The enzymeEndoglycosidase H -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- is an enzyme with systematic nameglycopeptide-D-mannosyl-N4-2-asparagine 1,4-N-acetyl-beta-glucosaminohydrolase. It is a highly specific endoglycosidase which cleaves asparagine-linked mannosericholigosaccharides, but not highly processed complex oligosaccharides from glycoproteins. It is used for research purposes to deglycosylate glycoproteins.
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. Endoglycosidase H cleaves the bond in the diacetylchitobiose core of the oligosaccharide between two N-acetylglucosaminesubunits directly proximal to the asparagine residue, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. 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. 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.
Biochemical Applications
Endoglycosidase 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 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.
