以電子自旋共振光譜分析敘利亞倉鼠普立昂胜肽序列 108-144 類澱粉纖維之結構特性

Abstract

普立昂蛋白原是位於神經細胞外膜上的醣蛋白，正常形態 (PrPC) 的結構以 α-螺旋為主，當結構上轉變為 β 結構為主之致病形態 (PrPSc) 時，會不正常聚集而堆疊為類澱粉纖維，此種形式的普立昂蛋白會造成腦細胞死亡，產生海綿樣組織空洞，最後導致死亡。目前仍不清楚普立昂蛋白進行結構轉變時，究竟是結構中的哪一段形成分子間 β 結構，本論文即探討普立昂胜肽形成的類澱粉纖維之堆疊特性及與其胺基酸序列的關係。 本論文使用敘利亞倉鼠之胜肽片段（序列108-144）為實驗材料，選取七個疏水性、非極性之胺基酸為接上自旋標記的位置，於胜肽合成時以半胱胺酸替代。以高效液相層析儀純化胜肽後，在半胱胺酸支鏈接上具有自由基電子的甲烷硫磺酸基團 MTSSL 自旋標記，再利用野生型普立昂胜肽形成的類澱粉纖維為晶種，誘發帶有自旋標記的胜肽形成類澱粉纖維，最後利用電子自旋共振技術解析各標記位點之間的訊號強度推斷出相對距離，來探討普立昂胜肽形成類澱粉纖維後之結構特性。另外本論文也利用穿透式電子顯微鏡技術檢視普立昂胜肽形成之類澱粉纖維，以確認類澱粉纖維是否成功生成。 電子自旋共振的結果顯示，具有不同替代位點的胜肽之間其結構差異非常明顯。在自發性生長的類澱粉纖維之光譜中，其代表電子間訊號強度之線寬大致可排序為：A118R1 ≈ V121R1 > M134R1 ≈ L125R1 > A113R1 > M129R1 ≈ M138R1；而在晶種誘發生長組別的光譜中，除位點 M129R1 外，其曲線形狀均較為寬胖。以此結果我們推斷，在胜肽片段 108-144 中，可能存在兩個結構較為緊密的澱粉樣核心區，分別位於A113 與 L125 以及 M134 與 M138 之間。在兩個澱粉樣核心區之間，縱然 M129 周圍區域的結構較為鬆散，卻可能對整體澱粉樣纖維結構產生決定性的影響。從倉鼠普立昂胜肽的實驗出發，我們希望可以解開普立昂蛋白結構上的堆疊關鍵區域，再進一步推展到人類普立昂蛋白上，以便作為未來疾病治療、預防與藥物開發之依據。

Prion protein is a glycoprotein anchored on the membrane of neuron cells. The normal, cellular form (PrPC) is rich in α-helices. When PrPC is transformed to disease-causing form (PrPSc), β structures appear to dominate in prion protein. PrPSc is prone to association, leading to the formation of amyloid fibrils. This aggregation form of prion protein can induce neuronal death in the brain, which results in sponge-like holes, and finally lead to fatal consequence. Little is known about the core regions where the structural conversion takes place and form intermolecular β structure (also known as cross-β structure). Our study aims to provide an insight in the structural features of the prion amyloid fibrils and the relationship with its amino acid sequence. Prion peptide (residue 108-144) from Syrian hamster is used as our target in this study. Seven hydrophobic, non-polar amino acid residues were picked out for substitution to cysteine in each mutant peptide respectively, serving as the spin labeling sites. The wild-type and mutant peptides were synthesized by solid-phase peptide synthesis and purified by HPLC. The mutant peptides were then labeled with MTSSL, featuring its methane thiosulfonate group and radical spin, on the side chain of cysteine residues (site-directed spin labeling). The spin-labeled peptide monomers were induced to form amyloid fibrils with or without adding seeds, which were prepared from the wild-type amyloid fibrils. The spin-labeled amyloid fibrils were further analyzed by electron spin resonance (ESR) spectroscopy for obtaining the information of relative proximity between spins. The morphologies of the amyloid fibrils were also examined by TEM to confirm the presence of fibrils. Our ESR results revealed distinct features between peptides of different mutated sites. In the spectrograms of spontaneously formed fibrils, the linewidths could be ranked as A118R1 ≈ V121R1 > M134R1 ≈ L125R1 > A113R1 > M129R1 ≈ M138R1, and the curves of seeded fibrils were all relatively broadened except for M129R1. It is assumed that there might be two amyloid cores within the peptide 108-144, ranging from A113 to L125 and from M134 to M138 respectively. Between these two cores the residues around M129 stood out as a key region, which might be loose in structure but packed between the two cores. Our study can provide useful information to explain the location of the amyloid core in the process of prion protein propagation, and we hope to develop drugs against prion disease progression.