title: Cooperative role of calnexin and TigA in Aspergillus oryzae glycoprotein folding.
abstract: Calnexin (CNX), known as a lectin chaperone located in the endoplasmic reticulum (ER), specifically recognizes G1M9GN2-proteins and facilitates their proper folding with the assistance of ERp57 in mammalian cells. However, it has been left unidentified how CNX works in Aspergillus oryzae, which is a filamentous fungus widely exploited in biotechnology. In this study, we found that a protein disulfide isomerase homolog TigA can bind with A. oryzae CNX (AoCNX), which was revealed to specifically recognize monoglucosylated glycans, similarly to CNX derived from other species, and accelerate the folding of G1M9GN2-ribonuclease (RNase) in vitro. For refolding experiments, a homogeneous monoglucosylated high-mannose-type glycoprotein G1M9GN2-RNase was chemoenzymatically synthesized from G1M9GN-oxazoline and GN-RNase. Denatured G1M9GN2-RNase was refolded with highest efficiency in the presence of both soluble form of AoCNX and TigA. TigA contains two thioredoxin domains with CGHC motif, mutation analysis of which revealed that the one in N-terminal regions is involved in binding to AoCNX, while the other in catalyzing protein refolding. The results suggested that in glycoprotein folding process of A. oryzae, TigA plays a similar role as ERp57 in mammalian cells, as a partner protein of AoCNX.
authors.affiliation: Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Synthetic Cellular Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Ning
authors.initials: N
authors.lastname: Wang
authors.affiliation: Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Seko
authors.affiliation: Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Takeda
authors.affiliation: Department of Biotechnology, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kikuma
authors.affiliation: Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Synthetic Cellular Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan yukito@riken.jp.
authors.forename: Yukishige
authors.initials: Y
authors.lastname: Ito
mesh_terms: D001236:Aspergillus oryzae / Q000378:metabolism*; D037281:Calnexin / Q000502:physiology*; D002240:Carbohydrate Sequence; D005656:Fungal Proteins / Q000737:chemistry / Q000502:physiology*; D006023:Glycoproteins / Q000737:chemistry / Q000502:physiology*; D006031:Glycosylation; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000502:physiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D017510:Protein Folding; D011499:Protein Processing, Post-Translational
chemical_list: D005656:Fungal Proteins; D006023:Glycoproteins; D006360:Heat-Shock Proteins; D037281:Calnexin
keywords: Aspergillus oryzae; N-linked glycoprotein; calnexin; protein disulfide isomerase; ribonuclease