New England Biolabs, P0768S, α1-2,3,6 Mannosidase

Related Categories Exoglycosidases,, Proteome Analysis Applications Glycan Sequencing,, Recombinant Glycoprotein Expression,, Glycoprotein Analysis Specification Unit Definition One unit is defined as the amount of enzyme required to cleave > 95% of the terminal mannose from 1 nmol of Man(α1,3)-Man(β1,4)-GlcNAc-7-amino-4-methyl-coumarin (AMC), in 1 hour at 37°C in a total reaction volume of 10 μl. Reaction Conditions 1X GlycoBuffer 4 Supplement with 1X Zinc Incubate at 37°C 1X GlycoBuffer 4 50 mM sodium acetate (pH 4.5 @ 25°C) Storage Buffer 20 mM Tris-HCl 50 mM NaCl pH 7.5 @ 25°C Heat Inactivation 95°C for 10 minutes Molecular Weight Theoretical: 110 kDa Unit Assay Conditions Two fold dilutions of α1-2,3,6 Mannosidase are incubated with 1 nmol AMC-labeled substrate, 1X GlycoBuffer 4 and 1X Zinc in a 10 μl reaction. The reaction mix is incubated at 37°C for 1 hour. Separation of reaction products are visualized via thin layer chromatography (1). FAQ Q: What is the difference between α1-2,3 Mannosidase, α1-6 Mannosidase and α1-2,3,6 Mannosidase? A: A: α1-2,3 Mannosidase (NEB #P0729) and α1-6 Mannosidase (NEB #P0727) are cloned from Xanthomonas manihotis, and α1-2,3,6 Mannosidase (NEB #P0768) is cloned from Canavalia ensiformis (jack bean). α1-2,3 Mannosidase cleaves only α1-2 and α1-3 linked mannose residues, and α1-6 Mannosidase cleaves only α1-6 linked mannose residues. Whereas, α1-2,3,6 Mannosidase (JBM) cleaves all three α1-2, α1-3 and α1-6 linked mannose residues. Q: Can α1-2,3,6 Mannosidase (JBM) be a substrate itself for mannosidase activity? A: No. α1-2,3,6 Mannosidase is a glycosylated, high mannose protein. The majority of the glycosylation is removed during purification; however, a small amount of mannosylation still remains. The remaining mannosylated glycans appear resistant to auto-digestion. Q: What is a good positive control for α1-2,3 Mannosidase? A: pNP-α-mannopyranoside (4-Nitrophenyl α-D-mannopyranoside) Q: When digesting complex substrates that include α1-6 Mannose residue, can additional mannose enzymes be used in combination with α1-2,3,6 Mannosidase? A: Because α1-2,3,6 Mannosidase has a slightly reduced activity towards α1-6 mannose residues (in comparison to α1-2 and α1-3 mannose residues) it may be advantageous to use the enzyme in combination with α1-6 Mannosidase (NEB #P0727). A sequential digest is recommended, in which digestion with α1-2,3,6 Mannosidase is followed by α1-6 Mannosidase (NEB #P0727) treatment. Q: Does this enzyme require denaturing conditions to act on glycoproteins? A: No, in general exoglycosidases can remove sugar residues from native (folded) glycoproteins (hydrophilic glycans face away from the protein backbone). However, the protein folding around a glycan site might affect enzyme accessibility. Exoglycosidase digestion of some proteins will require longer incubation times, or in some cases a mild extent of denaturation. Q: What is a good method to re-purify a glycan or glycopeptide after exoglycosidase treatment? A: Filtration with micro-spin filters, or solid-phase extraction (for example: graphitized carbon cartridges). Q: How much exoglycosidase should be used? A: The amount of enzyme required varies when different substrates are used. Start with 1-2 µl for 1µg of glycoprotein or 100 nM of oligosaccharide for one hour in a 10-25 µl reaction. If there is still undigested material, let the reaction go overnight. Q: Do detergents inhibit glycosidases? A: At moderate levels (0.5-1.0% ionic and non-ionic detergents) most of the glycosidases show satisfactory activity or are unaffected. Exceptions include PNGase F, PNGase F Recombinant, Remove-iT PNGase F and O-Glycosidase which are inhibited by SDS. Q: What are Glycosidases and their uses? A: Glycosidases are used to get information about the carbohydrate groups attached to glycoproteins and glycopeptides. They come in two varieties, endoglycosidases that cleave entire carbohydrate groups from proteins and exoglycosidases that remove monosaccharides from the non-reduced ends of the carbohydrate. A reduced end is one generated by an endoglycosidase. Researchers frequently use an endoglycosidase followed by one or more exoglycosidases and then analyze the products using SDS-PAGE or various types of liquid chromatography.