How Sweet is Your Protein: Using Enzymes to Study Glycosylation

The simple way to look at a protein is as a polypeptide chain, which is folded and folded again until it becomes a 3D molecule. However, it’s not just a simple, but the simplistic way.

Most eukaryotic proteins exist as several isoforms, differing in posttranslational modifications, which allows them to perform slightly different functions or the same function  under slightly different conditions.

A hint that your protein is glycosylated

One day you notice that your purified protein runs as several bands on your western blot, even though you know your antibody is highly specific. Bands smaller than your predicted protein size can often be carelessly dismissed as degradation products, but larger bands are usually isoforms of your protein.

A common posttranslational modification of proteins is glycosylation; a process in which sugars, such as mannose, are attached to the polypeptide chain¹. Because these sugars are attached by enzymes inside the ER and Golgi, it’s worth checking if your protein is a glycoprotein² and if it has any connection to these organelles. (More on the reagents for the ER and Golgi modified proteins see here).

The helpful enzymes

Glycosylases are enzymes, which break bonds between mono- and oligosaccharides and other compounds. They are divided into two classes: enzymes that hydrolyze N-glycosyl compounds and enzymes that hydrolyze O- and S-glycosyl compounds. You can use glycosylases to study sugar additions to proteins.

For an initial check, I recommend using a mix of several different enzymes with varying specificities. For example, the NEB Protein Deglycosylation mix contains five different deglycosylation enzymes to remove all attached sugars. Follow the manufacturer’s instruction, but in my experience, it works best if you first denature the protein and use a long incubation time.

The critical point will be separating the native and deglycosylated variant of your protein after the reaction. Check in advance which of the denaturing gels gets the best resolution of proteins in your range. Load an enzyme treated sample and a mock treated control right next to each other, and then run the gel with a snail pace—otherwise you may not see the shift.

If it doesn’t work, your protein size-changing modification is probably not glycosylation (maybe look into phosphorylation?).

But if you do discover that your band shifts after deglycosylation treatment, congratulations! Now it’s time to look at your protein glycosylation in more detail.

Digging further with enzymes

Glycoproteins differ in how and what sugars are attached to it. And the same protein can have multiple different forms of sugars attached. Your protein could contain N- and/or O-linked sugars. N-glycosylation occurs at the amide nitrogen on the side-chain of the asparagine, O- at the hydroxyl oxygen on the side-chain of hydroxylysine, hydroxyproline, serine, or threonine.

You can differentiate between different types of attachments using specific enzymes.

PNGaseF

PNGaseF removes all types of N-linked glycosylation, from mono- to complex polysaccharides. Use it to distinguish if your protein has N-or O-linked sugars (O-linked won’t be touched by PNGaseF).

EndoH

Endoglycosidase H cleaves within the polymer of mannose and some hybrid oligosaccharides from N-linked glycoproteins. If the enzyme mix or PNGase work, you can use EndoH to learn more about your protein’s trafficking. If your protein remains in the ER, EndoH will work. If your glycoprotein goes through the Golgi, its sugars are modified by Golgi enzymes and they will become resistant to EndoH.

O-Glycosidase

This enzyme will remove O-linked disaccharides attached to Ser/Thr residues.

At least that’s the theory. But O-linked sugars are much more difficult to remove than N-linked sugars. In practice, removal of O-linked sugars might require a mixture of enzymes that trim the sugars off sequentially. So, it’s a little harder to prove your protein contains O-linked glycans.

And remember, if any of the above enzymes do not work, it does not mean that your protein lacks specific modifications. As always, absence of evidence does not mean evidence of absence. Do try several conditions such as denatured and native protein and increasing incubation time.

Do you have any more tips about detecting glycosylation? Please, share them with us.

 References

1. Packer N.H.., et al. 1997.  Proteome analysis of glycoforms: A review of strategies for the microcharacterisation of glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis. Electrophoresis. 18 (3-4): 452–460.
2. Ghaderi, D., et al. 2012. Production platforms for biotherapeutic glycoproteins.  Occurrence, impact, and challenges of non-human sialylation. Biotechnology and Genetic Engineering Reviews. 28 (1): 147-176.