Endo-β-N-acetylglucosaminidase M digestion (Endo-M)
Endoglycosidases cleave at defined sites within an oligosaccharide chain of glycoproteins/glycolipids. They are the most easy-to-use enzymes for elucidating the function and structure of oligosaccharides, because they can separate both intact oligosaccharide chains and proteins/lipids from glycoconjugates under mild conditions without causing damage.
Endo-β-N-acetylglucosaminidase (EC 220.127.116.11) catalyzes the hydrolysis of the N, N’-diacetylchitobiose moiety in the core region of asparagine-linked oligosaccharides of various glycoproteins. The enzyme has a characteristic to remain one N-acetyl-D-glucosamine residue on the protein. On the other hand, the deglycosylation method using PNGase F cannot remove oligosaccharides unless the protein is denatured. Thus, only endo-β-N-acetylglucosaminidases can be used for deglycosylation of native glycoproteins.
Endo-β-N-acetylglucosaminidase M (Endoglycosidase M or Endo-M) has broad substrate specificity for various N-linked oligosaccharides, and can cleave high-mannose type, hybrid- type and bi-antennary complex-type oligosaccharides (Fig.1) [Yamamoto, et al., 1994, Kadowaki, et al., 1990, Fujita, et al., 2004]. This enzyme can release the bi-antennary complex-type oligosaccharides from native glycoproteins like human asialotransferrin and sialotransferrin without the addition of a detergent [Kadowaki, et al., 1990]. This enzyme has exceptionally high transglycosylation activity and can transfer the oligosaccharides of glycopeptides to GlcNAc or the compound having GlcNAc moiety [Yamamoto, et al., 1994]. Therefore, Endo-M is usually used as a tool for the addition of oligosaccharides to proteins/peptides rather than for the digestion of glycoproteins/glycopeptides.
bi-antennary complex type oligosaccharide
Endo-β-N-acetylglucosaminidase M (Endo-M) from Mucor hiemalis.
5X Reaction buffer : 500 mM sodium acetate buffer (pH 6.0)
2X SDS-PAGE sample buffer: 0.125 M Tris-HCl buffer (pH 6.8), 10% β-ME, 4% SDS, 10% sucrose, 0.004% Bromophenol blue
Reaction incubator or water bath (100°C, 37 °C)
Release of oligosaccharides from glycoproteins by using Endo-M.
Transfer 20 μL of glycoprotein sample (10 μg/μL), 10 μL of 5X reaction buffer, 2.5 μL of 10 U/mL Endo-M, and 17.5 μL of deionized water into a microtube.
Incubate at 30°C for 20 h.
To examine the release of oligosaccharide, mix 5 μL of the reaction sample and 5 μL of 2X SDS-PAGE sample buffer, and then heat at 100°C for 3 min.
Load 10 µL of the sample mixture on SDS-PAGE gel and run the electrophoresis. Perform either Coomassie blue staining or silver staining.
Figure & Legends
Fig. 1. Specificity of Endo-M.
1 Yamamoto, K., Kadowaki, S., Fujisaki, M., Kumagai, H. and Tochikura, T. (1994) Novel specificities of Mucor hiemalis endo-beta-N-acetylglucosaminidase acting complex asparagine-linked oligosaccharides. Biosci. Biotechnol. Biochem. 58, 72-77. [PMID : 7509658]
2 Kadowaki, S., Yamamoto, K., Fujisaki, M., Izumi, K., Tochikura, T. and Yokoyama, T. (1990) Purification and characterization of a novel fungal endo-beta-N-acetylglucosaminidase acting on complex oligosaccharides of glycoproteins. Agric. Biol. Chem. 54, 97-106. [PMID : 1368528]
3 Fujita, K., Kobayashi, K., Iwamatsu, A., Takeuchi, M., Kumagai, H. and Yamamoto, K. (2004) Molecular cloning of Mucor hiemalis endo-beta-N-acetylglucosaminidase and some properties of the recombinant enzyme. Arch Biochem Biophys. 432, 41-49. [PMID : 15519295]