Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-α-d-mannosidase

Maras, Marleen; Callewaert, Nico; Piens, Kathleen; Claeyssens, Marc; Martinet, Wim; Dewaele, Sylviane; Contreras, Hans; Dewerte, Isabelle; Penttilä, Merja; Contreras, Roland

doi:10.1016/s0168-1656(99)00222-9

Cited by 55 publications

(39 citation statements)

References 28 publications

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“…Trichoderma reesei ␣-1,2-mannosidase at 30 g͞ml was prepared from Pichia pastoris (7). APTS (8-Amino-1,3,6-pyrenetrisulfonic acid) was from Molecular Probes.…”

Section: Methodsmentioning

confidence: 99%

“…Preparation of APTS-labeled Man5GlcNAc2. Bovine RNaseB N-glycans [Man (5)(6)(7)(8)(9) GlcNAc 2 ] (30 nmol) were treated with 50 mM APTS (1,000-fold excess) in a reaction mixture (60 l) containing citric acid (0.6 M), NaCNBH 3 (0.5 M), and DMSO (50% vol͞vol). Incubation was for 4 h at 55°C followed by 16 h at 37°C.…”

Section: Identification Of Hek293s Cell Lines With Altered N-glycosylmentioning

confidence: 99%

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Structure and function in rhodopsin: High-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N -acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line

Reeves

Callewaert

Contreras

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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603

538

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An HEK293S cell line resistant to ricin was prepared by mutagenesis by using ethyl methanesulfonate. It was shown to lack N-acetylglucosaminyltransferase I (GnTI) activity, and consequently unable to synthesize complex N-glycans. The tetracycline-inducible opsin expression system was assembled into this GnTI ؊ HEK293S cell line. Stable cell lines were isolated that gave tetracycline͞sodium butyrateinducible expression of the WT opsin gene at levels comparable with those observed in the parent tetracycline-inducible HEK293S cell line. Analysis of the N-glycan in rhodopsin expressed by the HEK293S GnTI ؊ stable cell line showed it to be Man5GlcNAc2. In a larger-scale expression experiment (1.1 liter) a WT opsin production level of 6 mg͞liter was obtained. Further, the toxic constitutively active rhodopsin mutant, E113Q͞E134Q͞M257Y, previously shown to require inducible expression, has now been expressed in an HEK293S GNTI ؊ -inducible cell line at levels comparable with those obtained with WT rhodopsin.

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“…Trichoderma reesei ␣-1,2-mannosidase at 30 g͞ml was prepared from Pichia pastoris (7). APTS (8-Amino-1,3,6-pyrenetrisulfonic acid) was from Molecular Probes.…”

Section: Methodsmentioning

confidence: 99%

Section: Identification Of Hek293s Cell Lines With Altered N-glycosylmentioning

confidence: 99%

Structure and function in rhodopsin: High-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N -acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line

Reeves

Callewaert

Contreras

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

603

538

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“…The secreted version of the Trichoderma reesei ␣-1,2-mannosidase was purified by using a combination of hydrophobic interaction chromatography, anion-exchange chromatography, and gel filtration chromatography, as described previously (22,29). All sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) experiments were performed with 10% polyacrylamide gels under standard running conditions.…”

Section: Methodsmentioning

confidence: 99%

“…To evaluate whether the heterogeneity of secreted glycoproteins was decreased in the GlycoSwitchM8 strain, we heterologously expressed the hyperglycosylated T. reesei ␣-1,2-mannosidase as a secreted protein in wild-type strain GS115 and the GlycoSwitchM8 strain, as described by Maras et al (22). The supernatant was separated by SDS-PAGE (Fig.…”

Section: Vol 70 2004 N-glycan Engineering In P Pastoris 2641mentioning

confidence: 99%

In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans inPichia pastoris

Vervecken

Kaǐgorodov

Callewaert

et al. 2004

Appl Environ Microbiol

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167

124

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The Pichia pastoris N-glycosylation pathway is only partially homologous to the pathway in human cells. In the Golgi apparatus, human cells synthesize complex oligosaccharides, whereas Pichia cells form mannose structures that can contain up to 40 mannose residues. This hypermannosylation of secreted glycoproteins hampers the downstream processing of heterologously expressed glycoproteins and leads to the production of protein-based therapeutic agents that are rapidly cleared from the blood because of the presence of terminal mannose residues. Here, we describe engineering of the P. pastoris N-glycosylation pathway to produce nonhyperglycosylated hybrid glycans. This was accomplished by inactivation of OCH1 and overexpression of an ␣-1,2-mannosidase retained in the endoplasmic reticulum and N-acetylglucosaminyltransferase I and ␤-1,4-galactosyltransferase retained in the Golgi apparatus. The engineered strain synthesized a nonsialylated hybrid-type N-linked oligosaccharide structure on its glycoproteins. The procedures which we developed allow glycan engineering of any P. pastoris expression strain and can yield up to 90% homogeneous protein-linked oligosaccharides.Most protein-based therapeutic agents produced in heterologous expression systems are glycosylated, a modification that is crucial for correct folding, stability, and bioactivity of the protein and influences its pharmacokinetic properties, such as tissue distribution and blood clearance. Glycoproteins with terminal sialic acids on their glycans persist longer in the blood than glycoproteins with terminal galactose, N-acetylglycosamine, or mannose residues because the latter compounds are cleared rapidly via receptors in the liver and on reticuloendothelial cells (e.g., the asialoglycoprotein receptor and the mannose receptor) (10,20,30,31). In addition, glycan structures produced in nonhuman cells can cause immune reactions, as exemplified by the reaction against xenografts of porcine origin; these reactions are primarily caused by the presence of ␣-galactose on the glycoproteins (7). Another example is the immune reaction against glycoproteins from yeast, which results from the presence of ␣-1,3-mannose, ␤-linked mannose, and/or phosphate residues in either a phosphomonoester or phosphodiester linkage (1, 32). Consequently, recombinant glycoproteins produced for therapeutic applications should be expressed in heterologous hosts that produce protein-linked oligosaccharides that closely resemble those of humans.

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“…Class II a-mannosidases contain the lysosomal mannosidases, Golgi mannosidase II, yeast vacular mannosidase (Yoshihisa & Anraku, 1989, 1990, and ER amannosidase II (Weng & Spiro, 1993, 1996. Several Golgi a-mannosidases have been cloned and characterized from Pencillium citrinum (Yoshida & Ichishima, 1995;Yoshida et al, 1993), A. saitoi (Ichishima et al, 1999), Aspergillus oryzae (Akao et al, 2006), T. reesei (Maras et al, 2000), and Aspergillus nidulans (Eades & Hintz, 2000). These enzymes are monomeric, with a molecular mass of 50-60 kDa, and they show maximal activity in the semi-acidic condition (pH 4-6).…”

Section: Introductionmentioning

confidence: 99%

Deletion of the msdS/AfmsdC gene induces abnormal polarity and septation in Aspergillus fumigatus

Zhang

Wang

et al. 2008

Microbiology

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a-Mannosidases play an important role in the processing of mannose-containing glycans in eukaryotes. A deficiency in a-mannosidase is lethal in humans and cattle. In contrast to mammals, Saccharomyces cerevisiae does not require the endoplasmic reticulum a-mannosidase gene for growth. However, little is known of the consequence of loss of function of class I a-mannosidases in filamentous fungi. In this study, the msdS/AfmsdC gene was identified to encode 1,2-amannosidase MsdS in Aspergillus fumigatus. Soluble MsdS expressed in Escherichia coli was characterized as a typical class I a-mannosidase. The msdS gene was deleted by replacement of the msdS gene with a pyrG gene. Although the mutant showed a defect in N-glycan processing, as well as a reduction of cell wall components and a reduced ability of conidiation, it appeared that the rate of hyphal growth was not affected. Morphology analysis revealed abnormal polarity and septation at the stages of germination, hyphal growth and conidiation. Although the mechanism by which the N-glycan processing affects polarity and septation is unclear, our results show that msdS is involved in polarity and septation in A. fumigatus.

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Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-α-d-mannosidase

Cited by 55 publications

References 28 publications

Structure and function in rhodopsin: High-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N -acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line

Structure and function in rhodopsin: High-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N -acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line

In Vivo Synthesis of Mammalian-Like, Hybrid-Type N-Glycans inPichia pastoris

Deletion of the msdS/AfmsdC gene induces abnormal polarity and septation in Aspergillus fumigatus

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