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
2005
Geysens, Steven | Pakula, Tiina | Uusitalo, Jaana | Dewerte, Isabelle | Penttilä, Merja | Contreras, Roland
We describe isolation and characterization of the gene encoding the glucosidase II alpha subunit (GII[alpha]) 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[alpha] (gls2[alpha]) 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 [alpha]-1,3-linked glucose residue but not the innermost [alpha]-1,3-linked glucose residue from the Glc₂Man₉GlcNAc₂ N-glycan ER structure. Transformation of the Rut-C30 strain with a repaired T. reesei gls2[alpha] 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 GII[alpha] 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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