Chemical synthesis of erythropoietin glycoforms for insights into the relationship between glycosylation pattern and bioactivity

The role of sialyloligosaccharides on the surface of secreted glycoproteins is still unclear because of the difficulty in the preparation of sialylglycoproteins in a homogeneous form. We selected erythropoietin (EPO) as a target molecule and designed an efficient synthetic strategy for the chemical synthesis of a homogeneous form of five EPO glycoforms varying in glycosylation position and the number of human-type biantennary sialyloligosaccharides. A segment coupling strategy performed by native chemical ligation using six peptide segments including glycopeptides yielded homogeneous EPO glycopeptides, and folding experiments of these glycopeptides afforded the correctly folded EPO glycoforms. In an in vivo erythropoiesis assay in mice, all of the EPO glycoforms displayed biological activity, in particular the EPO bearing three sialyloligosaccharides, which exhibited the highest activity. Furthermore, we observed that the hydrophilicity and biological activity of the EPO glycoforms varied depending on the glycosylation pattern. This knowledge will pave the way for the development of homogeneous biologics by chemical synthesis.