In a previous blog we discussed N-glycans, their structure and methods of analysis. However, N-glycans are only one form of glycosylation that we may have to deal with on glycoproteins, the other major type being O-glycosylation.
O-glycosylation is so named since the glycans are found attached to the hydroxyl (-OH) side chains of the amino acids Serine and Threonine, hence the term “O-glycosylation”. So, first let’s take a look at the structural similarities and differences between mammalian O-glycans and N-glycans. At the moment we won’t consider the biological roles of these O-glycans, but will elaborate on this point in a future blog.
Despite both N- and O-glycans being forms of glycosylation found in many organisms, including humans, there are numerous differences between the two. O-glycans are relatively small, only about 3-6 monosaccharides in size, compared to N-glycans which can be between ~8 and 20 monosaccharides. O-glycans use the monosaccharide N-Acetylgalactosamine (GalNAc) as the residue attaching the glycan to the Serine or Threonine side chain, whereas N-glycans use the monosaccharide N-Acetylglucosamine (GlcNAc) as the attaching residue to the Asparagine side chain. There is also no conserved single core structure for O-glycans compared to the conserved pentasaccharide structure found in the core of N-glycans. Monosaccharides are simply added to the O-glycan “core” GalNAc residue sequentially one at a time. The attachment of each monosaccharide is nonetheless not haphazard, but under biosynthetic control.
It is also important to note that the actual site of O-glycan attachment, the Serine or Threonine residue, is not found in a linear consensus sequence in the protein chain. This is unlike N-glycosylation where the Asparagine that is glycosylated is found in the consensus tripeptide Asn-X-Ser/Thr, where X can be any amino acid except Proline. This means that any known protein sequence can be examined for sites where N-glycans may be found (although there is no guarantee that an identified site will be glycosylated) but that unfortunately, the same pointer is not valid for locating potential O-glycans. Clues may exist in the amino acid sequence for the likelihood of O-glycosylation, such as O-glycans often being found in Ser/Thr rich regions with Proline and Glutamic acid also often being found in the vicinity. Sequences with these features are not guaranteed to be O-glycosylated however and even where O-linked glycans are present in a Ser/Thr rich region the question “which amino acid(s) in this region will be O-glycosylated?” is an important one which still needs to be answered.
So, there are numerous differences between N-glycan and O-glycan structures but how does this translate into how we analyze them? Are there differences in the techniques we use to study O-glycans compared to those we use for N-glycans? Well, since O-glycans are fundamentally chains of monosaccharides, just as N-glycans are, we can certainly use some of the same techniques to investigate their structures. Just as with N-glycans, we need to release and isolate O-glycans prior to analysis. There is no broad specificity enzyme for the release of O-glycans comparable to the enzyme peptide N-glycosidase F for release of N-glycans. Release of O-glycans is best achieved using the chemical process of reductive elimination followed by purification of the released O-glycans.
Once we have isolated the released O-glycans, we can derivatize (e.g. permethylate) and analyze the derivatives just as we can do for N-glycans, e.g. using Matrix Assisted Laser Desorption Ionization mass spectrometry (MALDI-MS) to generate compositional and fragment ion structural information. MALDI data of permethylated O-glycans can be used to give relative quantitation of the species present. Unlike N-glycans though, O-glycans are not readily amenable to fluorescence analysis by on-line liquid chromatography mass spectrometry, which is a common analytical tool for N-glycans. This is due to the reductive nature of the O-glycan release process not being amenable to the fluorescent labeling chemistry. In practice this is not a major drawback, since O-glycans have limited heterogeneity due to their small size, thus population spread is not significant.
Linkage analysis can also be performed on O-glycans, just as it is performed on N-glycans. It needs to be borne in mind that the GalNAc residue is reduced during the initial release of the O-glycan from the protein and thus, whilst the chemistry will still work, this reduced GalNAc will give rise to unique fragment ions in the mass spectrometric data not found in any monosaccharides derived from N-glycans. It is therefore important to remember that should GalNAc be detected in a monosaccharide analysis experiment, being essentially limited to O-glycans in the therapeutic glycoprotein we are dealing with, it would trigger the need for further O-glycan analytical procedures to be performed for confirmation of the structures.
Identification of site(s) of O-glycosylation within the protein chain can be achieved using peptide mapping, in the same way as we do for N-glycans. The nature of O-glycans does add a further layer of potential complexity in that, if O-glycans are found in clustered regions of the protein, which is quite possible based on the protein structure and functionality of the O-glycans themselves, then mass spectrometric investigations into the O-glycopeptide may not be limited to intact mass information. In these instances, there is a need to generate and assess higher energy O-glycopeptide fragmentation data and use the generated fragment ions to investigate sites of O-glycan attachment within the peptide. Judicious use of proteases can help this by producing different peptides across the region of the O-glycan attachment site(s). This does of course depend on the protein structure and what can reasonably be achieved through proteolysis. Experience in this area is essential to ensure correct experimental design and interpretation of results in what can be a complex series of analyses.
In summary, whilst N-glycans and O-glycans may appear on the surface as simply two different forms of glycosylation, they have developed for specific biological needs and thus have their own unique structures and properties. This means we need to apply glycan analytical techniques that, whilst having some overlap between analysis of the two types, will have specific approaches and considerations based on the differences that exist between these two classes of structure.
