Direct assay for endo-α-mannosidase substrate preference on correctly folded and misfolded model glycoproteins.
We previously reported a unique assay system for UDP-glucose glycoprotein glucosyltransferase (UGGT) toward glycoprotein folding intermediates during the folding process. The assay involved the in vitro folding of both high-mannose type oligosaccharyl crambin, which yielded only the correctly folded glycoprotein form (M9-glycosyl-native-crambin), and its mutant, which yielded misfolded glycoproteins (M9-glycosyl-misfolded-crambin), in the presence of UGGT. The process successfully yielded both mono-glucosylated M9-glycosyl-native-crambin (G1M9-glycosyl-native-crambin) and M9-glycosyl-misfolded-crambin (G1M9-glycosyl-misfolded-crambin). Here, we report the use of our in vitro folding system to evaluate the substrate preference of Golgi endo-α-mannosidase against G1M9-native and -misfolded glycoprotein forms. In our assay Golgi endo-α-mannosidase removed Glc-α-1-3-Man unit from G1M9-native and -misfolded-crambins clearly proving that Golgi endo-α-mannosidase does not have specific preference for correctly folded or misfolded protein structure.
Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
Simone
S
Dedola
Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka, 560-0043, Japan.
Masayuki
M
Izumi
Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
Yutaka
Y
Makimura
Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
Akira
A
Seko
Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
Akiko
A
Kanamori
Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
Yoichi
Y
Takeda
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. Electronic address: yukito@riken.jp.
Yukishige
Y
Ito
Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan; Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka, 560-0043, Japan. Electronic address: kajihara@chem.sci.osaka-u.ac.jp.
Yasuhiro
Y
Kajihara
D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics; D015394:Molecular Structure; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry; D017510:Protein Folding; D013379:Substrate Specificity; D043323:alpha-Mannosidase / Q000378:metabolism*
D006023:Glycoproteins; D010940:Plant Proteins; C024934:crambin protein, Crambe abyssinica; D043323:alpha-Mannosidase
Endo-α-mannosidase; Glycoproteins; Highmannose type oligosaccharide; Misfolded glycoprotein