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Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger

Author: Ngiam, C. · Jeenes, D.J. · Punt, P.J. · Hondel, C.A.M.J.J. van den · Archer, D.B.
Type:article
Date:2000
Institution: Centraal Instituut voor Voedingsonderzoek TNO
Source:Applied and Environmental Microbiology, 2, 66, 775-782
Identifier: 56522
doi: doi:10.1128/AEM.66.2.775-782.2000
Keywords: Nutrition · Egg white · Glucan 1,4 alpha glucosidase · Lysozyme · Messenger RNA · Microsome enzyme · Protein disulfide isomerase · Ribonuclease · Aspergillus niger · Endoplasmic reticulum · Enzyme activity · Enzyme analysis · Gene overexpression · Immunoblotting · Nonhuman · Protein folding · Protein induction · Protein secretion · Renaturation · Saccharomyces cerevisiae · Actins · Aspergillus niger · Calcimycin · Dithiothreitol · Down-Regulation · Endoplasmic Reticulum · Fungal Proteins · Gene Expression Regulation, Fungal · Heat-Shock Proteins · HSP70 Heat-Shock Proteins · Microsomes · Protein Denaturation · Protein Disulfide-Isomerase · Protein Folding · Recombinant Proteins · Ribonuclease, Pancreatic · RNA, Messenger · Saccharomyces cerevisiae · Transcription, Genetic · Transformation, Genetic · Aspergillus niger · Eukaryota · Saccharomyces cerevisiae

Abstract

Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes. A gene, pdiA, encoding PDIA was previously isolated from Aspergillus niger, and we report its functional characterization here. Functional analysis of PDIA showed that it catalyzes the refolding of denatured and reduced RNase A. pdiA also complemented PDI function in a Saccharomyces Cerevisiae Δpdi1 mutant in a yeast-based killer toxin assay. Levels of pdiA mRNA and PDIA protein were raised by the accumulation of unfolded proteins in the endoplasmic reticulum. This response of pdiA mRNA levels was slower and lower in magnitude than that of A. niger bipA, suggesting that the induction of pdiA is not part of the primary stress response. An increased level of pdiA transcripts was also observed in two A. niger strains overproducing a heterologous protein, hen egg white lysozyme (HEWL). Although overexpression of PDI has been successful in increasing yields of some heterologous proteins in S. cerevisiae, overexpression of PDIA did not increase secreted yields of HEWL in A. niger, suggesting that PDIA itself is not limiting for secretion of this protein. Downregulation of pdiA by antisense mRNA reduced the levels of microsomal PDIA activity by up to 50%, lowered the level of PDIA as judged by Western blots, and lowered the secreted levels of glucoamylase by 60 to 70%. Chemicals/CAS: Actins; BipA protein, Aspergillus; Calcimycin, 52665-69-7; Dithiothreitol, 3483-12-3; Fungal Proteins; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Protein Disulfide-Isomerase, EC 5.3.4.1; Recombinant Proteins; Ribonuclease, Pancreatic, EC 3.1.27.5; RNA, Messenger; TigA protein, Aspergillus niger