and an additional one that is partially responsible for the ER localization of the enzyme and for the enhancement of the deglucosylation rate as its mannose 6-phosphate receptor homologous domain presents the substrate to the catalytic site. This review deals with our present knowledge on the glucosyltransferase and the glucosidase.
Journal of Biological Chemistry, 2006
Glucosidase II is essential for sequential removal of two glucose residues from N-linked glycans during glycoprotein biogenesis in the endoplasmic reticulum. The enzyme is a heterodimer whose ␣-subunit contains the glycosyl hydrolase active site. The function of the -subunit has yet to be defined, but mutations in the human gene have been linked to an autosomal dominant form of polycystic liver disease. Here we report the identification and characterization of a Saccharomyces cerevisiae gene, GTB1, encoding a polypeptide with 21% sequence similarity to the -subunit of human glucosidase II. The Gtb1 protein was shown to be a soluble glycoprotein (96 -102 kDa) localized to the endoplasmic reticulum lumen where it was present in a complex together with the yeast ␣-subunit homologue Gls2p. Surprisingly, we found that ⌬gtb1 mutant cells were specifically defective in the processing of monoglucosylated glycans. Thus, although Gls2p is sufficient for cleavage of the penultimate glucose residue, Gtb1p is essential for cleavage of the final glucose. Our data demonstrate that Gtb1p is required for normal glycoprotein biogenesis and reveal that the final two glucose-trimming steps in N-glycan processing are mechanistically distinct.
Scientific reports, 2016
The endoplasmic reticulum (ER) has a sophisticated protein quality control system for the efficient folding of newly synthesized proteins. In this system, a variety of N-linked oligosaccharides displayed on proteins serve as signals recognized by series of intracellular lectins. Glucosidase II catalyzes two-step hydrolysis at α1,3-linked glucose-glucose and glucose-mannose residues of high-mannose-type glycans to generate a quality control protein tag that is transiently expressed on glycoproteins and recognized by ER chaperones. Here we determined the crystal structures of the catalytic α subunit of glucosidase II (GIIα) complexed with two different glucosyl ligands containing the scissile bonds of first- and second-step reactions. Our structural data revealed that the nonreducing terminal disaccharide moieties of the two kinds of substrates can be accommodated in a gourd-shaped bilocular pocket, thereby providing a structural basis for substrate-binding specificity in the two-step...
The Journal of Cell Biology, 1990
Glucosidase II, an asparagine-linked oligosaccharide processing enzyme, is a resident glycoprotein of the endoplasmic reticulum. In kidney tubular cells, in contrast to previous findings on hepatocytes, we found by light and electron microscopy immunoreactivity for glucosidase II predominantly in post-Golgi apparatus structures. The majority of immunolabel was in endocytotic structures beneath the plasma membrane. Immunoprecipitation confirmed presence of the glucosidase II subunit in purified brush border preparations. Kidney glucosidase II contained species carrying endo H-sensitive, high mannose as well as endo H-resistant oligosaccharide chains. Some species of glucosidase II contained sialic acid. The sialylated species were enzymatically active. This study demonstrates than an enzyme presumed to be a resident of the endoplasmic reticulum may show alternative localizations in some cell types.
Immunolocalization of the oligosaccharide trimming enzyme glucosidase II
The Journal of cell biology, 1986
We used immunoelectron microscopy to localize glucosidase II in pig hepatocytes. The enzyme trims the two inner alpha 1,3-linked glucoses from N-linked oligosaccharide precursor chains of glycoproteins. Immunoreactive enzyme was concentrated in rough (RER) and smooth (SER) endoplasmic reticulum but not detectable in Golgi apparatus cisternae. Transitional elements of RER and smooth membraned structures close to Golgi apparatus cisternae contained labeling for glucosidase II. Specific labeling was also found in autophagosomes. These results indicate strongly that glucosidase II acts on glycoproteins before their transport to, and processing in Golgi apparatus cisternae, and suggest that an important transitional region for glucosidase II exists between RER and Golgi apparatus cisternae. Degradation in autophagolysosomes could form a normal catabolic pathway for glucosidase II.
Journal of Biological Chemistry, 1998
Several lines of evidence suggest that soluble peptide: N-glycanase (PNGase) is involved in the quality control system for newly synthesized glycoproteins in mammalian cells. Here we report the occurrence of a soluble PNGase activity in Saccharomyces cerevisiae. The enzyme, which was recovered in the cytosolic fraction, has a neutral pH optimum, and dithiothreitol is required for activity. All of these properties were similar to those of earlier described for mammalian PNGases. Interestingly, the yeast enzyme activity was found to be present almost exclusively in cells in stationary phase; little activity was detected in logarithmic growth phase cells. Upon incubation of a glycosylatable peptide R-Asn-X-Thr-R with permeabilized yeast spheroplasts, we detected formation of both glycosylated peptide and the peptide product expected from PNGase-mediated deglycosylation of this glycopeptide, namely, R-Asp-X-Thr-R. Recent findings that yeast have an active system for the retrograde transport of unfolded (glyco)proteins and glycopeptides out of the endoplasmic reticulum (ER) into the cytosol raise the possibility that this PNGase may participate in an early step in degradation of these molecules following their export from the ER.
FEMS Yeast Research, 2004
Proteins entering the endoplasmic reticulum (ER) have to acquire an export-competent structure before they are delivered to their final destination. This folding process is monitored by an ER protein quality control system. Folding-incompetent conformers are eliminated via a mechanism called ER-associated protein degradation (ERAD). Genetic studies in the yeast Saccharomyces cerevisiae have revealed that carbohydrate modification plays a crucial role in these processes. Here we show that a previously isolated der mutant (der7-1) is defective in ERAD. We identify DER7 as the gene encoding N -glycan-processing a-glucosidase I (EC 3.2.1.106) of the ER and demonstrate that its inactivity, due to a substitution of the conserved glycine residue at position 725 by arginine (G725R) in the der7-1 mutant, leads to ER-stress.
Demonstration of a peptide:N-glycosidase in the endoplasmic reticulum of rat liver
The Biochemical journal, 1997
Prompted by previous observations that polymannose oligosaccharides are released from newly synthesized glycoproteins [Anumula and Spiro (1983) J. Biol. Chem. 258, 15274-15282], we examined rat liver endoplasmic reticulum (ER) for the presence of endoglycosidases that could be involved in an event presumed to be a function of the protein quality control machinery. Our investigations indicated that a peptide:N-glycanase (PNGase) is present in ER membranes that has the capacity to release from radiolabelled glycopeptides glucosylated as well as non-glucosylated polymannose oligosaccharides terminating at their reducing end in a di-N-acetylchitobiose sequence (OS-GlcNAc2). This enzyme, which was found to be luminal in orientation, was most active in the pH range 5.5-7.0 and although it had no exogenous bivalent-cation requirements it was inhibited by EDTA. Detailed studies with Man9GlcNAc2-peptides demonstrated that in addition to the free oligosaccharide (Man9GlcNAc2) an additional ne...