Mammalian α-1,2-mannosidases are involved in the processing of asparagine-linked oligosaccharides in endoplasmic reticulum (ER) and Golgi body. In some fungi the similar enzyme is secreted to the medium. These all belong to Glycoside Hydrolase Family 47. The enzyme has a strict specificity toward α-1,2-mannose. Preferred substrates are α-1,2-mannobiose and high-mannose type oligosaccharides with terminal α-1,2-mannoses (e.g. Man9GlcNAc2). Synthetic α-mannosides, such as pNP-α-mannoside, are not the substrate to the enzyme. Note that the enzyme in ER has a limited specificity to a single α-1,2-mannose residue of Man9GlcNAc2, while Golgi α-1,2-mannosidase removes all the four α-1,2-mannoses. |
Category | Biosynthesis & Metabolism |
Protocol Name | Enzyme assay of fungal α-1,2-mannosidase |
Authors
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Yoshida, Takashi
Faculty of Agriculture and Life Science, Hirosaki University
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KeyWords |
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Reagents
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Pyridylaminated high-mannose type oligosaccharide (Man9GlcNAc2-PA) 1)
(PA-Labeled mannooligosaccharides are commercially provided by Takara Bio Inc., Otsu, Japan) |
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Instruments
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HPLC system with a fluorescent detector. |
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Methods |
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Mix 5 μL of Man9GlcNAc2-PA (total 50 pmol), 5 μL of 200 mM sodium acetate buffer, pH 5.0, water, and the enzyme to make total 50 μL of solution. |
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Incubate at 30°C for an appropriate period of time. |
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Stop the reaction by chilling in an iced bath. |
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Put the reaction mixture into a centrifugal membrane-filter (0.2 μm), then spin down. |
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Analyze aliquots of the solution by HPLC with an amide column (TSKgel Amide 80, 4 × 250 mm) which is run in 3% acetic acid-triethylamine, pH 7.3/acetonitrile/water (8:55:37) at a flow rate of 0.4 mL/min.2) |
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Monitor the fluorescence of the effluent at an excitation 315 nm and emission 380 nm (Fig. 1). |
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Notes | The reaction buffer should be at neutral pH in determining mammalian enzymatic activity. |
Figure & Legends |
Figure & Legends
Fig. 1. Trimming of high-mannose oligosaccharides by α-1,2-mannosidase.
Number in each panel is the reaction period (min).
This figure was originally published in Biosci. Biotechnol. Biochem. Akao F, Yoshida T et al. "Cloning and Expression of 1,2-α-Mannosidase Gene (fmanIB) from Filamentous Fungus Aspergillus oryzae: in Vivo visualization of the FmanIBp-GFP Fusion Protein" 2006, 70(2) 471–479. |
Copyrights |
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This work is released underCreative Commons licenses
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Date of registration:2015-02-16 11:37:22 |
- Hase, S., Natsuka, S., Oku, H., and Ikenaka, T. (1987) Identification method for twelve oligomannose-type sugar chains thought to be processing intermediates of glycoproteins. Anal Biochem. 167, 321–6 [PMID : 3442327]
- Yoshida, T., Kato, Y., Asada, Y., and Nakajima, T. (2000) Filamentous fungus Aspergillus oryzae has two types of α-1,2-mannosidases, one of which is a microsomal enzyme that removes a single mannose residue from Man9GlcNAc2. Glycoconj J. 17, 745–8 [PMID : 11443275]
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