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

Steven Geysens, Tiina Pakula, Jaana Uusitalo, Isabelle Dewerte, Merja Penttilä, Roland Contreras

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Abstract

We describe isolation and characterization of the gene encoding the glucosidase II alpha subunit (GIIα) 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 GIIα (gls2α) 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 α-1,3-linked glucose residue but not the innermost α-1,3-linked glucose residue from the Glc2MaH 9GlcNAc2 N-glycan ER structure. Transformation of the Rut-C30 strain with a repaired T. reesei gls2α gene changed the glycosylation profile significantly, decreasing the amount of monoglucosylated structures and increasing the amount of high-mannose N-glycans. Full conversion to high-mannose carbohydrates was not obtained, and this was probably due to competition between the endogenous mutant subunit and the introduced wild-type GIIα 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.

Original languageEnglish
Pages (from-to)2910-2924
Number of pages15
JournalApplied and Environmental Microbiology
Volume71
Issue number6
DOIs
Publication statusPublished - 1 Jun 2005
MoE publication typeA1 Journal article-refereed

Fingerprint

frameshift mutation
Trichoderma reesei
Frameshift Mutation
glucosidases
Trichoderma
glycosylation
Glycosylation
Organism Cloning
molecular cloning
mutation
Endoplasmic Reticulum
endoplasmic reticulum
t-plasminogen activator
Polysaccharides
brefeldin A
Brefeldin A
protein
polysaccharides
dithiothreitol
gene

Cite this

@article{e549846a500e453fa3e3db26bb72cce5,
title = "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",
abstract = "We describe isolation and characterization of the gene encoding the glucosidase II alpha subunit (GIIα) 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 GIIα (gls2α) 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 α-1,3-linked glucose residue but not the innermost α-1,3-linked glucose residue from the Glc2MaH 9GlcNAc2 N-glycan ER structure. Transformation of the Rut-C30 strain with a repaired T. reesei gls2α gene changed the glycosylation profile significantly, decreasing the amount of monoglucosylated structures and increasing the amount of high-mannose N-glycans. Full conversion to high-mannose carbohydrates was not obtained, and this was probably due to competition between the endogenous mutant subunit and the introduced wild-type GIIα 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.",
author = "Steven Geysens and Tiina Pakula and Jaana Uusitalo and Isabelle Dewerte and Merja Penttil{\"a} and Roland Contreras",
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T1 - Cloning and characterization of the glucosidase II alpha subunit gene of Trichoderma reesei

T2 - A frameshift mutation results in the aberrant glycosylation profile of the hypercellulolytic strain Rut-C30

AU - Geysens, Steven

AU - Pakula, Tiina

AU - Uusitalo, Jaana

AU - Dewerte, Isabelle

AU - Penttilä, Merja

AU - Contreras, Roland

PY - 2005/6/1

Y1 - 2005/6/1

N2 - We describe isolation and characterization of the gene encoding the glucosidase II alpha subunit (GIIα) 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 GIIα (gls2α) 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 α-1,3-linked glucose residue but not the innermost α-1,3-linked glucose residue from the Glc2MaH 9GlcNAc2 N-glycan ER structure. Transformation of the Rut-C30 strain with a repaired T. reesei gls2α gene changed the glycosylation profile significantly, decreasing the amount of monoglucosylated structures and increasing the amount of high-mannose N-glycans. Full conversion to high-mannose carbohydrates was not obtained, and this was probably due to competition between the endogenous mutant subunit and the introduced wild-type GIIα 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.

AB - We describe isolation and characterization of the gene encoding the glucosidase II alpha subunit (GIIα) 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 GIIα (gls2α) 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 α-1,3-linked glucose residue but not the innermost α-1,3-linked glucose residue from the Glc2MaH 9GlcNAc2 N-glycan ER structure. Transformation of the Rut-C30 strain with a repaired T. reesei gls2α gene changed the glycosylation profile significantly, decreasing the amount of monoglucosylated structures and increasing the amount of high-mannose N-glycans. Full conversion to high-mannose carbohydrates was not obtained, and this was probably due to competition between the endogenous mutant subunit and the introduced wild-type GIIα 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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EP - 2924

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JF - Applied and Environmental Microbiology

SN - 0099-2240

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