Endoglycosidase H

The enzyme Endoglycosidase H (EC 3.2.1.96, Endo-β-N-acetylglucosaminidase H, N,N'-diacetylchitobiosyl beta-N-acetylglucosaminidase, mannosyl-glycoprotein endo-beta-N-acetylglucosamidase, di-N-acetylchitobiosyl beta-N-acetylglucosaminidase, endo-beta-acetylglucosaminidase, endo-beta-(1->4)-N-acetylglucosaminidase, mannosyl-glycoprotein 1,4-N-acetamidodeoxy-beta-D-glycohydrolase, endoglycosidase S, endo-N-acetyl-beta-D-glucosaminidase, endo-N-acetyl-beta-glucosaminidase, endo-beta-N-acetylglucosaminidase D, endo-beta-N-acetylglucosaminidase F, endo-beta-N-acetylglucosaminidase H, endo-beta-N-acetylglucosaminidase L, glycopeptide-D-mannosyl-4-N-(N-acetyl-D-glucosaminyl)2-asparagine 1,4-N-acetyl-beta-glucosaminohydrolase, endoglycosidase H) is an enzyme with systematic name glycopeptide-D-mannosyl-N4-(N-acetyl-D-glucosaminyl)2-asparagine 1,4-N-acetyl-beta-glucosaminohydrolase.[1][2][3][4][5][6] It is a highly specific endoglycosidase which cleaves asparagine-linked mannose rich oligosaccharides, but not highly processed complex oligosaccharides from glycoproteins. It is used for research purposes to deglycosylate glycoproteins.

Mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase
Identifiers
EC number3.2.1.96
CAS number37278-88-9
Databases
IntEnzIntEnz view
BRENDABRENDA entry
ExPASyNiceZyme view
KEGGKEGG entry
MetaCycmetabolic pathway
PRIAMprofile
PDB structuresRCSB PDB PDBe PDBsum

Structure and Activity

Endoglycosidase H is isolated from Streptomyces plicatus or Streptomyces griseus. Its molecular weight is 29 000 Daltons. The primary structure is described by Robbins 1984.[7]

Endoglycosidase H cleaves the bond in the diacetylchitobiose core of the oligosaccharide between two N-acetylglucosamine (GlcNAc) subunits directly proximal to the asparagine residue, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine.[8]

It deglycosylates mannose glycoproteins, but the extent and rate of the deglycosylation depends to a high degree on the nature of the glycoproteins. The deglycosylation rate can be increased by denaturation of the glycoproteins (e.g., by carboxymethylation, sulfitolysis or by heating in the presence of sodium dodecyl sulfate). The addition of 0.1 M 2-mercaptoethanol highly increases enzyme activity against glycoproteins containing inter- or intra-molecular disulfide bridges, unlike detergents like Triton X-100, n- Octylglucoside, or zwitterionic detergents.[9]

Biochemical Applications

Endoglycosidase H (Endo H) is commonly used by cell biologists to monitor posttranslational modification in the Golgi apparatus. Most proteins destined for the cell surface are translated by ribosomes into the rough endoplasmic reticulum (ER) and translocated into the Golgi. Upon entering the ER a molecule containing 14 sugar subunits is linked en bloc to an asparagine in a selective manner by the enzyme oligosaccharyl transferase. It is this oligosaccharide molecule which is modified by a series of enzymes as the protein moves through the different compartments of the Golgi apparatus. Endoglycosidase H is able to cleave each structure of this oligosaccharide as it is processed until the enzyme Golgi alpha-mannosidase II removes two mannose subunits. Since all later oligosaccharide structures are resistant to Endo H cleavage the enzyme is widely used to report the extent of oligosaccharide processing a protein of interest has undergone.[10]

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References
  1. Chien S, Weinburg R, Li S, Li Y (1977). "Endo-β-N-acetylglucosaminidase from fig latex". Biochem. Biophys. Res. Commun. 76: 317–323. doi:10.1016/0006-291x(77)90727-6.
  2. Koide N, Muramatsu T (August 1974). "Endo-beta-N-acetylglucosaminidase acting on carbohydrate moieties of glycoproteins. Purification and properties of the enzyme from Diplococcus pneumoniae". The Journal of Biological Chemistry. 249 (15): 4897–904. PMID 4152561.
  3. Pierce RJ, Spik G, Montreuil J (June 1979). "Cytosolic location of an endo-N-acetyl-beta-D-glucosaminidase activity in rat liver and kidney". The Biochemical Journal. 180 (3): 673–76. doi:10.1042/bj1800673. PMC 1161109. PMID 486141.
  4. Pierce RJ, Spik G, Montreuil J (January 1980). "Demonstration and cytosolic location of an endo-N-acetyl-beta-D-glucosaminidase activity towards an asialo-N-acetyl-lactosaminic-type substrate in rat liver". The Biochemical Journal. 185 (1): 261–4. doi:10.1042/bj1850261. PMC 1161293. PMID 7378051.
  5. Tai T, Yamashita K, Ogata-Arakawa M, Koide N, Muramatsu T, Iwashita S, Inoue Y, Kobata A (November 1975). "Structural studies of two ovalbumin glycopeptides in relation to the endo-beta-N-acetylglucosaminidase specificity". The Journal of Biological Chemistry. 250 (21): 8569–75. PMID 389.
  6. Tarentino AL, Plummer TH, Maley F (February 1974). "The release of intact oligosaccharides from specific glycoproteins by endo-beta-N-acetylglucosaminidase H". The Journal of Biological Chemistry. 249 (3): 818–24. PMID 4204553.
  7. Robbins PW, Trimble RB, Wirth DF, Hering C, Maley F, Maley GF, Das R, Gibson BW, Royal N, Biemann K (June 1984). "Primary structure of the Streptomyces enzyme endo-beta-N-acetylglucosaminidase H". The Journal of Biological Chemistry. 259 (12): 7577–83. PMID 6429133.
  8. Endoglycosidase H, North Star
  9. Trimble RB, Maley F (September 1984). "Optimizing hydrolysis of N-linked high-mannose oligosaccharides by endo-beta-N-acetylglucosaminidase H". Analytical Biochemistry. 141 (2): 515–22. doi:10.1016/0003-2697(84)90080-0. PMID 6437277.
  10. Alberts B (2002). Molecular Biology of the Cell (4th ed.). New York: Garland. ISBN 978-0-8153-4072-0.

Close enzymes

Endoglycosidases F and D, cleave Glc-Nac

PNGase F (Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine_amidase)

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