73NISR Research GRANT
2005 Young Investigator Research Grant

Cellular Response to the Overexpression of Mutated or Exogenous Secretory Proteins in Aspergillus oryzae
Takahiro SHINTANIDivision of Bioscience and Biotechnology for Future Bioindustries, Graduate School of Agricultural Science, Tohoku University

Research aims
Filamentous fungi have been used as hosts that produce
useful proteins because of their ability to secrete proteins ata very high level. The protein expression systems have beendeveloped in several filamentous fungi and had a great success in secreting the recombinant proteins derived from eukaryotic microbes. However, the efficiency of secretion ofhigher eukaryotic proteins was quite poor in many cases.
One of the reasons for a decreased production could be theinstability of exogenously expressed proteins in the secretory pathway. In eukaryotic cells, it is known that aberrant
secretory proteins are subjected to a protein quality control
and degraded intracellularly to maintain their cellular
homeostasis. Therefore, the protein production, a situation
where exogenous proteins are overexpressed, might put
stresses on host cells. In this research, I used Aspergillus
oryzae as a host strain to analyze the cellular response tothe overexpression of a mutated secretory protein.
Methods
The plasmids used in this study were listed in Table 1.
Each plasmid was integrated at the niaD locus of A. oryzae
NS4 to generate the strain expressing the wild-type or mutant 1,2-a-mannosidase. A. oryzae cells were grown in
YPM medium (1% yeast extract, 1% peptone, 2% maltose)
at 30°C for 24 hours. Northern blot analysis and cDNA microarray were performed as described by Maeda et al.1)
Results
Tatara et al.2) showed that the cystein residue-443 of 1,2a-mannosidase from Aspergillus saitoi (msdS) was important to maintain its conformation and the substitution ofCys-443 to Phe (C443F) resulted in a marked decrease inits secretion from A. oryzae cells, suggesting that the mutant enzyme was subjected to a protein quality control inTable 1.

The plasmids used in this study.

Name

PromoterTarget cDNA for expression

pNE
pNE-AM1
pNE-AM1-C443F
pNAN8142
pNAN-AM1
pNAN-AM1-C443F

enoA
enoA
enoA
No. 8142
No. 8142
No. 8142none
wild-type f-msdS
C443F f-msdS
nonewild-type f-msdS
C443F f-msdS

the host cells. Therefore, I decided to express the mutant
version of msdS in A. oryzae to analyze the cellular response to aberrant proteins loaded into the secretary pathway. Since the expression level is an important consideration to analyze the response to the expression of aberrant
proteins, two different promoters were used in this study,i.e. one was the strong No. 8142 promoter, and the otherwas a more moderate enoA promoter. Northern blot analysis verified that the transcription level of msdS from No.
8142 promoter was much higher than that from enoA promoter.
Using these strains, I examined the production of thewild-type and C443F msdS proteins with SDS-PAGE andimmunoblotting. The wild-type msdS proteins were efficiently secreted to the medium with both promoters, although amount of msdS protein was more than tenfold
higher from the No. 8142 promoter than the enoA promoter.
On the other hand, the mutant msdS protein was not detected in the medium by Coomassie Brilliant Blue staining.
Therefore, I tried to detect the mutant protein by immunoblotting. The mutant enzyme was still undetectableeven by immunoblot analysis when expressed from theenoA promoter. In contrast, when overexpressed, the mutantenzymes were found in the medium and extensively subjected to the hyperglycosylation. To test the possibility thatthe mutant enzyme was accumulated inside the cells, thetotal cell extract was subjected to immunoblot analysis. At
the moderate expression level, only a faint band of theC443F enzyme was detected, although a significant amountof the wild-type enzyme was observed, suggesting that themutant enzyme was degraded rather than accumulated inside the cells. No obvious difference in amounts of enzymes was seen when overexpressed. Interestingly, the mutant enzyme observed in the cell extract was not hyperglycosylated, suggesting that the mutant enzymes with the hyperglycosylation were efficiently secreted to the medium.
Next, I examined the cellular response to the overexpression of the mutant msdS proteins at the transcription level.
Using cDNA microarrays comprising about 5,000 non-redundant EST clones from A. oryzae, the transcriptions werecompared between the strains expressing wild-type and mutant enzymes. The transcriptions of bipA and pdiA, whichmight be involved in protein folding in ER, were increasedin the cells expressing the mutant enzyme, suggesting thatthe overexpression of the mutant msdS induced the unfolded protein response (UPR). Northern analysis confirmed that the overexpression of the mutant enzyme induced the transcriptions of ER chaperons, while the moder-74

ate expression did not.
Conclusion
Only one amino acid substitution (Cys-443 to Phe)caused instability of 1,2-a -mannosidase in A. oryzae. Atthe moderate expression level, the mutant protein might bedegraded through the secretory pathway, although themechanism of how it is degraded is unclear. The hyperglycosylation of secretory proteins occurs at the Golgi apparatus in yeast. Therefore, I reasoned that the degradation
might occur at ER because no hyperglycosylated mutant
protein was detected in the cell lysate. When overproduced,
the mutant proteins could bypass the ER-associated degradation and transport to Golgi apparatus where the hyperglycosylation occurs. The hyperglycosylated mutant proteins
were efficiently secreted to the medium. The mutant protein

detected in the cell lysate might be localized at ER because
they were not hyperglycosylated, and the accumulated aberrant proteins could induce the UPR.
References
1) Maeda H., Sano M., Maruyama Y., Tanno T., Akao T., Totsuka Y.,
Endo M., Sakarada R., Yamagata Y., Machida M., Akita O.,
Hasegawa F., Abe K., Gomi K., Nakajima T., and Iguchi Y. (2004)
Transcriptional analysis of genes for energy catabolism and hydrolytic enzymes in the filamentous fungus Aspergillus oryzae usingcDNA microarrays and expressed sequence tags. Appl. Microbiol.
Biotechnol. 65, 74–83.
2) Tatara Y., Yoshida T., and Ichishima E.. (2005) A single free cysteine
residue and disulfide bond contribute to the thermostability of Aspergillus saitoi 1,2-a -mannosidase. Biosci. Biotechnol. Biochem. 69,2101–2108.