I'm not too familiar with the mechanisms of glycosylation but I recall that the main residues that undergo N-glycosylation are arginine and asparagine, from which the latter I think is more common. N-glycosylation in general is nowadays outlined very nicely in some basic books of biochemistry and molecular biology (like Lodish et al. Molecular Cell Biology).

A quick search at Google actually brings up a few hits, which describe the strong preference of N-glycosylation of asparagine over glutamine (see page 779 from http://pac.iupac.org/publications/pac/pdf/1999/pdf/7105x0777.pdf) and also results that present glycosylation of glutamine (http://www.jbc.org/content/285/21/16012.long).

Hope this helps!

Even though the side chain of glutamine is chemically very similar to that of asparagine (both have a carboxamide group at the end), to my understanding N-linked glycosylation of glutamine is uncommon. Therefore, it makes a good substitute for asparagine when you want to remove a glycosylation site. Alanine on the other hand is just a simple way to delete a side chain of interest. It's a better option than, say, glycine due to it still retaining chirality at the chosen residue and has a non-bulky apolar side chain.

Thanks Arne for this first answer. Did you know why Glutamin don't be N-glycosilate?

I'm not too familiar with the mechanisms of glycosylation but I recall that the main residues that undergo N-glycosylation are arginine and asparagine, from which the latter I think is more common. N-glycosylation in general is nowadays outlined very nicely in some basic books of biochemistry and molecular biology (like Lodish et al. Molecular Cell Biology).

A quick search at Google actually brings up a few hits, which describe the strong preference of N-glycosylation of asparagine over glutamine (see page 779 from http://pac.iupac.org/publications/pac/pdf/1999/pdf/7105x0777.pdf) and also results that present glycosylation of glutamine (http://www.jbc.org/content/285/21/16012.long).

Hope this helps!

Thanks Arne for this very helpfull links! Thanks also from my colleague!!!

The consensus N-linked glycosylation sequence is Asn-X-Ser/Thr. The enzymatic machinery responsible for addition of the core glycan recognizes the above sequence with few exceptions. One additional carbon in the side chain (Gln) is not readily tolerated.

Some proteins do crystallize with full N-linked glycans.

If one does not have enough protein to try everything, or if the glycosylated protein will not crystallize, deglycosylation should be tested. There are at least two things to consider when deglycosylating a protein. First, how easy or difficult it will be to deglycosylate the protein to homogeneity and second how much less soluble the protein may become after glycosidase treatment.

Try different glycosidases. Try PNGase first as it removes the whole N-linked glycan. Then try the endoglycosidases such as Endo F1, F3, Endo H, which leave a single GlcNAc on the Asn. If the endoglycosidases do not work, then consider exoglycosidases such as sialidase, galactosidase, hexaminidase, and mannosidase. In general, the smaller the resulting N-glycan residue, the less flexibility you will have left on the protein, meaning a higher degree of conformational homogeneity and a likely higher chance of getting crystals (that diffract).

The chemical and physical conditions one will need for an efficient cleavage with any certain enzyme can vary. This is most likely due to the accessibility of the cleavage site. In the most facile cases, treatment at 4 degrees Celsius with trace amount (1:10,000) of PNGase O/N can result in complete removal of the N-glycans. On the other extreme, for certain proteins one may need more enzyme than substrate protein and the digestion performed at 37 degrees Celsius. Varying incubation time, enzyme concentration, sample concentration and temperature can be used to achieve full deglycosylation.

Being the enzyme that cuts the deepest on the N-glycan, one problem with PNGase is that the cleavage site (the NH2-C linkage on the Asn side chain) on certain N-linked glycosylation site could be quite inaccessible. In many cases, one may find one has to heat the reaction to 37 degrees Celsius in order to achieve a certain level of PNGase cleavage. For highly N-glycosylated proteins (having more than 4 or 5 sites), PNGase treatment is almost sure to fail. The other thing is, PNGase treatment often results in poorly soluble proteins as the removal of the N-glycan exposes some surface patch for aggregation, or destabilizes the protein.

The Endo F1, F3, H, especially Endo H, can often result in better completeness of N-glycan removal, and better solubility of the protein, with the expense of one GlcNAc left on the protein. However, these can require the N-glycans to be of certain type. For example, Endo H requires the N-glycans to be high-mannose type, which has to be generated from certain cell lines, or by a series of exo-glycosidase treatment. Note: For insect cell produced proteins, endo H is very likely to work.

One can often achieve some degree of separation of the glycosylated form and un-glycosylated on ion-exchange, even when the N-glycans are not the charged type (for example, high mannose). This is likely due to a solubility difference of the differently glycosylated species.

In cases where one has charged N-glycans, for example, proteins made from normal mammalian cells, the negative charge of the sialic acid on the N-glycans could be significant, that it can mask the protein's own surface charge.

Consequently, the protein, regardless of its real PI, will bind to ion exchange resin, while the deglycosylated will still behave normally. This can result in the separation of the two or more species. Actually, highly glycosylated proteins with complex type glycans (with terminal sialic acid) often run as multiple peaks on ion exchange, due to the heterogeneity of the N-glycan.

Hydrophobic Interaction Chromatography (HIC) should also be considered as a way to separate the deglycosylated protein.

Lectin columns are another consideration when is comes to separating the deglycosylated protein. However the applicability of lectins depends on the N-glycan's type, and this can get complicated.

Proteins produced from insect cells are often high-mannose type. This means that the N-glycans from insect cells are likely susceptible to Endo H treatment, that the N-glycans are not charged, that the N-glycans themselves are more or less homogenous as they are likely to be Man3,4,5 only, and are also smaller than the complex type glycans made from mammalian cells. This would lead one to believe that such glycosylated proteins are more amenable to crystallization.

Keep in mind that 100% purity and homogeneity, although ideal is often not required for crystallization. Keep in mind that moderation is key and that one might be better off with more partially purified sample screening than less ultrapure sample for screening.

When starting our screening glycosylated and deglycosylated proteins it is generally a good idea to try a chemically diverse screen such as the Hampton Research Index screen. Based upon the results of Index, one may then proceed to the appropriate chemical space identified by Index, meaning salt based, polymer based, mixed polymer/salt, and so on.