Glycosylation is an incredibly complex post translational modification (PTM) of proteins that is, at least in part, controlled by the repertoire of expressed glycosidases and glycosyltransferases in the cell’s glycan processing pathway. These enzymes reflect the organism’s unique genome and therefore it cannot be expected that the glycosylation machinery will be the same between different organisms. Indeed, glycosylation profiles can differ between cell types from the same organism. There are a number of research papers and reviews covering this subject (i.e. analysis of glycoproteins from different species (1, 2) and from different tissues from the same species (3) showing significant differences in the glycan profiles).
So what considerations do we need in our approaches to glycan analysis in terms of methodology and data interpretation to ensure that key structural glycan features from cell lines derived from different species are not missed?
We are helped in our deliberations by the fact that, in biopharmaceutical manufacturing, the majority of cell lines with the capacity to glycosylate (which may be derived from either the animal, plant or fungal kingdoms) produce their differing glycan profiles through the way they combine the same basic monosaccharides. Thus, mammalian species use fucose, mannose, galactose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid (of one form or another) in various combinations for their N- and O-glycans (for a more detailed discussion of glycan structures see here). These monosaccharides are found to one degree or another on all mammalian cell lines. As we move down through the evolutionary tree (or across taxonomic classification, depending on your point of view) we find that the number of different types of monosaccharides used in glycans decreases. Thus, insects do not have sialic acid in their glycans and neither do plants.
Fungal species can exhibit variation in the monosaccharides they use depending on the species being considered. For example, Saccharomyces sp. use only mannose and N-acetylglucosamine in their glycans but Aspergillus sp. can use these same monosaccharides plus galactose, thus the range of monosaccharides used is simpler still. Mammalian species therefore have the most developed glycan processing pathway in terms of the types of monosaccharide used. One caveat to this is that plants can also use xylose in their N-glycans, a monosaccharide that is not seen in the N- or O-glycans of animals.
The structures of commonly encountered monosaccharides used by various organisms are shown in Figure 1 below.
Figure 1: Structures of the commonly encountered monosaccharides. Subsets of these monosaccharides are used variously by animals, plants and fungi in their glycosylation pathways
So, if we are looking at similar, or indeed simpler, monosaccharide compositions from non-mammalian derived cell lines, where should our considerations for significant differences in structure lie? The answer lies in the way these same monosaccharides are combined by the glycosyltransferases present in the various cell lines.
Unlike the linear arrangement of amino acids in proteins, glycans are branched structures where monosaccharides are attached to one another at defined positions around each monosaccharide ring. The position of attachment and nature of the monosaccharide attached is based on the specificity of the expressed glycosyltransferases and these vary depending on the cell type.
Fungal cells can generate very different glycan structures to those found in other cell types through making very extensive use of mannose and attaching it in superabundance to N-glycans as well as using it to produce O-glycans. This reflects not only linkage variation but also a much simpler overall form of glycosylation, albeit one that is significantly divergent from mammalian systems, through the use of a smaller repertoire of glycosyltransferases and monosaccharides.
This variation of structure can have a profound effect not only on the data coming from the analyses but on the analytical methodologies themselves. Certain linkages can preclude the action of enzymes that are routinely used for glycan release. For example, if fucose is attached in alpha1-3 linkage to the core N-Acetylglucosamine of an N-glycan, a linkage that is seen in insects and plants but not mammals (see Figure 2) then the standard N-glycan release enzyme PNGase F will not work. In this situation PNGase A must be used to release the N-glycan population instead. Unfortunately, PNGase A does not produce good release of larger glycans, such as those found in mammalian cells, meaning that this enzyme cannot be used as a release mechanism for all glycan analysis.
Figure 2: A commonly observed, relatively simple N-glycan structure found in plants.
Figure 3: An example of a mammalian biantennary N-glycan carrying a galactose residue alpha 1-3 linked to another galactose residue on each of the antennae.
A second common form of modification is that of galactose alpha1-3 linked to galactose on the termini of glycans (Figure 3). This is again produced as a result of a specific glycosyltransferase that is found in certain cell lines (e.g. murine and porcine) but not human. The absence of this epitope in humans means that it has the potential to be immunogenic, since the body may recognize this as a foreign antigen and trigger an immunologic response (see Figure 4).
Figure 4
It is important to have access to methods that can recognize this unit specifically for what it is. Linkage analysis (the analysis of the monosaccharide linkages within a glycan population used as part of glycan characterization) will identify the 3-linked galactose and the terminal galactose, but we also need to demonstrate that these are indeed two connected monosaccharides and not linkages derived from other normal but not connected monosaccharides. This can be achieved through the use of either specific exoglycosidases (enzymes that will digest specific linkages) or by looking for definitive fragment ions from a mass spectrometric analysis of the glycans.
Species and cell line specific variations of the “basic” mammalian glycan structural traits may at first appear to be problematic for glycan analysis. However, with knowledge of the cell line being used, it is perfectly feasible to develop and modify standard analytical strategies to encompass what could be present on the samples.
Knowledge of the biological source of the sample is essential to ensure an effective experimental approach. Using this knowledge to plan and effectively review generated data means key structures that could have significant biological repercussions, such as a potential to be immunogenic, will not go unidentified.
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