Endoglycosidase H
Encyclopedia
The enzyme Endoglycosidase H (Endo-β-N-acetylglucosaminidase H, 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.
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-acetylglucosamine
(GlcNAc) subunits 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 (e.g., by carboxymethylation, sulfitolysis or by heating in the presence of sodium dodecyl sulfate). Over 0.02% SDS may inactivate the enzyme.
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 .
in the Golgi apparatus
. Most proteins destined for the cell surface are translated
by ribosomes in 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.
s F and D, cleave Glc-Nac
PNGase F
(Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine_amidase)
Endoglycosidase
An Endoglycosidase is an enzyme that releases oligosaccharides from glycoproteins or glycolipids. Or it merely cleaves polysaccharide chains between residues that are not the terminal residue, although releasing oligosaccharides from conjugated protein and lipid molecules is more common.It breaks...
which cleaves asparagine
Asparagine
Asparagine is one of the 20 most common natural amino acids on Earth. It has carboxamide as the side-chain's functional group. It is not an essential amino acid...
-linked mannose rich oligosaccharides, 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-acetylglucosamine
N-Acetylglucosamine
N-Acetylglucosamine is a monosaccharide derivative of glucose. It is an amide between glucosamine and acetic acid...
(GlcNAc) subunits 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 (e.g., by carboxymethylation, sulfitolysis or by heating in the presence of sodium dodecyl sulfate). Over 0.02% SDS may inactivate the enzyme.
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 (Endo H) is commonly used by cell biologists to monitor posttranslational modificationPosttranslational modification
Posttranslational modification is the chemical modification of a protein after its translation. It is one of the later steps in protein biosynthesis, and thus gene expression, for many proteins....
in the Golgi apparatus
Golgi apparatus
The Golgi apparatus is an organelle found in most eukaryotic cells. It was identified in 1898 by the Italian physician Camillo Golgi, after whom the Golgi apparatus is named....
. Most proteins destined for the cell surface are translated
Translation (genetics)
In molecular biology and genetics, translation is the third stage of protein biosynthesis . In translation, messenger RNA produced by transcription is decoded by the ribosome to produce a specific amino acid chain, or polypeptide, that will later fold into an active protein...
by ribosomes in the rough endoplasmic reticulum
Endoplasmic reticulum
The endoplasmic reticulum is an organelle of cells in eukaryotic organisms that forms an interconnected network of tubules, vesicles, and cisternae...
(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
Golgi alpha-mannosidase II
Golgi α-mannosidase II is a key enzyme involved in N-linked Glycan processing. It is inhibited by small molecule swainsonine....
removes two mannose
Mannose
Mannose is a sugar monomer of the aldohexose series of carbohydrates. Mannose is a C-2 epimer of glucose. It is not part of human metabolism, but is a component of microbial cell walls, and is therefore a target of the immune system and also of antibiotics....
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.
External links
Close enzymes
EndoglycosidaseEndoglycosidase
An Endoglycosidase is an enzyme that releases oligosaccharides from glycoproteins or glycolipids. Or it merely cleaves polysaccharide chains between residues that are not the terminal residue, although releasing oligosaccharides from conjugated protein and lipid molecules is more common.It breaks...
s F and D, cleave Glc-Nac
PNGase F
Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase
In enzymology, a peptide-N4-asparagine amidase is an enzyme that catalyzes a chemical reaction that cleaves a N4-asparagine residue in which the glucosamine residue may be further glycosylated, to yield a N-acetyl-beta-D-glucosaminylamine and a peptide containing an aspartate residue...
(Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine_amidase)