Cloning and Characterization of the Glucosidase II Alpha Subunit Gene of Trichoderma reesei: a Frameshift Mutation Results in the Aberrant Glycosylation Profile of the Hypercellulolytic Strain Rut-C30 (original) (raw)
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Genetic Evidence for the Heterodimeric Structure of Glucosidase II
Journal of Biological Chemistry, 1999
It has been proposed that in rat and murine tissues glucosidase II (GII) is formed by two subunits, GII␣ and GII, respectively, responsible for the catalytic activity and the retention of the enzyme in the endoplasmic reticulum (ER). To test this proposal we disrupted genes (gls2␣ ؉ and gls2 ؉) encoding GII␣ and GII homologs in Schizosaccharomyces pombe. Both mutant cells (gls2␣ and gls2) were completely devoid of GII activity in cell-free assays. Nevertheless, N-oligosaccharides formed in intact gls2␣ cells were identified as Glc 2 Man 9 GlcNAc 2 and Glc 2 Man 8 GlcNAc 2 , whereas gls2 cells formed, in addition, small amounts of Glc 1 Man 9 GlcNAc 2. It is suggested that this last compound was formed by GII␣ transiently present in the ER. Monoglucosylated oligosaccharides facilitated glycoprotein folding in S. pombe as mutants, in which formation of monoglucosylated glycoproteins was completely (gls2␣) or severely (gls2 and UDP-Glc:glycoprotein:glucosyltransferase null) diminished, showed ER accumulation of misfolded glycoproteins when grown in the absence of exogenous stress as revealed by (a) induction of binding protein-encoding mRNA and (b) accumulation of glycoproteins bearing ER-specific oligosaccharides. Moreover, the same as in mammalian cell systems, formation of monoglucosylated oligosaccharides decreased the folding rate and increased the folding efficiency of glycoproteins as pulse-chase experiments revealed that carboxypeptidase Y arrived at a higher rate but in decreased amounts to the vacuoles of gls2␣ than to those of wild type cells.
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.
Applied and Environmental Microbiology, 2002
This paper describes the characterization of an intracellular -glucosidase enzyme BGLII (Cel1a) and its gene (bgl2) from the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). The expression pattern of bgl2 is similar to that of other cellulase genes known from this fungus, and the gene would appear to be under the control of carbon catabolite repression mediated by the cre1 gene. The BGLII protein was produced in Escherichia coli, and its enzymatic properties were analyzed. It was shown to be a specific -glucosidase, having no -galactosidase side activity. It hydrolyzed both cellotriose and cellotetraose. BGLII exhibited transglycosylation activity, producing mainly cellotriose from cellobiose and sophorose and cellobiose from glucose. Antibodies raised against BGLII showed the presence of the enzyme in T. reesei cell lysates but not in the culture supernatant. Activity measurements and Western blot analysis of T. reesei strains expressing bgl2 from a constitutive promoter further confirmed the intracellular localization of this -glucosidase.
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.
Gene, 2001
A gene (gluc78) encoding an antifungal glucan 1,3-b-glucosidase was cloned from strain P1 of the biocontrol fungus Trichoderma atroviride (formerly T. harzianum). A putative regulatory sequence upstream from the coding region was cloned using single-strand extension from a primer in the known portion of the gene, circularized with T4 ligase, and then reamplified with PCR to generate double-stranded DNA. The entire genomic DNA sequence consisted of 3440 bp, with 559 and 579 bp, respectively, in 5 0 and 3 0 untranslated regions. The transcription unit contains a single intron, positioned in the 5 0 untranslated region. The gene encodes for a protein of 770 aa, including a 40 aa signal peptide. Symmetry between the first and second halves of the mature protein was found. The gene is present as a single copy in T. atroviride and a similar gene also is present in T. harzianum and T. virens. The encoded protein has similarity to a small group of sequences from filamentous fungi and no significant similarity to 1,3-b-glucanases or glucosidases from other organisms. Northern analysis indicates that the gene is repressed in the presence of 3% glucose and expressed in media containing 0.1% of the sugar. Laminarin (0.1%) enhances expression after 18 h and other polymers such as scleroglucan and pustulan may enhance expression after 40 h of growth.
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...
Partial purification and some properties of α–glucosidase from Trichoderma longibrachiatum
2012
The use of hydrolase enzyme plays an important role in the industrial production of a-D-glucose from carbohydrate sources. This study investigated partial purification and characterization of a-glucosidase from Trichoderma longibrachiatum with a view to enhancing its potentials in biotechnological processes. Strains of Trichoderma longibrachiatum were cultured on rice bran medium at 30°C for 96 hour for the production of a–glucosidase. The enzyme was partially purified by eluting the ammonium sulphate (70%) saturation precipitated sample on Sephadex G-75 and Sephadex G-25. Enzyme assay was carried out using p-nitrophenyl-a-D-glucopyranoside (PNP- a-G) as the substrate and protein concentration was determined. Kinetic parameters, molecular weight, pH effect, temperature and thermostability were also determined. The activity of enzyme in the presence of arylglucosides and different cations were monitored. The partially purified protein migrated as a single band in 10% SDS-Polyacrylami...
Glycobiology, 2004
The glycosylation of Cel7A (CBH I) from Trichoderma reesei varies considerably when the fungus is grown under different conditions. As shown by ESI-MS and PAG-IEF analyses of both intact protein and the isolated catalytic core module, the microheterogeneity originates mainly from the variable ratio of single N-acetylglucosamine over high-mannose structures on the three N-glycosylation sites and from the presence or absence of phosphate residues. Fully N-and O-glycosylated Cel7A can only be isolated from minimal medium and probably reflects the initial complexity of the protein on leaving the glycosynthetic pathway. Extracellular activities are responsible for postsecretorial modifications in other cultivation conditions: a-(132)-mannosidase, a-(133)-glucosidase and an Endo H type activity participate in N-deglycosylation (core), whereas a phosphatase and a mannosidase are probably responsible for hydrolysis of O-glycans (linker). The effects are most prominent in corn steep liquor±enriched media, where the pH is closer to the pH optimum (5±6) of these extracellular hydrolases. In minimal medium, the low pH and the presence of proteases could explain for the absence of such activities. On the other hand, phosphodiester linkages in the catalytic module are only observed under specific conditions. The extracellular trigger is still unknown, but mannophosphorylation may be regulated intracellularly by a-(132)mannosidases and phosphomannosyl transferases competing for the same intermediate in the glycosynthetic pathway.
Using anion-exchange chromatography the catalytic domain of endoglucanase 1 (Cel7B) from Trichoderma reesei was resolved in multiple fractions with different isoelectric points, presumably related to different glyco-forms of the enzyme. The protein fractions were analysed using lectins and electrospray MS. Isolated N-glycans were analysed by fluorophore-assisted carbohydrate electrophoresis and amine-adsorption HPLC. The results show that this particular preparation contained at least 14 different glycoforms. The major isoform contained only one GlcNAc, presumably N-linked, and one mannose, most probably O-linked to serine/threonine at a separate site. Except for a small population containing Man 5 GlcNAc 2 +1–2 Man, the rest of the protein had negatively charged phosphate-containing N-glycans. All glycoforms contained at least one O-linked mannose residue. The increased negative charge of the protein, introduced by oligosaccharide phosphorylation, is the most probable reason for the different isoelectric points and the occurrence of multiple peaks during purification.