GLYCOSYLATION

A major post-translational modification is glycosylation. Glycosylation is the attachment of monosaccharides and oligosaccharides to a protein backbone via a glycosidic linkage. It is estimated over 80% of all plasma proteins are glycosylated.

There are two main types of glycosylation:

• N-linked - where the sugar units are attached via the amide nitrogen of an asparagine residue; and
• O-linked - where the sugar units are attached via the hydroxyl group of serine, threonine, hydroxylysine or hydroxyproline residues.

Other glycosidic linkages include S-linkage to cysteine and C-linkage to tryptophan.

The addition of specific sugar residues to individual amino acids in a polypeptide is not a random event. It is generally believed that each polypeptide glycosyltransferase recognizes specific amino acid sequence motifs. N-linked glycosylation occurs at asparagines in the consensus sequence -Asn-Xaa-Ser/Thr/Cys where Xaa is not Proline. Similarly, C-glycosylation occurs at tryptophan residues in the motifs -Trp-Xaa-Xaa-Trp or Trp-Ser/Thr-Xaa-Cys. There is no known motif for O-glycosylation, but it is known O-glycosylation is more probable in sequences with a high proportion of serine, threonine and proline residues.

If you want to find out more about the glycosylation characteristics of our proteins, click here to view summary data.

The presence of a potential glycosylation amino acid sequence motif does not guarantee the site is glycosylated. Furthermore, each glycosylated site may be fully or partially glycosylated, and one site may have many different glycan structures.

This protein glycosylation complexity results because the glycosylation process is not under direct genetic control. Instead glycosylation is a finely controlled process that is dependent upon the availability and activity of the various glycosyltransferases, monosaccharides and precursors.

More indirectly, levels of various hormones, such as thyrotropin and retinol (vitamin A), also regulate glycosylation through a variety of cell type dependent effects. The result is that the same protein derived at different times of development or from different tissues can possess different glycan chains.

Human cell expression results in human glycosylation

Recombinant proteins are dependent on the glycosylation machinery of the cell line to determine what glycan structures are attached. As E. coli does not possess the same type of cellular machinery used for glycosylation in higher organisms like humans, human proteins expressed in E. coli are not glycosylated.

Human proteins expressed in rodent cell lines e.g., CHO and NSO, and the insect cell line Sf21 exhibit glycosylation structures that are distinct from those found on the native protein. For example human erythropoietin (EPO) expressed in BHK-21 and CHO cells have elongated oligosaccharide branches that are not found on native EPO isolated from human plasma.

Figure 1

Figure 1
(a) N-linked glycan structure typically found on native human EPO
(b) N-linked glycan structure found on recombinant human EPO expressed in CHO cells.
Note that the Gal(b1-4)GlcNAc(b1-3) repeat found in CHO expressed EPO is not found in N-glycan structures on native human EPO.

Similarly, tissue plasminogen activator isolated from human Bowes melanoma cells has N-linked glycans with predominately GalNAc(b1-4)GlcNAc(b1-2/6)Man branches. The same protein made recombinantly in mouse C127 cells had N-linked glycans with mainly Gal(b1-4)GlcNAc(b1-2)Man branches and high-mannose structures, not found on the native human protein.

Figure 2

Figure 2
(a) N-linked glycan structure found on human tissue plasminogen activator isolated from human Bowes melanoma cells
(b) N-linked glycan structures found on recombinant human tissue plasminogen activator expressed in mouse C127 cells.

NB. Structures found on proteins taken from GlycoSuiteDB (www.glycosuite.com)

Find out more about the key differences:

• » HCX Proteins
• » Post-translational modifications
• » Tag-free Proteins
• » Glycosylation summary

View Apollo's hcx™ product range:

• » Recombinant Human Proteins
• » ELISA Kits

Top