- 學位論文
Structural difference between PF4/CXCL4 and PF4v1/CXCL4L1 revealed by NMR
利用核磁共振技術探討PF4/CXCL4和PF4v1/CXCL4L1的結構差異性
摘要
Chemokines were described for the ability to recruit leukocytes and affect tumor growth. Platelet factor-4 (PF4, also known as CXCL4) was the first chemokine demonstrated to be able to inhibit angiogenesis. The feature makes PF4/CXCL4 to be a potent target for tumor therapy. A nonallelic PF4/CXCL4 variant, designated PF4v1/CXCL4L1 was isolated from activated human platelets. PF4v1/CXCL4L1 differs in only three amino acids located in the carboxy-terminal part, but the variant contains higher potent in anti-angiogenesis. To more realize the structural basis in regulating the anti-angiogenic activity, we identified the structure difference between two molecules in the study. PF4/CXCL4 monomer carries two intramolecular disulfide bonds and forms a stable tetramer comprising two asymmetric dimers in solution. In contrast to PF4/CXCL4, the majority state of PF4v1/CXCL4L1 is a dimer and it shows the ability to form intermolecular disulfide bond, indicating the presence of free cysteines. Well-separated resonances in NMR 1H-15N HSQC spectrum reveal a folded symmetric dimer of PF4v1/CXCL4L1. However under reduced condition, PF4v1/CXCL4L1 behaves like a molten globule while PF4/CXCL4 still enables to spontaneously fold itself without the aid of intramolecular disulfide bonds. In final, the three mutations are equally important in keeping the structural difference. Reintroducing any PF4/CXCL4 sequence back to the three mutations won’t recovery the PF4/CXCL4 structural property in PF4v1/CXCL4L1. Taken together, the three mutations change PF4/CXCL4 oligomerization states, disulfide bond connectivity as well as protein folding property. The difference might cause different anti-angiogenic activity in vivo.
並列摘要
趨化激素(Chemokine)可吸引白血球至發炎部位，也會影響腫瘤的生長。第四型血小板因子(PF4，又稱CXCL4)是第一個被發現可以抑制血管新生 (angiogenesis)的趨化激素，此特性可使PF4/CXCL4做為治療腫瘤的標靶蛋白。從人體血小板中分離出變異型的PF4/CXCL4，稱為PF4v1或CXCL4L1。其與PF4/CXCL4的不同在於羧基端(carboxy-terminal)部位的三個胺基酸，此三個胺基酸的不同造成PF4v1/CXCL4L1比PF4/CXCL4更具有抑制血管新生的能力。為了更加了解這兩個蛋白質對於抗血管新生作用上的調節，我們針對其在結構上的差異性來進行討論。PF4/CXCL4的單體具有兩對分子內的雙硫鍵，會形成一個穩定的四聚體，此四聚體由兩個不對稱的二聚體所構成。反觀，PF4v1/CXCL4L1為二聚體，此二聚體由分子間的雙硫鍵所形成，此現象也說明了PF4v1/CXCL4L1可能具有游離的半胱氨酸(cysteine)。核磁共振1H-15N HSQC光譜中訊號的分散顯示了PF4v1/CXCL4L1為摺疊且結構對稱的二聚體。但在還原條件下，PF4v1/CXCL4L1顯示像熔球蛋白(molten globule)的結構特性，而少了分子內雙硫鍵的PF4/CXCL4其結構是可以自發性的摺疊。此外，此三個胺基酸對於保留結構差異性都是很重要的，在PF4v1/CXCL4L1中三個胺基酸任何一個置換成PF4/CXCL4的殘基，都使得其無法回復成PF4/CXCL4的結構特性。總而言之，此三個氨基酸的突變改變了PF4/CXCL4的聚合態、雙硫鍵的連接、甚至是摺疊的特性。這些差異也許是造成「抗血管新生」活性調節上不同的原因。