How to Scrutinize Your Glycosylated Proteins Without Using Glycosidases

You might have come across protein glycosylation before. Somewhere in the recesses of your memory you might even recall reading something about the protein you’re studying being glycosylated, but what does this mean and how do you analyze it?

Glycosylated proteins are molecules decorated with sugar groups as they pass through the ER and Golgi networks. And it’s not just proteins, lipids can also be glycosylated to give glycolipids.

You might want to analyze these sugary streamers to give you information about which glycan groups are attached to your protein, to examine your protein’s journey through the secretory system or even to change the clearance rate of a glycoprotein used as a therapeutic. While most people turn to glycosidase enzymes to examine glycoproteins, there is another way.

Antibodies vs. Lectins

If you don’t want to cut up your glycoproteins and decipher the meaning of small changes in molecular weight, you can either use standard antibodies directed against glycan groups or lectins to study glycosylated proteins.

Use either to:

• Examine glycoprotein localization or quantity
• Analyze glycosyltransferase or glycosidase activity
• Purify by affinity chromatography or
• Detect them in a simple western

Antibodies

Just like any other specific epitope, antibodies have been generated to the different glycan groups. Buy them from antibody suppliers, or go the cheaper route and ask for them from a lab that has produced and used the antibodies.

Lectins

You can also use lectins to examine glycosylated proteins. They work in a very similar way to antibodies. You don’t even need to make many changes to standard antibody protocols!

Lectins bind reversibly and specifically to different glycan groups. You can find them in both animals and plants where they function in cell adhesion and glycoprotein synthesis. Compared to antibodies lectins are:

• Not as specific as a good antibody, but specific enough for standard assays
• More stable
• Cheaper to manufacture so less expensive to buy
• Better characterized

If you can locate a lectin specific to the sugar group you want to study, then it is your first port of call. You can buy lectins with a range of tags from fluorophores to biotin so you’re ready to explore all sorts of methods with these sticky little proteins.

Selecting Lectins

First, you must pick the lectin that binds the carbohydrate chains you are interested in.

The most commonly used lectin is concanavalin A (ConA), which binds high mannose sugar structures. High mannose glycans are generated in the ER before the proteins are transported to the Golgi body where the sugars are added to and swapped about to create more complex branched chains.

But others lectins exist too. For example, you could use Ricinus communis agglutinin (RCA) that binds the more complex galactose structures of the later secretory system.

Directing Lectins

Once you’ve got your lectin and are ready to roll – experimentally speaking – be aware of these few simple tips to save you a lot of hassle in the long run.

1. Change Your Blocking Solution

Blocking is a limiting step in sugar binding analysis, because lots of blocking agents contain carbohydrates and generate high background.

Instead of your usual blocking solution, try Bio-Rad’s Synblock or a standard PVA solution. Clearly one of them has a higher price tag than the other, but the choice is yours.

Strangely, the very people that are renown in the lectin field, Vectorlabs, produce ‘Carbo-free’ block, but this can bind different lectins, so perhaps avoid that one?

2. Double Check the Specificity

Another problem with using lectins is that complex glycans are built on the high mannose core, so lectins like ConA, which bind the high mannose core, also identify complex glycans.

However, there are ways though to work around this problem. To begin, capture the total signal from your proteins using ConA to label all glycans. Then, use a lectin like Ricinus communis agglutinin (RCA) that binds galactosylated proteins. Normalize the RCA signal to ConA to give the relative amount of complex glycans compared to total glycan across your protein sample.

3. Use Proper Negative Controls

To assess your lectin’s specificity, preabsorb your lectin with the corresponding carbohydrate, in much the way you would preabsorb an antibody for use as a negative control.

You have two methods to preabsorb your lectin:

i.  Add the excess carbohydrate to the sample for 30-60 minutes at room temperature. Then add your lectin to the sample for binding, or

ii.  Add the excess carbohydrate to the lectin for 30-60 minutes at room temperature before applying to your study sample.

Although some manufacturers recommend the first method, it isn’t guaranteed to completely block your lectin. Since carbohydrates can interact with the sample in all sorts of ways during the incubation, negating this step. Given the choice, use the second method of incubating the sugar with the lectin before applying to your sample. This will give you a better chance of success.

And with these few facts in mind, you can transform your glycan research! You can visualize them inside the cell using immunofluorescence, localize them in tissues with immunohistochemistry, quantify them by flow or purify by chromatography. Finally, you can team lectins with glycosidase enzyme analysis to examine your glycosylated proteins from every angle.