The non-glycosylated N-terminal domain of human thrombopoietin is a molten globule under native conditions

Human thrombopoietin (hTPO) is a primary hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production. The non-glycosylated form of 1-163 residues of hTPO (hTPO(163)) including the N-terminal active site domain (1-153 residues) is a candidate for treating thrombocytopenia. However, the autoantigenicity level of hTPO(163) is higher than that of the full-length glycosylated hTPO (ghTPO(332)). In order to clarify the structural and physicochemical properties of hTPO(163), circular dichroism (CD) and differential scanning calorimetry (DSC) analyses were performed. CD analysis indicated that hTPO(163) undergoes an induced-fit conformational change (+19.0% for helix and -16.7% for beta-strand) upon binding to the neutralizing antibody TN1 in a manner similar to the coupled folding and binding mechanism. Moreover, DSC analysis showed that the thermal transition process of hTPO(163) is a multistate transition; hTPO(163) is thermally stabilized upon receptor (c-Mpl) binding, as indicated with raising the midpoint (T-m) temperature of the transition by at least +9.5 K. The conformational variability and stability of hTPO(163) indicate that hTPO(163) exists as a molten globule under native conditions, which may enable the induced-fit conformational change according to the type of ligands (antibodies and receptor). Additionally, CD and computational analyses indicated that the C-terminal domain (154-332 residues) and glycosylation assists the folding of the N-terminal domain. These observations suggest that the antibody affinity and autoantigenicity of hTPO(163) might be reduced, if the conformational variability of hTPO(163) is restricted by mutation and/or by the addition of C-terminal domain with glycosylation to keep its conformation suitable for the c-Mpl recognition.