單一色胺酸控制醣胺聚醣結合穿膜胜肽GBPECP進入細胞之機制

Glycosaminoglycan promoted cell entry of cell-penetrating peptide GBPECP depends on a single Tryptophan

洪莉鈞(Hung, Li Chun)

指導教授 ： 蘇士哲

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2016年)

摘要

在過去研究中，人類嗜酸性血球陽離子蛋白（human eosinophil cationic protein, ECP）中所發現的十個胺基酸所組成的小片段胜肽（GBP¬¬ECP）具有與肝素結合能力和細胞穿膜能力，但其機制尚未明確，而此片段中的色氨酸可能是胜肽與細胞膜表面結合的關鍵。本研究將先建立一個仿生模型，利用肝素作為細胞膜表面的醣類以及十二烷基磷酸膽鹼（Dodecylphosphocholine,DPC）所製造的單層微胞粒（micelle）來模擬細胞膜，並利用核磁共振技術，鑑定胜肽上單一胺基酸與其他物質之間的關係。除此之外，本研究也利用同源蛋白衍生出的其他十個胺基酸的小片段胜肽GBPEDN以及GBPECP(W4R)，探討胺基酸的差異所造成與肝素結合能力和穿膜效應的影響，同時，本研究也利用螢光實驗探討色胺酸在穿膜機制中所扮演的角色，證實此胺基酸的側鏈具有進入到微胞粒中的效應。本論文將此小片段胜肽與肝素以及微胞粒作用的胺基酸建立出來，探討其關係有助於未來開發及設計新型穿膜胜肽。

關鍵字

核磁共振 ； 穿膜胜肽 ； 色胺酸

並列摘要

A 10-residue glycosaminoglycan binding peptide derived from human eosinophil cationic protein, GBPECP, has been recently designated as a potent cell-penetrating peptide. Intracellular penetration of GBPECP highly depends on the existence of a tryptophan residue in the sequence. To realize the cell-penetrating mechanism in a model system mimicking peptide, glycan, and membrane environment, Dodecylphosphocholine (DPC) lipid micelle and heparin fragment were titrated into GBPECP solution to induce chemical shift perturbations in nuclear magnetic resonance (NMR) study. Our data showed that charged residues of R5 and K7 played substantial roles in recognizing heparin while R3 had less effect. The only aromatic residue W4, however, acted as an irreplaceable moiety for lipid membrane insertion, suggesting its critical role in related to GBPECP cell entry. Interestingly, although replacement of W4 with R4 significantly improved heparin binding activity of the peptide, such modification abolished cell penetration. Furthermore, in the absence of heparin, binding between GBPECP and DPC micelle still took place. The side chain of W4 adopts an opposite orientation from those of R5 and R7, with W4 responsible for cell penetrating and both R5 and K7 for GAG binding. These two effects appeared to synergistically promote GBPECP penetration through macropinocytosis and further modulate microenvironment of cell surface. More recently we have discovered that GBPECP treatment suppressed tumor cell migration and invasion. GBPECP, therefore, shows high potential as novel therapeutics through rapid and effective internalization, and interference with cell motility.

並列關鍵字

NMR ； CPP ； Tryptophan

參考文獻

1. Fang, S.L., et al., A novel cell-penetrating peptide derived from human eosinophil cationic protein. PLoS One, 2013. 8(3): p. e57318.

2. Dyer, K.D. and H.F. Rosenberg, The RNase a superfamily: generation of diversity and innate host defense. Molecular diversity, 2006. 10(4): p. 585-597.

3. Cho, S., J.J. Beintema, and J. Zhang, The ribonuclease A superfamily of mammals and birds: identifying new members and tracing evolutionary histories. Genomics, 2005. 85(2): p. 208-20.

4. Yang, D., et al., Human ribonuclease A superfamily members, eosinophil-derived neurotoxin and pancreatic ribonuclease, induce dendritic cell maturation and activation. The Journal of Immunology, 2004. 173(10): p. 6134-6142.

5. Boix, E., et al., Structural determinants of the eosinophil cationic protein antimicrobial activity. Biol Chem, 2012. 393(8): p. 801-15.