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

摘要: We describe isolation and characterization of the gene encoding glucosidase II alpha subunit (GIIalpha) industrially important fungus Trichoderma reesei. This is catalytic part heterodimeric enzyme involved in structural modification within endoplasmic reticulum (ER) N-linked oligosaccharides present on glycoproteins. The GIIalpha (gls2alpha) hypercellulolytic strain Rut-C30 contains a frameshift mutation resulting truncated product. Based peculiar monoglucosylated N-glycan pattern proteins produced by strain, we concluded that protein can still hydrolyze first alpha-1,3-linked glucose residue but not innermost from Glc2Man9GlcNAc2 ER structure. Transformation with repaired T. reesei gls2alpha changed glycosylation profile significantly, decreasing amount structures increasing high-mannose N-glycans. Full conversion to carbohydrates was obtained, this probably due competition between endogenous mutant introduced wild-type protein. Since also quality control nascent polypeptide chains, its transcriptional regulation studied producing recombinant tissue plasminogen activator (tPA) cultures treated stress agents dithiothreitol (DTT) brefeldin A (BFA), which are known block transport induce unfolded response. While mRNA levels were clearly upregulated upon tPA production or BFA treatment, no such enhancement observed after DTT addition.