Abstract
Hepatoma-derived growth factor (HDGF) was discovered from the conditional medium of HuH7 cell line. HDGF recognizes cell-surface heparan sulfate through binding to its conserved N-terminal HATH domain to promote protein internalization. Meanwhile, as a growth factor, N-terminal processing of the N-terminal first 10 residues and the presence of intramolecular disulfide bond between residues C12 and C108 has been reported critical in protein secretion. In this study, we aimed to exanimate the structural and functional relationship between heparin binding and the N-terminal processing of HATH domain. We defined the contribution of positively charged residues in heparin binding based on nuclear magnetic resonance (NMR) titration experiments and surface plasmon resonance (SPR) that residue K19 together with surrounding basic residues constitute the major heparin-binding site. By unambiguously labeling paramagnetic spin label on C12 of HATH mutant C108S, we observed the transient interaction between HATH N- and C-terminus by NMR paramagnetic relaxation enhancement (PRE) method. The result well explained the spontaneity of the disulfide linkage between C12 and C108. Subsequently, comparison of heparin binding between oxidized (-S-S-) and reduced (-SH) HATHs revealed similar KD values. Hence, the N-terminal processing caused ignorable effect in heparin recognition. Considering heparin-binding affecting protein internalization while N terminus process on secretion, these two effects are decoupled when HDGF acts on cell membrane.