Endoglycosidases

Glycobiology Analysis Manual, 2nd Edition

Native and Sequential N-Linked Glycan Strategies

For some glycoproteins, no cleavage by PNGase F occurs unless the protein is denatured. For others, some or all of the oligosaccharides can be removed from the native protein after extensive incubation (three days or longer) with PNGase F. PNGase F will remain active under reaction conditions for at least three days, making it suitable for extended incubations of native glycoproteins. Some particular residues, due to their location in the native protein structure, are resistant to PNGase F cleavage and cannot be removed unless the protein is denatured.

Endoglycosidases F1, F2, and F3 are less sensitive to protein conformation than PNGase F and are more suitable for deglycosylation of native proteins. Endoglycosidase F1 (Endo F1) cleaves asparaginelinked or free high mannose (oligomannose) and hybrid structures, while Endoglycosidase F2 (Endo F2) and Endoglycosidase F3 (Endo F3) have the ability to cleave complex structures. The linkage specificities of Endoglycosidases F1, F2, and F3 suggest a general strategy for deglycosylation of proteins that may remove all classes of N-linked oligosaccharides without denaturing the protein. As discussed previously, complex oligosaccharides can be reduced to the trimannosyldiacetylchitobiose (Man3GlcNAc2) core using neuraminidase, β-galactosidase, N-acetylglucosaminidase, and fucosidases as required. The remaining trimannosyldiacetylchitobiose core structures can be removed with Endoglycosidase F3.

The Native Protein Deglycosylation Kit (Cat. No. NDEGLY) supplies all three of these enzymes (Endo F1, Endo F2 and Endo F3) with reaction buffers and detailed interactions.

Endoglycosidase F1

Synonyms: Endo F1; Endo-β-N-acetylglucosaminidase F1

Endo F1 cleaves between the two N-acetylglucosamine residues in the N-linked diacetylchitobiose glycan core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine (see Figure 1).

Cleavage site and structural requirements for Endoglycosidase F1

Figure 1. Cleavage site and structural requirements for Endoglycosidase F1.
R1 = H or Asn
R2 = Oligomannose or hybrid configuration
R3 = H or α(1→6)fucose

Endo F1 cleaves asparagine-linked or free high mannose (oligomannose) and hybrid structures, but not complex oligosaccharides (see Figure 2). Core fucosylation of hybrid structures reduces the rate of cleavage by Endo F1 more than 50‑fold. Endo F1 will cleave sulfated high mannose oligosaccharides whereas Endoglycosidase H will not. Endo F1 may be used under native or non-denaturing deglycosylation conditions.

Cleavage site for Endoglycosidase F1

Figure 2. Cleavage site for Endoglycosidase F1 in (A) high mannose glycans and (B) hybrid glycans.

Endoglycosidase F2

Synonyms: Endo F2; Endo-β-N-acetylglucosaminidase F2

Endo F2 cleaves between the two N-acetylglucosamine residues in the N-linked diacetylchitobiose glycan core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine (see Figure 3).

Cleavage site and structural requirements for Endoglycosidase F2

Figure 3. Cleavage site and structural requirements for Endoglycosidase F2.
R1 = H or Asn
R2 = Biantennary or oligomannose configurations
R3 = H or α(1→6)fucose

Endo F2 cleaves biantennary complex oligosaccharides (see Figure 4). High mannose oligosaccharides are cleaved at a rate reduced 20‑fold compared to that of complex structures. Endo F2 will not cleave hybrid structures. Fucosylation has little effect on Endo F2 cleavage of biantennary structures. Endo F2 is useful under native or non-denaturing deglycosylation conditions.

Cleavage site for Endoglycosidases F2 and F3

Figure 4. Cleavage site for Endoglycosidases F2 and F3 in a complex biantennary glycan. Sequential degradation using the exoglycosidic enzymes neuraminidase and β- galactosidase to remove terminal monosaccharides is shown.

Endoglycosidase F3

Synonyms: Endo F3; Endo-β-N-acetylglucosaminidase F3

Endo F3 cleaves between the two N-acetylglucosamine residues in the N-linked diacetylchitobiose glycan core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine (see Figure 5).

Cleavage site and structural requirements for Endoglycosidase F3

Figure 5. Cleavage site and structural requirements for Endoglycosidase F3.
R1 = N- and C-substitution by groups other than H
R2 = Biantennary or triantennary complex oligosaccharide or trimannosyl(Man3) core
R3 = H or α(1→6)fucose
R4 = Asn (Asn or H if fucosylated at R3)

Endo F3 has no activity on oligomannose and hybrid molecules. Endo F3 cleaves non-fucosylated biantennary and triantennary complex oligosaccharides at a slow rate, but only if peptide-linked (see Figures 4 and 6). Core fucosylation of biantennary structures increases activity up to 400‑fold. Core fucosylated biantennary structures are efficient substrates for Endo F3, even as free oligosaccharides. Endo F3 will also cleave fucosylated trimannosyl core structures on free and protein-linked oligosaccharides.

Native deglycosylation of complex tetraantennary glycans requires sequential hydrolysis to the trimannosyldiacetylchitobiose (Man3GlcNAc2) core prior to cleavage using Endo F3 (see Figure 7).

Cleavage site for Endoglycosidases F3

Figure 7. Cleavage site for Endoglycosidases F3 in complex triantennary glycan. Sequential degradation using the exoglycosidic enzymes neuraminidase and β-galactosidase to remove terminal monosaccharides is shown.

Sequential deglycosylation of complex tetraantennary glycan

Figure 7. Sequential deglycosylation of complex tetraantennary glycan. The terminal monosaccharides are sequentially removed using the exoglycosidic enzymes neuraminidase, β- galactosidase, and N-acetylglucosaminidase, until the trimannosyldiacetylchitobiose core remains for subsequent cleavage by Endo F3.

Endoglycosidase H

Synonyms: β-N-Acetylglucosaminidase H

Endoglycosidase H cleaves between the N-acetylglucosamine residues of the chitobiose core of N-linked glycans, leaving one N-acetylglucosamine residue attached to the asparagine (see Figure 8). The specificity of this enzyme is such that oligomannose and most hybrid types of glycans, including those that have a fucose residue attached to the core structure, are cleaved whereas complex type glycans are not released. Thus, this enzyme is extremely useful for selective release of high mannose (oligomannose) or hybrid type glycans from glycoproteins. The enzyme is also active against dolichol-linked glycans containing these structures.

The functional pH range for Endoglycosidase H is 5.0 to 6.0, with the optimum pH at 5.5. No loss of activity was observed during incubation at 37 °C for 48 hours over the pH range 4.5 to 8.5. However, below pH 4.5, activity is rapidly lost.

Cleavage site and structural requirements for Endoglycosidase H

Figure 8. Cleavage site and structural requirements for Endoglycosidase H.
R1 = Oligomannose (2‑150 mannose residues)
R2 = H-, mono- or oligosaccharide at the C-2 or C-4 position
R3 = H or α(1→6)fucose
R4 = Asn or dolichol pyrophosphate

Materials

     
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