N-Linked Glycan Strategies

Glycobiology Analysis Manual, 2nd Edition

Use of the endoglycosidic enzymes such as PNGase F (N-Glycosidase F) is the most effective method of removing virtually all N-linked oligosaccharides from glycoproteins. A variety of endoglycosidases are available, each with slightly different properties. PNGase F, for example, is most commonly used for enzymatic deglycosylation of N-linked glycoproteins but cannot be used universally.  Other endoglycosidases may be required depending on the substrate and the application. 

Oligosaccharides containing a fucose α(1→3)-linked to the asparagine-linked N-acetylglucosamine (GlcNAc), commonly found in glycoproteins from plants or parasitic worms, are resistant to PNGase F. PNGase A (NGlycosidase A), isolated from almond meal, must be used in this situation. However, PNGase A is ineffective when sialic acid is present on the N-linked oligosaccharide.

Steric hindrance slows or inhibits the action of PNGase F on certain residues of glycoproteins. Denaturation of the glycoprotein by heating with sodium dodecyl sulfate (SDS) and 2‑mercaptoethanol greatly increases the rate of deglycosylation.

Through sequential deglycosylation of monosaccharides using exoglycosidases, all complex oligosaccharides can be reduced to the trimannosyldiacetylchitobiose (Man3GlcNAc2) core. Complex N-linked glycans can be selectively hydrolyzed with a neuraminidase, β-galactosidase, and N-acetylglucosaminidase, available as part of the Enzymatic Deglycosylation Kit (Cat. No. EDEGLY). Additional cleavage using fucosidases may be required in some situations.

PNGase F

PNGase F cleaves all asparagine-linked complex, hybrid, or high mannose oligosaccharides unless the core contains an α(1→3)-fucose. A tripeptide with the oligosaccharide-linked asparagine as the central residue is the minimal substrate for PNGase F (see Figure 1). The asparagine residue from which the glycan is removed is deaminated to aspartic acid (see Figure 2). The oligosaccharide is left intact and is suitable for further analysis. Detergent and heat denaturation increase the rate of cleavage up to 100 times. Most native proteins can still be completely N-deglycosylated, but incubation time must be increased. The optimal pH is 8.6 and the enzyme is active in the pH range of 6 to 10.

Cleavage site and structural requirements for PNGase F

Figure 1. Cleavage site and structural requirements for PNGase F.
R1 = N- and C-substitution by groups other than H
R2 = H or the rest of an oligosaccharide structure
R3 = H or α(1→6)fucose

Cleavage products from PNGase treatment of N-glycans

Figure 2. Cleavage products from PNGase treatment of N-glycans.

Proteomics Grade PNGase F

Proteomics Grade PNGase F (Cat. No. P7367) is extensively purified and lyophilized from dilute potassium phosphate buffer to produce a stable product. The product is free from glycerol and other stabilizers that may interfere in sensitive glycoprotein analysis methods.

Features include:

  • Excellent for applications requiring N-linked deglycosylation (see Figure 3).
  • Superior performance for on-blot, in-gel, and in-solution digestion methods.
  • High specific activity − ≥25,000 units/mg.
  • Compatible for use in MALDI-TOF mass spectrometry.
SDS-PAGE analysis of native and PNGase F-treated alpha-1antitrypsin

Figure 3. SDS-PAGE analysis of native and PNGase F-treated α-1antitrypsin. The test sample (Lane 5) was deglycosylated in solution with 5 units of PNGase F for 1 hour at 37 °C prior to separation on SDS-PAGE. Note the shift in the mobility of the band upon deglycosylation.
Lanes
1: Molecular weight marker
2, 3, 4: Control, native α1 antitrypsin
5: In-solution deglycosylated α1 antitrypsin.

Glycopeptidase A

Glycopeptidase A (Cat. No. G0535) hydrolyzes oligosaccharides containing a fucose residue α(1→3)-linked to the asparagine-linked N-acetylglucosamine (see Figure 4). These types of glycans are resistant to PNGase F. Like PNGase F, the asparagine residue from which the glycan is removed is deaminated to aspartic acid. However, PNGase A is ineffective when sialic acid is present on the N-linked oligosaccharide.

 

Cleavage site and structural requirements for Glycopeptidase A (PNGase A)

Figure 4. Cleavage site and structural requirements for Glycopeptidase A (PNGase A).
R1 = N- and C-substitution by groups other than H
R2 = H or the rest of an oligosaccharide structure

Endoglycosidase H

Endoglycosidase H (Cat. No. A0810) cleaves the β-1,4 glycosidic linkage of high-mannose N-linked glycans between GlcNac1 and GlcNac2, also known as the chitobiose (see Figure 5).  This will leave a glycoconjugate on the protein but remove the bulk of the glycan.  This is useful especially for deglycosylation under native conditions, but the glycan may need to be processed with exoglycosidases to form a high mannose glycoforms.  Because the cleavage site is further removed from the amino acid sequence, it is thought to be more accessible to the enzyme.

Cleavage site and structural requirements for Endoglycosidase H

Figure 5. Cleavage site and structural requirements for Endoglycosidase H.
R1 = N- and C-substitution by groups other than H
R2 = H or the rest of an oligosaccharide structure
R3 = H or α(1→6)fucose

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 asparagine-linked 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.

Materials

     
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