Abstract
Hepatoma-derived growth factor (hHDGF) stimulates cell proliferation on both sides of plasma membrane by either binding to membrane receptor as a growth factor or binding to DNA in nucleus as a transcriptional factor. Secreted hHDGF enables to recognize cell surface heparan sulfate to promote protein internalization and N-terminal PWWP/HATH domain consisting amino acid residue 1-100 have been proved to be responsible for heparin binding. HDGF-related proteins (HRPs) all consist a highly conserved HATH/PWWP domain in their N-terminus where the sequence identity is higher than 70%. We firstly used ITC and SPR methods to reveal the universal heparin affinity in all HRPs. The conserved HATH/PWWP domains demonstrated a broad range of binding affinity to heparin that HRP4 is the strongest heparin binder while LEDGF shows a relatively weak binding. The affinities (KD) are with more than 20-fold difference in magnitude. Using hHDGF HATH/PWWP domain as a prototype, a general heparin-binding site were defined by NMR chemical-shift perturbation where fourteen basic residues (Lys/Arg) with significant perturbations were identified. We tested the individual contributions from the identified basic residues by SPR measurement combining with alanine mutagenesis. The residue K19 together with residues K21, K61, K70, K72 and R79 demonstrates the higher potent in recognition, reflected in the weaker responses in SPR when they were mutated to alanine. Since the positive charges are conserved in all HRPs HATH/PWWP domains, we suspect that the variation in HRP sequence might remotely influences heparin binding by either perturbing protein structural stability or mediating molecular arrangement on a heparin polysaccharide chain.

中文摘要
肝癌衍生生長因子(HDGF)從肝癌細胞株的培養液發現後，目前已經在很多組織中被發現，細胞外提供的HDGF能藉由與細胞膜上的受器或硫酸乙醯肝素(heparan sulfate)結合後進入細胞質，且可被運送到細胞核內促進細胞分裂及增生，具有生長因子的功能；也能進入細胞核與DNA結合，具有轉錄因子的功能。分泌型hHDGF的N端PWWP/HATH domain含有100個胺基酸，過去研究已經證實會和肝素 (heparin)結合。HDGF相關蛋白(HRPs)在N端皆具有高度保留的PWWP/HATH domain，且序列相似性高於70%，我們利用核磁共振(NMR)化學位移擾動(chemical-shift perturbation)的方法，找出肝素可能結合的14個正電胺基酸位置，利用點突變的方法將正電胺基酸(Lys/Arg)突變成不帶電的丙胺酸(Alanine)，此14個點突變Ala分子結合表面電漿共振(SPR)技術發現K19、K21、K61、K70、K72、R79應為影響肝素結合的胺基酸位置。為了了解HRPs家族與肝素的結合能力，分別使用恆溫滴定微卡計(ITC)及表面電漿共振(SPR)，竟發現肝素在序列相似性這麼高的HRPs PWWP/HATH domain中有不同的結合能力，HRP-4是結合能力最強的蛋白質，而LEDGF是結合能力最差的蛋白質，兩者的結合能力(KD)相差高於20倍。在此正電胺基酸具有高度保留的PWWP/HATH domain卻有如此大的不同，推測在序列上局部的變異可能會影響結構穩定或調節肝素多醣鏈的分子排列。

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