title: Measurement of endo-α-mannosidase activity using a fluorescently labeled oligosaccharide derivative.
abstract: Endo-α-mannosidase, a GH99-family glycoside hydrolase, cleaves α-mannoside linkages with glucose residues. This enzyme is proposed to play a critical role in N-glycan processing for deglucosylation. To measure endo-α-mannosidase activity, we synthesized a fluorescently labeled tetrasaccharide derivative (Glcα1-3Manα1-2Manα1-2Manα1-O-C3H6-NH-Dansyl) in a stereocontrolled manner. The tetrasaccharide skeleton was prepared by step-wise coupling using mannose donors 4 and 7. The 1,2-cis α-glycosidic linkage on the non-reducing end of the glucose residue was constructed by inversion of the stereochemistry of the C-2 hydroxyl group in the α-mannose residue. Finally, the dansyl group was introduced at the reducing end via an aminopropyl linker. This probe successfully measured endo-α-mannosidase activity.
authors.affiliation: a Division of Molecular Science, Gunma University , Kiryu , Japan.
authors.forename: Shogo
authors.initials: S
authors.lastname: Iwamoto
authors.forename: Yuta
authors.initials: Y
authors.lastname: Kasahara
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Kamei
authors.forename: Akira
authors.initials: A
authors.lastname: Seko
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Takeda
authors.forename: Yukishige
authors.initials: Y
authors.lastname: Ito
authors.forename: Ichiro
authors.initials: I
authors.lastname: Matsuo
mesh_terms: D002240:Carbohydrate Sequence; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008358:Mannose / Q000737:chemistry; D008361:Mannosidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D009844:Oligosaccharides; D008361:Mannosidases; C051635:endo-alpha-D-mannosidase; D008358:Mannose
keywords: N-glycan processing; endo-α-mannosidase; fluorescently labeled glycan; glycan synthesis