Hydrogen deuterium exchange reveals changes to protein dynamics of recombinant human erythropoietin upon N- and O- desialylation

Recombinant human erythropoietin (EPO) is a therapeutic glycoprotein widely used for treating anemia. EPO glycans carry extensive sialylation and the level of the modification is known to affect receptor binding, protein stability and pharmacokinetics. Nonetheless, a detailed understanding of the effects of sialylation on EPO conformation and dynamics is still lacking. Here we investigate the changes to EPO dynamics following enzymatic trimming of terminal sialic acid by amide hydrogen deuterium exchange mass spectrometry (HDX-MS). The results revealed that desialylation enhances structural flexibility near the glycosylation sites, with greater effects observed around the O-glycosylation site relative to the N-glycosylation sites. The affected regions are surface-exposed loops connecting the helix bundle, which do not appear to reduce the thermostability of the molecule as revealed from melting measurement. Our findings demonstrate the feasibility of HDX-MS technique in deciphering the function of specific type of glycosylation that can provide novel insights into the role of sialylation on protein therapeutics.