Cellular responses to the expression of aberrant secretory proteins in Aspergillus oryzae

Takahiro Shintani, Daisuke Shiro, Jun-ichi Yokota, Katsuya Gomi

Author address: 

Graduate School of Agricultural Science, Tohoku University, Sendai, Miyagi, Japa


Filamentous fungi have been used as hosts that produce useful proteins because of their ability to secrete proteins at a very high level. Despite a great success in producing the proteins derived from eukaryotic microbes, the efficiency of secretion of higher eukaryotic proteins was quite poor in many cases. One of the reasons for the decreased production could be the instability of heterologously expressed proteins in the secretory pathway. In this research, we used Aspergillus oryzae as a host strain to analyze the cellular response to the overexpression of an aberrant secretory protein. We expressed the mutant version of 1,2-alpha-mannosidase from Aspergillus saitoi, whose cystein residue at the position 443 was substitute to phenylalanine (C443F MsdS). It has been reported that this amino acid residue is crucial to maintain the proper folding of MsdS and this mutation therefore causes the decreased production of this protein in A. oryzae. We integrated the mutant msdS allele in a niaD locus as well as the wild type allele whose expressions were controlled under a moderate enoA promoter or a strong No.8142 promoter. When expressed from the enoA promoter, the mutant MsdS was not secreted whereas the wild type was detected in a medium fraction. The decreased production of the mutant was not due to the defect in its secretion, because it was not seen even in a cellular fraction. Moreover, it was stabilized in the hrdA deletion strain, suggesting that the mutant MsdS was degraded via the ER-associated degradation pathway. When expressed from the No.8142 promoter, although the mutant MsdS was secreted at a level comparable to the wild type, it was hyperglycosylated, which is rarely seen in Aspergillus. Since the mutant MsdS in the intracellular fraction did not receive hyperglycosylation, which occurs in Golgi apparatus, the proteins might be accumulated in the ER. DNA microarray analysis revealed that the transcriptions of ER chaperones and components of the ER-Golgi transport were specifically upregulated in the strain overexpressing the mutant MsdS, suggesting that the accumulated mutant MsdS induced the unfolded protein response. These findings demonstrate that aberrant secretory proteins are primarily degraded by the ERAD but also the ER-Golgi transport and the hyperglycosylation might relieve the stresses caused by their accumulation in the secretory pathway.

abstract No: 


Full conference title: 

    • ECFG 9th (2008)