研究肝素結合凝血附著素c之蛋白質運送能力

邱予芹

doi:10.6342/NTU.2010.01293

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研究肝素結合凝血附著素c之蛋白質運送能力

Investigation on the protein delivery of heparin-binding haemagglutinin adhesin c（HBHAc）

邱予芹 , Masters　　Advisor：沈麗娟　　

繁體中文 DOI： 10.6342/NTU.2010.01293

肝素結合凝血附著素；細胞色素c ；基因重組精胺酸去亞胺酶連接體；雙硫鍵；硫醚鍵；融合蛋白；蛋白質遞送； heparin-binding haemagglutinin adhesin ； cytochrome c ； recombinant arginine deiminase ； conjugates ； disulfide ； thioether ； fusion protein ； protein delivery

Abstract │ Reference (315) │ Times Cited (1) │ Altmetrics

Abstract 〈TOP〉

肝素結合凝血附著素（heparin-binding haemagglutinin adhesin，HBHA）的c-terminal區域已被認為是主掌HBHA蛋白與宿主肺部細胞附著作用的主要區段，此c-terminal區域主要含lysine（K）、alanine（A）、proline（P）三種胺基酸，且由KKAAPA（R1）和KKAAAKK（R2）這兩段片段重複排列而成。過去本實驗室將R1、R2這兩種片段依不同組合合成出六段胜肽：HBHAa （3R1）、HBHAb（3R1+1R2）、HBHAc（1R1+2R2）、HBHAd（2R1+2R2）、HBHAe（2R2）和全長HBHA的c-terminal區域（3R1+2R2），以人類肺部上皮癌細胞（A549）與人類結腸腺癌細胞（Caco-2）作為研究穿胞運送的模式細胞。結果顯示HBHAc 為具有結合、攝取及穿胞能力之最短HBHA相關胜肽序列。在本研究中，使用cytochrome c（Cyt c）及基因重組精胺酸去亞胺酶（recombinant arginine deiminase，rADI）其分子量分別是12 kDa及92 kDa當作模式蛋白質，以化學鍵結的方式或是分生技術，研究HBHAc運送不同大小蛋白質的能力，進一步探究不同的鍵結方法對於運送的效果是否有不同的影響。
首先將HBHAc標記上FITC螢光分子，從共軛焦螢光影像發現，投予HBHAc一小時後，HBHAc進入細胞內且分布於細胞質及細胞核中。Cyt c為細胞凋亡中的一個重要中間者，從粒線體釋出至細胞質中的Cyt c 會誘發細胞的凋亡反應。我們利用SPDP及Sulfo-SMPB linker以化學鍵結的方式將Cyt c和HBHAc以可還原的雙硫鍵及不可還原的硫醚鍵連接在一起，並純化之。投予Cyt c-HBHAc conjugates一小時後，共軛焦螢光影像發現兩種Cyt c-HBHAc conjugates不僅分布於細胞質也在細胞核中；在生物活性方面，兩種Cyt c-HBHAc conjugates皆顯著地抑制A549、MCF-7、Caco-2的細胞存活率，且兩種Cyt c-HBHAc conjugates彼此間無顯著差異。然而， 使用流式細胞儀配合Propidium iodide 評估細胞凋亡現象， Cytc-SPDP-HBHAc conjugates對於細胞週期並無顯著的影響。
另一方面，使用分子量較大的rADI 當作模式蛋白，rADI 可將精胺酸（Arginine）反應成瓜胺酸（Citrulline）和胺（ammonia），被認為是有潛力的抗癌藥物。使用分生技術製備HBHAc-rADI 質體，並用管柱純化此融合蛋白，MTT 發現HBHAc-rADI 融合蛋白相較於rADI 單獨投予，顯著地抑制MCF-7 的細胞生長；在單劑量或多劑量模式的比較中，多劑量投予HBHAc-rADI 融合蛋白比單劑量投予更明顯地抑制細胞生長。使用Ultra Performance Liquid Chromatography（UPLC）評估HBHAc-rADI 融合蛋白對於精胺酸和瓜胺酸含量的影響。投予10 mU/mL rADI或HBHAc-rADI 融合蛋白四天後，rADI 與HBHAc-rADI 融合蛋白皆使得細胞外的精胺酸下降，瓜胺酸增加，兩者間無顯著差異；控制組、rADI、HBHAc-rADI 融合蛋白投予下的細胞內精胺酸含量彼此間皆無顯著差異；但細胞內的瓜胺酸在HBHAc-rADI 融合蛋白投予下顯著地高於rADI 單獨投予（p＜0.05）；以細胞內瓜胺酸與精胺酸的比值（citrulline/arginine）來看，HBHAc-rADI 融合蛋白投予下顯著地高於rADI（p＜0.01）及控制組（p＜0.01），但單獨投予rADI 與控制組間無顯著差異。由這樣的結果推論rADI 被HBHAc已融合蛋白的方式運送進入細胞後，將細胞內的精胺酸水解成瓜胺酸。
總結，我們發現HBHAc 可以運送大分子進入細胞質及細胞核；利用化學鍵結形成conjugates 或是分生技術製備融合蛋白，皆可以有效的連接HBHAc 和蛋白質並成功地應用在蛋白質遞輸上。

Parallel Abstract 〈TOP〉

The c-terminal domain of heparin-binding haemagglutinin adhesin（HBHA） hasbeen considered as the primary domain to adhere to hosts’ pulmonary cells. Thec-terminal domain contains lysine（K）, alanine（A）, and proline（P）, and is identifiedwith two different repeats, KKAAPA motif (R1) and KKAAAKK motif (R2). In thepast, we evaluated the characteristics of binding, uptake, and transport in six modifiedsequences of HBHA-related peptides, including HBHAa (3R1), HBHAb (3R1+1R2),HBHAc(1R1+2R2), HBHAd(2R1+2R2), HBHAe(2R2) and HBHA(3R1+2R2).HBHAc is the shortest peptide of HBHA related peptides with ability of binding, uptake, and transport in A549 and Caco-2 cells. Therefore, in this study, we demonstrated that HBHAc could deliver model proteins, cytochrome c（Cyt c）and recombinant arginine deiminase（rADI）, into cells by chemical linkages and molecular biological techniques. Firstly, HBHAc was labeled with fluorescein isothiocyanate (FITC) to investigate the intracellular distribution. After 1 h incubation of HBHAc in A549 cells, HBHAc was found within cytoplasm and nucleus by confocal image. The conjugations of HBHAc and a model protein Cyt c, Cyt c-SPDP-HBHAc and Cyt c-SMPB-HBHAc, were prepared by disulfide linkage and thioether linkage, respectively. Both of conjugates were not only detected within cytoplasm and nucleus after 1 h incubation, but also significantly inhibited cell proliferation in A549, MCF-7, and Caco-2 at 3.2 μM of Cyt c conjugates compared to Cyt c treatment alone by MTT assay. There was no significant difference in the effect of different conjugates on the inhibition of cell proliferation. We also used flow cytometry to evaluate the apoptotic effect of Cyt c-HBHAc conjugates. However, the Cyt c-SPDP-HBHAc conjugates had no significant effect on cell cycle. rADI is an enzyme which hydrolyze arginine to citrulline and ammonia, and has been studied as a potential anti-cancer drug for arginine-auxotrophic tumors. We constructed HBHAc-rADI plasmid and purified HBHAc-rADI fusion protein. HBHAc-rADI fusion protein significantly inhibited cell proliferation in rADI-resistant MCF-7 cells by MTT assay. Subsequently, the effect of intracellular delivery of HBHAc-rADI on the amount of arginine and citrulline in culture medium and intracellular fluid was investigated by Ultra Performance Liquid Chromatography (UPLC). After four-day treatment of rADI and HBHAc-rADI fusion protein at 10 mU/mL, respectively, the depletion of extracellular arginine and proportional increase of citruline were observed, and there was no significant difference between each treatment. The concentration of intracellular arginine was similar in the cells under the treatment of rADI and HBHAc-rADI fusion protein compared to the controls. However, the concentration of intracellular citrulline under the treatment of HBHAc-rADI fusion protein was significantly higher than the one under the treatment of rADI. The ratio of intracellular citrulline to intracellular arginine under the treatment of HBHAc-rADI fusion protein was significant higher than the one under the treatment of rADI. Therefore, the results indicate that rADI was successfully delivered into cells in the form fusing with HBHAc and reserved the biological activity in the hydrolysis of intracellular arginine to citrulline.  
In summary, we demonstrated that HBHAc can be used as a carrier for macromolecular delivery into cytoplasma and nucleus. Using chemical linkages and molecular biotechnology to produce fusion proteins of biological active proteins and HBHAc are both successfully applied in the delivery of proteins.

Reference ( 315 ) 〈TOP〉

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Times Cited (1) 〈TOP〉

康竣崴(2012)。含穿胞胜肽HBHAc之重組綠色螢光蛋白於肝癌細胞株的胞內運輸之研究。臺灣大學藥學研究所學位論文。2012。1-140。

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