An important way to study glycosylation of proteins is to take those sugars off!
It would be nice if you could just dissolve them into a sugary sweet solution, but that’s not how it works with these covalently attached protein modifications. To take those glycans off, you are going to need enzymes that are up to the task!
Glycosidases are enzymes that hydrolyze glycosidic linkages, in other words, proteins that remove sugars. This is a large enzyme family, but two that will be your friends when studying glycoproteins are Endoglycosidase H (EndoH) and Peptide-N-Glycosidase F (PNGase F). Both of these enzymes work on N-linked sugars; no one enzyme will remove all O-linked glycans.
EndoH is a recombinant glycosidase sold by many manufacturer’s. It cleaves high mannose and hybrid glycans – the kind that are attached to a protein in the endoplasmic reticulum
In contrast, recombinant PNGaseF removes sugars that are sensitive and resistant to EndoH, including those added or modified in the cis and medial stacks of the Golgi. PNGaseF will take off high mannose, hybrid and complex sugars.
Using glycosidases to study trafficking of proteins
If a protein is glycosylated, it will receive simple sugars in the ER. As the protein travels to the Golgi, these sugars will become more complex as they are modified by the resident Golgi enzymes. Therefore, EndoH and PNGaseF can be used to study transit of a protein through the secretory system of the cell.
To follow this movement of the protein, treat labeled protein lysates that have been collected over time in a pulse–chase format with EndoH or PNGaseF. When the protein is first made in the ER, it will be EndoH and PNGaseF sensitive. However, as the protein travels into the Golgi, it will become EndoH resistant but remain PNGaseF sensitive. When you run these samples on a gel, you will see shifts in molecular weight corresponding to removal of the sugars and/or resistance to EndoH cleavage.
Other uses for glycosidases
Glycosidases aren’t just for studying trafficking of proteins. They can also be used to:
- Study the types of sugars added to a protein
- Strip sugars off a protein to reveal antibody epitopes masked by the sugars
- To make a protein homogenous for X-ray crystallography
- To study the role of the glycans in protein activity and/or ligand binding
- For separate mass spectrometry analysis of the core protein and the glycan component
- To change the clearance rate of a glycoprotein used as a therapeutic
Also, although I only specifically mentioned EndoH and PNGaseF, there are many other glycosidases used to study protein modifications. You might just have to try them all to find your favorite!
Photo courtesy of~Pawsitive~Candie_N