Endo-β-N-acetylglucosaminidase

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Endoglycosidase is an enzyme that cleaves intact glycans from glycoproteins. These enzymes are highly stable in cleaning formulations that do not contain glycerol, NaCl or other additives (such as EDTA). Test all enzymes for no proteolytic or unexpected glycoside activity. Endoglycosidase is the most widely used enzyme in glycoprotein analysis. These enzymes release complete glycans from glycoproteins, while exoglycosidase releases individual monosaccharides (ie, sialic acid, galactose, β-N-acetylglucosamine, mannose, etc.).

Figure 1. Structure of endoglycosidase.

Introductions

Although PNGase F is actually an amidase, it is usually included in enzymes due to its similar activity of cleaving intact N-linked glycans. It cleaves between the asparagine amino acid and the first β-N-acetylglucosamine sugar (GlcNAc) of the chitobiose core of the N-linked glycan. Endo F enzyme and Endo H cleave N-linked glycans between the first GlcNAc and the second GlcNAc, leaving behind charged residues, which helps increase the solubility of the deglycosylated protein, thereby reducing the protein Possibility of aggregate precipitation. PNGase F cleaves all N-linked glycans, while Endo H and Endo F enzymes remove specific types of N-linked glycans. Endoglycosidases cleave entire glycan groups from glycoproteins. The most commonly used endoglycosidase, Peptide-N-Glycosidase F is actually an amidase, which catalyzes the deglycosylation of most N-linked glycoproteins. The enzyme catalyzes the cleavage between the innermost N-Acetylglucosamine (GlcNAc) of the chitobiose core and the amine group of the asparagine residue.

Classifications

There is a variety of endoglycosidases that are active on N-linked glycans. They all have identical cleavage sites between the two GlcNAc residues of the chitobiose core, but they recognize and cleave different types of N-linked glycans. Endoglycosidase H cleaves within the chitobiose core of high mannose and some hybrid oligosaccharides from N-linked glycoproteins. Endoglycosidase S is highly specific for removing N-linked glycans from the heavy chain of native IgG. While Endoglycosidase D cleaves paucimannose N-linked glycans. There are fewer endoglycosidases also called α-N-Acetylgalactosaminidases active on O-linked glycans. The majority of O-glycosidases found to date are active only on a disaccharide Galβ1,3GalNAcα-O-Ser/Thr requiring the removal of Sialic acid and any other attached sugar residues before the enzyme can catalyze the removal of this Core 1 disaccharide from the glycoprotein. However, there is one enzyme with a slightly broader O-Glycosidase specificity, and it is able to catalyze the removal of Core 1 (Galβ1,3GalNAc) or Core 3 (GlcNAcβ1,3GalNAc) O-linked disaccharides from glycoproteins.

Mechanism

The mechanism is enzymatic hydrolysis. There are two forms of endoglycosidase mechanism; the acid-catalyzed protonation of glycoside oxygen can retain the stereochemistry at the anomeric carbon, or the acid-catalyzed protonation of glycoside oxygen is accompanied by the attack of water molecules activated by base residues to produce stereochemistry. Conversion. The two mechanisms have the same distance between the proton donor and the glycoside oxygen, so that the proton donor is close enough to the glycoside oxygen for hydrogen bonding. Since the retention mechanism is close to the two carboxyl groups, it undergoes a double displacement mechanism, resulting in a covalent glycosylase intermediate. Exoglycosidase will remove each carbohydrate monomer (x) one by one starting from xn and one end, while endoglycosidase can cut at any glycosidic bond (-), and combine certain carbohydrates with certain proteins. The linked characteristic "linked oligosaccharides" are cleaved.

Application

The use of endoglycosidase for mutagenesis has great potential. When this new mutant enzyme comes into contact with the appropriate compound, oligosaccharide synthesis will proceed and the newly formed polymer chain will not be hydrolyzed. This is a very useful tool because oligosaccharides have great therapeutic potential. For example, the hexavalent sugar H sugar will indicate malignant cell transformation associated with breast, prostate and ovarian cancer. Endoglycosidase may also play a role in fighting autoimmune diseases such as arthritis and systemic lupus erythematosus. In 2008, a group of researchers demonstrated that injection of endoglycosidase S "can effectively remove IgG-related sugar domains in the body and interfere with the pro-inflammatory process mediated by autoantibodies in a variety of autoimmune models. Obviously, this enzyme manipulation and mutation have broad prospects for being able to resist many diseases in the body.

Reference

1. Davies, G.; et al. Structures and mechanisms of glycosyl hydrolases. Structure. 1995, 3 (9): 853–59.