肝癌衍生生長因子其HATH區域的肝素結合及N端修飾的研究

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研究生：	林瑋賢 Lin, Wei-Hsien
論文名稱：	肝癌衍生生長因子其HATH區域的肝素結合及N端修飾的研究 Heparin Binding and N-Terminal Processing of HATH domain of Hepatoma Derived Growth Factor
指導教授：	蘇士哲 Sue, Shih-Che
口試委員:	程家維 CHENG, JYA-WEI 鄒德里 Tzou, Der-Lii M.
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2012
畢業學年度：	101
語文別：	英文
論文頁數：	58
中文關鍵詞：	肝癌衍生生長因子、核磁共振、肝素結合蛋白
外文關鍵詞：	Hepatoma-derived growth factor, Paramagnetic relaxation enhancement
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HDGF最早發現於肝癌衍生的HuH 7 細胞株培養液，為肝素 (heparin) 結合蛋白的一種，主要透過N端保留性的HATH區域來辨識細胞膜表面上的硫酸乙醯肝素 (heparan sulfate)，進而促進蛋白質其內吞作用。HDGF也是分泌性的生長因子，其蛋白質分泌受N端前十個胺基酸的修飾，伴隨C12和C108形成分子內的雙硫鍵作調控。於研究中，我們希望闡明肝素與HDGF的HATH 區域之結合交互作用與HATH區域其N端的修飾，彼此之間的關係。首先，我們利用NMR的方法，將肝素滴定至HDGF的樣品，觀察蛋白質與不同醣鏈之肝素的交互結合作用，並且結合先前實驗室HDGF與肝素反應的SPR數據，進而分析正電性胺基酸對於肝素結合的貢獻度。我們發現K19和其周圍的正電性胺基酸構成了主要的肝素結合位置。其次，我們在HATH區域標定了順磁性的分子，觀察蛋白質其分子內的PRE，並且發現HDGF瞬間交互作用(transient interaction)主要影響於N端和C端。更進一步從實驗中證實，N端和C端的變動，是因為HDGF自發性的形成C12和C108分子內的雙硫鍵，將HATH區域的N端和C端靠在一起。隨後由SPR的方法比較氧化態 (-S-S-) 和還原態 (-SH) 的HDGF之肝素結合能力，氧化態 (-S-S-) 和還原態 (-SH)擁有相似的解離平衡常數值 (KD)，意謂氧化態 (-S-S-) 的HATH區域同樣具有辨識細胞表面上硫酸乙醯肝素的能力。因此HDGF HATH區域其N端的修飾，對於細胞表面上的硫酸乙醯肝素結合是不容忽視。考慮肝素結合影響了蛋白質的內吞作用；然而N端的修飾則影響了蛋白質的分泌作用。當HDGF作用在細胞膜上，似乎這兩個效應是分開的。

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.

Contents
ABSTRACT    
中文摘要    
CONTENTS    
ABBREVIATIONS    
INTRODUCTION    
1 Background of Hepatoma-derived growth factor
2 Function of HATH domain    
3 A module HATH Structure of HDGF for function    
4 Secretion of HDGF    
5 Form of heteromer between HDGF and HRP-2    
6 Aim of research    
MATERIALS AND METHODS    
1 Cloning constructs of HDGF HATH domain (HATH100 and HATH110)    
2 Point mutation of hHDGF HATH110 to HATH110C108S by the method of site-directed mutagenesis    
3 sequences of HATH100, HATH110 and HATH110C108S    
4 Protein expression and purification    
5 Preparation of heparin-derived oligosaccharides    
6 15N-labeled and 15N-, 13C-labeled proteins    
7 NMR HSQC experiments and heparin titrations    
8 15N NMR phase-modulated CLEAN chemical EXchange (CLEANEX- PM) experiments with a fast-HSQC (FHSQC) detection    
9 SDS page analysis of HATH110    
10 Preparation of spin label conjugated proteins    
11 NMR analysis of PRE effect (HATH100 and HATH110C108S)    
12 Circular Dichroism (CD)    
13 CD measurements    
14 HSQC analysis of HATH110 (-SH) and HATH110    
15 Backbone assignment of HATH110 (-SH) and HATH110    
16 Surface Plasmon Resonance (SPR) experiments for heparin binding to HATH110 (-SH), HATH110 (-S-S-) and poHATH    
RESULTS    
1 Heparin chain-length dependent effect of HATH binding in NMR titration experiment    
2 Binding affinities of HDGF HATH in heparin chain-length-dependent effect    
3 Heparin binding induced conformational change of HDGF HATH in CLEANEX-PM experiment    
4 Polymerization in HDGF HATH110    
5 Spontanleously oxidized (-S-S-) form in HDGF HATH110    
6 Structural differences of HDGF HATH domain between reduced (HATH110 (-SH)) and oxidized (HATH110 (-S-S-)) form    
7 Heparin-binding affinities of poHATH, HATH110 (-SH) and HATH110 (-S-S-) in SPR measurements    
DISSCUSSION    
1 Heparin-binding of HDGF    
2 N-terminal processing of HDGF    
3 A purposed model for processed form of HDGF trafficking    
REFERENCES    

                                

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