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Articles by K Totani
Total Records ( 3 ) for K Totani
  T Watanabe , K Totani , I Matsuo , J. i Maruyama , K Kitamoto and Y. Ito
 

Glucosidase II (G-II) is a glycoprotein-processing enzyme that successively cleaves two 1,3-linked glucose residues from N-linked oligosaccharides in the endoplasmic reticulum. G-II is a heterodimer whose -subunit contains a glycosidase active site, but the function(s) of the β-subunit remain poorly defined. We report here an in vivo enzymatic analysis using gene disruptants lacking either the G-II - or β-subunit in the filamentous fungus Aspergillus oryzae. Using synthetic oligosaccharides as probes, G-II activity of the membranous fraction of the gene disruptants was investigated. The fraction lacking the β-subunit retained hydrolytic activity toward p-nitrophenyl -d-glucopyranoside but was inactive toward both Glc2Man9GlcNAc2 and Glc1Man9GlcNAc2. When the fraction containing the β-subunit was added to the one including the -subunit, the glucosidase activity was restored. These results suggested that the β-subunit confers the substrate specificity toward di- and monoglucosylated glycans on the glucose-trimming activity of the -subunit.

  Y Haga , K Totani , Y Ito and T. Suzuki
 

During N-glycosylation of proteins, significant amounts of free unconjugated glycans are also generated in the lumen of the endoplasmic reticulum (ER). These ER-derived free glycans are translocated into the cytosol by a putative transporter on the ER membrane for further processing. However, the molecular nature of the transporter remains to be determined. Here, we report the establishment of a novel assay method for free oligosaccharide transport from the ER lumen using chemically synthesized fluorescence-labeled N-glycan derivatives. In this method, fluorescence-labeled glycan substrates were encapsulated inside mouse liver microsomes, followed by incubation with the cytosol and a fluorescence-quenching agent (anti-fluorophore antibody). The rate of substrate efflux was then monitored in real time by the decrease in the fluorescence intensity. The present data clearly demonstrated that the oligosaccharide transport activity under the current assay conditions was both ATP and cytosol dependent. The transporter activity was also found to be glycan structure specific because free glucosylated glycans were unable to be transported out of the microsomes. This new assay method will be a useful tool for identifying the transporter protein on the ER membrane.

  D Hu , Y Kamiya , K Totani , D Kamiya , N Kawasaki , D Yamaguchi , I Matsuo , N Matsumoto , Y Ito , K Kato and K. Yamamoto
 

Glucosidase II (GII) is a glycan-processing enzyme that trims two 1,3-linked glucose residues from N-glycan on newly synthesized glycoproteins. Trimming of the first 1,3-linked glucose from Glc2Man9GlcNAc2 (G2M9) is important for a glycoprotein to interact with calnexin/calreticulin (CNX/CRT), and cleavage of the innermost glucose from Glc1Man9GlcNAc2 (G1M9) sets glycoproteins free from the CNX/CRT cycle and allows them to proceed to the Golgi apparatus. GII is a heterodimeric complex consisting of a catalytic subunit (GII) and a tightly associated β subunit (GIIβ) that contains a mannose 6-phosphate receptor homology (MRH) domain. A recent study has suggested a possible involvement of the MRH domain of GIIβ (GIIβ-MRH) in the glucose trimming process via its putative sugar-binding activity. However, it remains unknown whether GIIβ-MRH possesses sugar-binding activity and, if so, what role this activity plays in the function of GII. Here, we demonstrate that human GIIβ-MRH binds to high-mannose-type glycans. Frontal affinity chromatography revealed that GIIβ-MRH binds most strongly to the glycans with the 1,2-linked mannobiose structure. GII with the mutant GIIβ that lost the sugar-binding activity of GIIβ-MRH hydrolyzes p-nitrophenyl--glucopyranoside, but the capacity to remove glucose residues from G1M9 and G2M9 is significantly decreased. Our results clearly demonstrate the capacity of the GIIβ-MRH to bind high-mannose-type glycans and its importance in efficient glucose trimming of N-glycans.

 
 
 
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