Glycoproteins - Hughes, R. 1985

Structure
Endoglycosidases

As briefly discussed in Section 2.2.1, endoglycosidases hydrolyze internal bonds within carbohydrate chains, leading to the release of Oligosaccharides from Glycoproteins or glycopeptides generated by the proteolysis of glycoproteins. A large number of endoglycosidases are now known (Table 2.4) [8]. To characterize their Specificity, it is necessary to have oligosaccharides or glycopeptides of known structure, such as Ovalbumin fragments. In turn, specific endoglycosidases serve as an exceptionally valuable tool for determining the Structural Features of glycoprotein carbohydrate chains. These Enzymes are capable of reacting with intact glycoproteins and even with those located On the surface of undamaged Cells. Therefore, they may help directly elucidate the Biological Role of the carbohydrate components in Glycoconjugates.

Class="center">Table 2.4. Endoglucosidases [8]

Fig. 2.12. Action specificity of endo-β-N-acetylglucosaminidases. R is a hydrogen atom or a sugar residue.

All type A endo-β-N-acetylglucosaminidases cleave the glycosidic bond between two N-acetylglucosamine residues directly attached to the aspartate residue of the polypeptide (Fig. 2.12). Enzymes D and CI (but not H or CII) hydrolyze this bond even when the chitobiose is substituted with fucose, as occurs in immunoglobulin G glycopeptides (Fig. 2.9). These enzymes also exhibit other differences in specificity. The 3a-substituted mannose residue located at the non-reducing end of a glycopeptide sensitive to enzymes D and CI must be unsubstituted, such as in ovalbumin glycopeptides VI or V. Glycopeptide IV is not a substrate for these enzymes. These enzymes also hydrolyze oligosaccharides, such as the products obtained by the action of type B endo-β-N-acetylglucosaminidase. Glycosidase H requires a more complex chain of a-mannose residues attached to C-6 of the ß-mannosyl residue in the trisaccharide core region of glycopeptides. This enzyme is inactive if fucose is attached to the core region. Glycosidases H and CII rapidly cleave ovalbumin glycopeptides IIIA, IIIB, IV, V, and VI. However, There are two fundamental differences between these two enzymes. First, glycosidase CII requires a branched structure in which both the C-3 and C-6 positions of the ß-mannose residue are substituted with a-mannose residues. Second, enzyme CII does not act on chains containing a 4-O-substituted mannose residue linked by an a3-bond to the core region, because the hydroxyl group at position 4 must be unsubstituted for its activity to be manifested (Fig. 2.12) [11].

Type B endo-β-N-acetylglucosaminidase (e.g., from almond emulsin [15]) is capable of acting on both intact glycoproteins and glycopeptides. The bond cleaved in this process is located between N-acetylglucosamine and asparagine (Fig. 2.12).

Streptococcus endo-β-N-acetylgalactosaminidase exhibits high specificity toward the galß1→3galNAc disaccharide attached to a Serine or Threonine residue of the polypeptide chain, which is characteristic of many glycoprotein O-glycans. However, if this disaccharide is substituted, the enzyme ceases to act, which naturally limits its utility. A similar enzyme has been isolated from the culture medium of Clostridium perfringens; the reaction it catalyzes yields a free galß1→3galNAc disaccharide and a polypeptide chain.

Fig. 2.13. Substrate specificity of endo-β-galactosidases. R₁ is a hydrogen atom or a sugar residue. R₂ is a hydrogen atom, a sugar residue, or a ceramide residue.

Endo-β-galactosidases are also specific toward extended carbohydrate sequences. The enzyme from E. freundii hydrolyzes the internal galactoside bond in glycoprotein carbohydrate chains if the galactose is linked via a (ß1→4) bond to N-acetylglucosamine, or in Glycolipids if the galactose is linked by the same bond to glucose, which in turn is attached to a ceramide moiety. The Cleavage of such internal bonds occurs even in very long oligosaccharide chains formed by The addition of sugar residues to the non-reducing terminal N-acetylglucosamine residue (Fig. 2.13). When the C-6 position of the galactose residue, which is part of an important trisaccharide unit, is substituted, susceptibility to the enzyme is completely abolished. However, the attachment of sulfate to N-acetylglucosamine does not affect enzyme activity. Such a sulfated structure is found, in particular, in keratan sulfate, a component of Proteoglycans. The latter are hydrolyzed by the enzyme with the accumulation of free neuNAc→galNAc(SO₄)→gal chains. A similar specificity is possessed by the enzyme from Flavobacterium. Both of these enzymes have been obtained in homogeneous form, and their molecular mass was found to be 35,000. The difference between the discussed enzymes is that the enzyme from Flavobacterium is constitutive, whereas the Synthesis and Secretion of the enzyme from Escherichia freundii are induced only when the bacterium is grown on a medium containing keratan sulfate or glycoproteins with corresponding bonds in their glycans.

Streptococcus endo-β-galactosidase hydrolyzes the branched oligosaccharides shown in Fig. 2.13. These CARBOHYDRATES represent the antigenic determinants of Blood group A substances (with a terminal N-acetylgalactosamine residue) or B substances (with a terminal galactose residue). N-Acetylglucosamine is a necessary part of the substrate, and accordingly, the enzyme hydrolyzes galß1→4glcNAc bonds, but not galß1→3glcNAc bonds. Both of these bonds are present in different chains substituted either with fucose, or with galactose or N-acetylglucosamine. As a result, chains with blood group A and B antigenic activities are produced. Interestingly, the Streptococcus enzyme can not only distinguish between these antigenic structures, but its specificity is even higher than that of the corresponding Antibodies.