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.
We describe isolation and characterization of the gene encoding the glucosidase II alpha subunit (GIIalpha) 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 GIIalpha (gls2alpha) 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 gls2alpha 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 GIIalpha 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.
Geysens
Steven
S
Fundamental and Applied Molecular Biology, Department for Molecular Biomedical Research, Ghent University and VIB (Flemish Interuniversity Institute for Biotechnology), Ghent-Zwijnaarde, Belgium.
Pakula
Tiina
T
Uusitalo
Jaana
J
Dewerte
Isabelle
I
Penttilä
Merja
M
Contreras
Roland
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D016368:Frameshift Mutation; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D006031:Glycosylation; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D000520:alpha-Glucosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D021122:Protein Subunits; C034251:4-nitrophenyl-alpha-glucosidase; D000520:alpha-Glucosidases