Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

1. Enzymatic deglycosylation

1. Use PCR tubes to minimize water loss due to evaporation. Label one set of tubes 1 to 7.
2. Thaw the 10X G7 buffer, the 10X glycoprotein denaturing buffer, and the 10% NP-40 and gently tap the tubes to mix the contents. Keep at room temperature.
3. Place the enzyme-containing vials on ice. Try to minimize thaw/freeze cycles.
4. Dissolve the contents of the hCGβ vial (150 μg) in 600 μl of dH₂O and keep on ice.
5. Prepare 1 ml of 1X G7 buffer by diluting the 10X stock in dH₂O.
6. Dilute 0.5 μl of PNGase F in 25 μl 1X G7 buffer and keep on ice.
7. Prepare the exoglycosidase mix (EG mix) by combining 2 μl each of α-N-Acetylgalactosaminidase, α1-2 Fucosidase, β1-3 Galactosidase, and α1-3, 6 Galactosidase.
8. Set up PCR tubes as indicated:

9. Cap the tubes, mix gently and place in the thermocycler, close the lid and denature the proteins by incubating 10 minutes at 94°C, followed by a 4°C hold (using PCR tubes in a thermocycler greatly prevents evaporation in small volume samples).
10. Remove the tubes from thermocycler and centrifuge to remove any visible condensation.
11. Add the following reagents as indicated:

12. Close the PCR tubes using new caps (discard the used ones since they do not fit properly after one incubation cycle).
13. Mix the tubes by gently tapping 4 times and then spin the contents down.
14. Place the tubes in the thermocycler and incubate at 37 °C for 4 hours then cool the samples to 4 °C.

2. SDS-PAGE of deglycosylated samples

1. Prepare 130 μl fresh 3X reducing SDS loading buffer by adding 4 μl of 1.25M DTT.
2. Add 12.5 μl of the prepared 3X reducing SDS loading buffer to each sample.
3. Close the tubes with new caps and gently tap the tubes to mix.
4. Incubate the tubes in a thermocycler at 94°C for 5 minutes and then cool to 4 °C.
5. Load 30 μl of each sample and 10 μl of the protein marker on a 10-20% Tris-Glycine gel. Save the remainder of the sample for part 3.1. Load 10μl of ColorPlus Prestained Protein Marker.
6. Electrophorese the gel at 130 volts at room temperature until the dye front is near the bottom of the gel.
7. When the gel has finished running, remove the gel from the cast and place it in a small plastic box with enough Coomasie blue stain to cover the gel.
8. Stain the gel for 1 hour with gentle agitation.
9. Wash the gel three times for 30 minutes in 50 ml of destain solution.
10. Record the images using a white light transilluminator or scanner. Alternatively, the gel can be dried between sheets of cellophane in a frame.

3. Pro-Q Emerald 300 for detection of glycosylated proteins in SDS-PAGE gels

1. In parallel with the gel in 2.1), load the remainder of the samples on a 10-20% Tris-Glycine gel. Load 10μl of ColorPlus Prestained Protein Marker.
2. While the gel is running dissolve the Pro-Q Emerald reagent with DMF and prepare the stock Fix, Wash and Oxidizing solutions for the Pro-Q Emerald 300 stain following the product manual provided with the kit.
3. When the electrophoresis is complete, remove the gel from cast and place it in a plastic box.
4. Fix the gel by adding 100 ml of the Fix solution and leaving it overnight at room temperature with gentle agitation.
5. Wash the gel with 100 ml of the Wash solution for 10 to 20 minutes at room temperature with gentile agitation. Repeat the wash with fresh Wash solution.
6. Oxidize the carbohydrates by incubating the gel with gentle agitation for 30 minutes in 25 ml of Oxidizing solution.
7. Wash the gel as described in step 3.5.
8. While the gel is washing prepare fresh Pro-Q Emerald 300 stain by adding 500 μl of the Pro-Q Emerald 300 reagent solution dissolved in step 3.2 to 25 ml of staining buffer provided in the kit.
9. Stain the gel by adding 25 ml of the stain prepared in step 3.8 and incubating in the dark with gentle agitation for 90 to 120 minutes.
10. Repeat the two wash steps described in step 3.5.
11. Record the images with an UV transilluminator at 300nm. Use the 80 kDa marker, which is labeled with Pro-Q Emerald Green, to overlap the UV image to a white light picture of the gel showing the prestained ladder.
12. Compare the images of the Coomasie stained gel in step 2.10 with the Pro-Q Emerald stained gel from step 3.11.

4. Representative results

The changes in protein migration after enzymatic deglycosylation are shown in Figure 2. Compare the control sample (panel A, lane 1) with the PNGase F treatment (removal of N-glycans, lane 2), and Deglycosylation Mix (PNGase F; plus endo-and exoglycosidases to remove O-glycans, lane 3). No further reduction in size is seen after digesting with additional glycosidases (lane 4). Besides a change in mass, bands become sharper as glycans are removed. A band running under the 17 kDa marker (asterisk) represents the fully deglycosylated hCGβ polypeptide (MW: 16 kDa). Other bands might derive from incomplete deglycosylation or from the multiple unidentified proteins present in the hCGβ sample (see lane 1). Lanes 5 to 7 (controls) show the bands corresponding to the glycosidases.

The glycoprotein staining by Emerald Green is shown in panel B. This reagent oxidizes and stains all glycans present in a protein molecule. Therefore the intensity of the signal decreases as hCGβ is enzymatically deglycosylated (lane 1 to lane 4). The residual signal in lanes 3 and 4 indicate the presence of glycan motifs, which are resistant to the enzymes used. The additional glycosidases used in lane 4 remove a few extra sugar residues: the protein migration is the same, but a slight reduction in intensity in staining can be seen. Resistant sugar moieties were not present in all protein species: some bands were not detected by Emerald Green (absent in the UV image, in brackets), indicating they were extensively deglycosylated. Additional data support the conclusion that hCGβ is heterogeneously glycosylated. The lower band on lane 2 (arrow) is faint on the Emerald Green image, while the upper band on lane 2 is bright, indicating that many glycans groups are still present. These data support the conclusion that recombinant hCGβ expressed in mouse cells contains multiple glycoforms⁹ . These different glycoforms are due to the inherent heterogeneity of glycosylation where some polypeptides do not receive a glycan in each consensus site and/or some glycans are extended while others even on the same protein are not.

Figure 1. Typical glycosylation patterns for secreted or cell-surface glycoproteins.

Figure 2. SDS-PAGE gels showing enzymatic deglycosylation of hCGβ. Panel A shows a Coomasie blue staining while Panel B shows the results of Pro-Q Emerald 300 for visualization of glycosylated proteins. Sample number: 1, hCGβ control; 2, PNGase F digestion; 3, Deglycosylation Mix digestion; 4, Deglycosylation Mix plus exoglycosidases digestion, samples 5 to 7 are the reagent controls (O-Glyc, O-Glycosidase; GNA, β-N-Acetylglucosaminidase; Gal/Fuc, β1-3 Galactosidase, α1-3,6 Galactosidase, and α1-2 Fucosidase; GalNA, α-N-Acetylglalactosaminidase; Neu, Neuraminidase; PNGase; PNGase F)