Expression of a cDNA encoding the glucose trimming enzyme glucosidase II in CHO cells and molecular characterization of the enzyme deficiency in a mutant mouse lymphoma cell line

Flura, Thomas; Brada, Daniela; Ziak, Martin; Roth, Jürgen

doi:10.1093/glycob/7.5.617

Cited by 38 publications

(38 citation statements)

References 46 publications

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“…For preparation of genomic DNA or RNA for 5Ј rapid amplification of cDNA ends (5Ј-RACE) cloning, T. reesei strains Rut-C30 (ϭ ATCC 56765), Rut-NG14 (ϭ ATCC 56765), and QM9414 (ϭ ATCC 26921) were grown in minimal medium. This medium contained (per liter) 20 2 O and supplemented with essential minerals, and the pH was adjusted to 5.2 by addition of KOH. The precultures were cultivated for 2 days at 28°C on a rotary shaker at 200 rpm, transferred into fresh medium (1:10, vol/vol), and cultivated for another day.…”

Section: Methodsmentioning

confidence: 99%

“…Since then, glucosidase II has been partially or completely purified from a wide variety of both lower and higher eukaryotic organisms (2,6,7,11,20,38,78,80,88). Following some initial controversy, it is now generally accepted that the protein is a rather asymmetric nonglobular heterodimer consisting of a catalytic alpha subunit with a molecular mass of about 110 kDa (GII␣) and a beta subunit with a molecular mass of 60 kDa (GII␤) (6,15,65,85,86,88).…”

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confidence: 99%

“…Disruption of the latter ORF yielded a yeast strain devoid of glucosidase II activity (88). Shortly after this, GII␣-encoding sequences were cloned from mouse T lymphocytes (6), pig liver (20), the slime mold Dictyostelium discoideum (22), and Schizosaccharomyces pombe (15). All derived protein sequences carry a conserved WX DLNE motif, which is the typical consensus sequence for the active site of glycosidase family 31 (29).…”

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confidence: 99%

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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

Geysens

Pakula

Uusitalo

et al. 2005

Appl Environ Microbiol

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We describe isolation and characterization of the gene encoding the glucosidase II alpha subunit (GII␣) of the industrially important fungus Trichoderma reesei. This subunit is the catalytic part of the glucosidase II heterodimeric enzyme involved in the structural modification within the endoplasmic reticulum (ER) of N-linked oligosaccharides present on glycoproteins. The gene encoding GII␣ (gls2␣) in the hypercellulolytic strain Rut-C30 contains a frameshift mutation resulting in a truncated gene product. Based on the peculiar monoglucosylated N-glycan pattern on proteins produced by the strain, we concluded that the truncated protein can still hydrolyze the first ␣-1,3-linked glucose residue but not the innermost ␣-1,3-linked glucose residue from the Glc 2 Man 9 GlcNAc 2 N-glycan ER structure. Transformation of the Rut-C30 strain with a repaired T. reesei gls2␣ gene changed the glycosylation profile significantly, decreasing the amount of monoglucosylated structures and increasing the amount of high-mannose N-glycans. Full conversion to highmannose carbohydrates was not obtained, and this was probably due to competition between the endogenous mutant subunit and the introduced wild-type GII␣ protein. Since glucosidase II is also involved in the ER quality control of nascent polypeptide chains, its transcriptional regulation was studied in a strain producing recombinant tissue plasminogen activator (tPA) and in cultures treated with the stress agents dithiothreitol (DTT) and brefeldin A (BFA), which are known to block protein transport and to induce the unfolded protein response. While the mRNA levels were clearly upregulated upon tPA production or BFA treatment, no such enhancement was observed after DTT addition.

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confidence: 99%

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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

Geysens

Pakula

Uusitalo

et al. 2005

Appl Environ Microbiol

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“…Co-translational removal of the terminal glucose residue occurs rapidly by the action of ␣-glucosidase I, followed by the successive removal of the second and third glucose residues by ␣-glucosidase II (15)(16)(17)(18)(19). The monoglucosylated oligosaccharide can be regenerated by a glucosyltransferase that acts posttranslationally on unfolded proteins (20).…”

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confidence: 99%

Calnexin Interaction with N-Glycosylation Mutants of a Polytopic Membrane Glycoprotein, the Human Erythrocyte Anion Exchanger 1 (Band 3)

Popov¹,

Reithmeier²

1999

Journal of Biological Chemistry

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The interaction of the endoplasmic reticulum chaperone calnexin with N-glycosylation mutants of a polytopic membrane glycoprotein, the human erythrocyte anion exchanger (AE1), was characterized by cell-free translation and in transfected HEK293 cells, followed by co-immunoprecipitation using anti-calnexin antibody. AE1 contains 12-14 transmembrane segments and has a single site of N-glycosylation at Asn-642 in the fourth extracytosolic loop. This site was mutated (N642D) to create a nonglycosylated protein. Calnexin showed a preferential interaction with N-glycosylated AE1 relative to nonglycosylated AE1 both in vitro and in vivo. This interaction could be blocked by inhibition of glucosidases I and II with castanospermine. Calnexin had access to novel N-glycosylated sites created in other extracytosolic loops in AE1 by site-directed or insertional mutagenesis. The interaction with AE1 was enhanced when multiple sites were introduced into the same loop or into two different loops. An association of calnexin with truncated versions of N-glycosylated AE1 was detected after release of the nascent chains from ribosomes with puromycin. The results show that the interaction of calnexin with the polytopic membrane glycoprotein AE1 was dependent on the presence but not the location of the oligosaccharide. Furthermore, calnexin was associated with AE1 after release of AE1 from the translocation machinery.Calnexin, an integral membrane protein, and its soluble homolog, calreticulin, are endoplasmic reticulum (ER) 1 chaperones that bind transiently to newly synthesized glycoproteins, thereby promoting their folding and oligomerization (1-4). These chaperones also display a prolonged interaction with misfolded and aggregated proteins, leading to their retention in the ER (5-7). Nascent polypeptides are targeted to the ER membrane by a signal recognition particle-dependent mechanism. The subsequent translocation of the polypeptide into the ER lumen and the integration of transmembrane segments into the ER membrane are mediated by a protein channel that forms the translocon (8). Calnexin is part of a multimeric protein complex, including calreticulin, BiP, GRP94, and Erp57, that "proofreads" the protein folding process in the ER (7, 9 -11).N-Glycosylation is a co-translational event involving the en bloc transfer of a Glc 3 Man 9 GlcNAc 2 oligosaccharide from a dolichol donor to a lumenal acceptor site (Asn-X-Ser/Thr) in the nascent chain by the ER oligosaccharyl transferase (12-14). Co-translational removal of the terminal glucose residue occurs rapidly by the action of ␣-glucosidase I, followed by the successive removal of the second and third glucose residues by ␣-glucosidase II (15-19). The monoglucosylated oligosaccharide can be regenerated by a glucosyltransferase that acts posttranslationally on unfolded proteins (20). Calnexin and calreticulin are lectins that interact with the Glc 1 Man 9 -5 GlcNAc 2 oligosaccharide (21-26), and calnexin-bound monoglucosyl glycoproteins are protected from ␣-glucosidase II removal of ...

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“…More specifically, removal of the first ␣1,3-linked glucose by GII creates a substrate for calnexin/calreticulin binding (14 -17), whereas removal of the second glucose causes dissociation of calnexin/calreticulin from the polypeptide (15, 18 -20). The hydrolysis of the second ␣1,3-linked glucose is necessary for the progression of properly folded glycoproteins from the ER to the Golgi (21, 22).The GII enzyme is composed of a 116-kDa ␣-subunit that contains a catalytic motif of the Family 31 glucosidases (11,23) and an 80-kDa ␤-chain of unknown function (24). We and others hypothesize that the ␤-chain is involved in enzyme localization (11, 24).…”

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confidence: 99%

Specific Isoforms of the Resident Endoplasmic Reticulum Protein Glucosidase II Associate with the CD45 Protein-tyrosine Phosphatase via a Lectin-like Interaction

Baldwin

Gogela-Spehar

Ostergaard

2000

Journal of Biological Chemistry

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We have previously demonstrated that CD45 physically associates with the endoplasmic reticulum processing enzyme glucosidase II (GII). GII consists of the catalytic ␣-chain and an associated ␤-chain. To gain insight into the basis of the association between CD45 and GII, we examined the biochemical requirements for the interaction. We show that the ␣-subunit is essential for the interaction. Interestingly, only a higher molecular weight form of GII␣ is capable of associating with CD45 in a competitive situation where multiple GII␣ isoforms are expressed. Further, transfection studies demonstrate that only isoforms containing the alternatively spliced sequence Box A1 are capable of binding CD45, although all isoforms are catalytically active. The interaction between CD45 and GII is dependent on the active site of GII, is mediated through the carbohydrate on CD45, and can be inhibited with mannose. Taken together, these results suggest that GII␣ acts as a lectin and binds to CD45 in an exon-dependent manner. This lectin activity of GII may be a novel mechanism for the regulation of CD45 biology and play a role in immune function, possibly by regulating CD45 glycosylation.CD45 is a highly abundant, transmembrane, protein-tyrosine phosphatase expressed on all cells of hematopoietic origin (1). The cytoplasmic phosphatase activity of CD45 has been shown to be essential for the early signal transduction events leading to both thymocyte maturation and T cell activation (1). There is substantial evidence to suggest that CD45 regulates the tyrosine phosphorylation of Src family kinases (2, 3). The external domain of CD45 is extremely heterogeneous with respect to size and carbohydrate content primarily because of three alternatively spliced exons that encode for potential Olinked glycosylations (4). The usage of these exons appears to be developmentally regulated (4). As well, the extracellular domain encodes attachment sites for numerous N-linked glycans, and these glycosylations appear to be important for cell surface expression and protein stability of CD45 (5). Finally, although CD45 is a cell surface protein, no specific ligand for the extracellular domain has been definitively identified. Perhaps relevant to the present study, there have been studies suggesting that some lectins such as CD22 (6, 7), galectin-1 (8, 9), and the mannan-binding protein (10) are able to bind to CD45 carbohydrate, although the biological significance of these interactions are largely not understood.Recently, our laboratory has demonstrated that the carbohydrate processing enzyme ␣-glucosidase II (GII) 1 physically interacts with CD45 (11). GII is found within the ER and catalyzes the hydrolysis of the inner two ␣1,3-linked glucose residues present on all N-linked immature oligosaccharides (12, 13). This processing of glucose in the ER has been shown to be intimately involved in protein folding by regulating the interaction between the nascent polypeptide and the lectin chaperones calnexin and calreticulin. More specifically, removal of t...

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Expression of a cDNA encoding the glucose trimming enzyme glucosidase II in CHO cells and molecular characterization of the enzyme deficiency in a mutant mouse lymphoma cell line

Cited by 38 publications

References 46 publications

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

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

Calnexin Interaction with N-Glycosylation Mutants of a Polytopic Membrane Glycoprotein, the Human Erythrocyte Anion Exchanger 1 (Band 3)

Specific Isoforms of the Resident Endoplasmic Reticulum Protein Glucosidase II Associate with the CD45 Protein-tyrosine Phosphatase via a Lectin-like Interaction

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