Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.
Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified (“high mannose N-glycans”) or are further processed in the Golgi (“complex N-glycans”). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCGβ), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), β1-4 Galactosidase, and β-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (α-N-Acetylgalactosaminidase, α1-2 Fucosidase, α1-3,6 Galactosidase, and β1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 μg), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.
The method described here using enzymatic deglycosylation and SDS-PAGE can provide valuable information about the glycosylation state of a protein of interest, while glycan-specific reagents facilitate the interpretation of the data. This protocol is intended for the initial studies of protein glycosylation and it is particularly suited for secretory and membrane glycoproteins from mammalian cells: the enzymes chosen in this case will specifically remove all N-glycans, and or N-glycans plus and the most common sugars extending and forming the core of O-glycans. Glycosidases have the additional advantage of being mild, compared with chemical deglycosylation methods, preserving the integrity of both sugars and protein backbone.
To elucidate the rate of occupancy (which amino acids are glycosylated), extent of glycosylation, or to determine the fine structure of glycans, more sophisticated techniques such as mass spectrometry, liquid chromatography or NMR are required.
Because of its simplicity, several steps in this protocol can be adjusted, substituted, and/or combined to accommodate various experimental needs. However, in order to obtain results that can be clearly interpreted it is important to understand its strengths and limitations. First, the specificity and purity of the glycosidases are crucial: only well-characterized enzymes tested to be free of proteases and other contaminating activities should be used. Unfortunately, there is no standard enzyme unit definition for glycosidases; the user should determine the appropriate substitution according to the manufacturer’s specifications. Second, a careful choice of detection is necessary: a) Protein staining reagents are useful only if deglycosylation results in a significant shift in molecular mass. Such a clear result as shown here is not always obtained. In other cases, we have seen abnormal migration after deglycosylation (far from the predicted molecular weight, or even slower migration). This phenomenon is not well understood, but it can be said that any change in migration is evidence that the protein has been deglycosylated. b) Sugar detection with antibodies presents unique challenges limiting their applicability. It has been very difficult to generate general anti-glycan antibodies; they are usually raised against a complex glycan target, which restricts their use. Moreover, several monoclonal anti-glycan antibodies display undesired cross reactivity10. c) Lectins (proteins with intrinsic sugar affinity) are well suited for sugar detection, plus they give insight on glycan structure. However, not all of them have a narrow specificity; and many are only partially characterized (meaning that they could have unknown affinities). As a consequence positive lectin staining provides indication, not proof, of the presence of a given sugar. d) Chemical labeling kits (based on periodate oxidation of sugars) are the method of choice to stain all glycoproteins, and thus well suited to follow deglycosylation.
When processing an unknown sample, it is a good practice to include glycoprotein controls. Fetuin is a readily available N– and O-glycoprotein; chorionic gonadotropin (both subunits) is also a good choice. Bovine serum albumin (BSA) can be used as a negative control. However, it should be noted that some non-glycosylated proteins react slightly with the Pro-Q Emerald 300, particularly when used at high concentrations. Non-glycosylated molecular weight standards, such as the prestained protein marker used in this protocol, have the advantage of displaying sharp bands. Only by chance is one of these proteins (80 kDa) reactive to Pro-Q Emerald 300. Therefore, the user might want to run a glycoprotein standard ladder instead, such as Candy-Cane from Invitrogen.
Finally, simple in-gel detection of nucleocytosolic glycoproteins (which are modified with a single O-GlcNAc) is possible with the use of a monoclonal antibody, along β-N-Acetylglucosaminidase for specificity control 11.The description of this technique is beyond the scope of this article, but it should be mentioned as glycosidases are useful tools for the study of many other glycoproteins and glycoconjugates present in cells. A comprehensive treatment of all known forms of glycosylation can be found in the second edition of the “Essentials of Glycobiology”; available free online at the NCBI Bookshelf (Bookshelf ID: NBK1908; PMID: 20301239)12.
The authors have nothing to disclose.
Don Comb
Name of the reagent | Company | Catalogue number | Comments |
Human chorionic gonadotropin β | Sigma Aldrich | C6572 | |
PCR Tubes | VWR | 20170-004 | |
PCR Thermocycler | Applied Biosystems | 4359659 | |
PNGase F | New England Biolabs | P0704 | Supplied with buffers |
Protein Deglycosylation Mix | New England Biolabs | P6039 | Supplied with buffers |
α-N-Acetylglalactosaminidase | New England Biolabs | P0734 | Supplied with buffer |
α1-2 Fucosidase | New England Biolabs | P0724 | Supplied with buffer |
α1-3,6 Galactosidase | New England Biolabs | P0731 | Supplied with buffer |
β1-3 Galactosidase | New England Biolabs | P0726 | Supplied with buffer |
10X Buffer G7 (500 mM Sodium Phosphate) | New England Biolabs | — | Supplied with PNGaseF or Degl Mix |
10X Glycoprotein Denaturing Buffer (5% SDS, 400 mM DTT) | New England Biolabs | — | Supplied with PNGaseF or Degl Mix |
10% NP-40 | New England Biolabs | — | Supplied with PNGaseF or Degl Mix |
3X SDS loading buffer (187.5 mM Tris-HCl pH 6.8, 6% SDS, 30% glycerol, 0.03% bromophenol blue) | New England Biolabs | B7703 | Supplied with 1.25M DTT |
ColorPlus Prestained Protein Marker | New England Biolabs | P7709 | |
10-20% Tris-Glycine Multigel | Cosmo Bio Co. | DCB-414893 | |
Cassette Electrophoresis Unit | Cosmo Bio Co. | DCB-303111 | |
Electrophoresis Power Supply EPS 301 | GE Healthcare | 18-1130-01 | |
Pro-Q Emerald 300 Glycoprotein Stain Kit | Invitrogen | P-21857 | |
Brilliant Blue R | Sigma Aldrich | B0149 | |
AlphaImager HP System | Cell Biosciences | 92-13823-00 |