1. Measurement of endo-α-mannosidase activity using a fluorescently labeled oligosaccharide derivative
- Author
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Ken-ichi Kamei, Ichiro Matsuo, Yoichi Takeda, Shogo Iwamoto, Akira Seko, Yukishige Ito, and Yuta Kasahara
- Subjects
Stereochemistry ,Molecular Sequence Data ,Oligosaccharides ,Mannose ,Applied Microbiology and Biotechnology ,Biochemistry ,Analytical Chemistry ,Residue (chemistry) ,chemistry.chemical_compound ,N-glycan processing ,Mannosidases ,Tetrasaccharide ,Organic chemistry ,Glycoside hydrolase ,Molecular Biology ,Enzyme Assays ,Fluorescent Dyes ,chemistry.chemical_classification ,Organic Chemistry ,General Medicine ,Oligosaccharide ,Carbohydrate Sequence ,chemistry ,Linker ,Derivative (chemistry) ,Biotechnology - 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.
- Published
- 2014