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

Study on the ability and mechanism of intracellular delivery of a recombinant enhanced green fluorescence protein(EGFP)fused with a cell-penetrating peptide, HBHAc, in a hepatocellular carcinoma cell line

康竣崴(Jyun-wei Kang)

指導教授 ： 沈麗娟

國立臺灣大學/醫學院/藥學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10950

摘要

肝素結合凝血附著素 (heparin-binding haemaglutinin adhesin，HBHA) 是一個源自於分枝桿菌的表面抗原蛋白，其中C端的片段負責著HBHA黏附於肺部表皮細胞與肺外擴散的功能，此C端的片段主要是由兩個片段重複排列組合而成：KKAAPA (R1) 和KKAAAKK (R2) ，本實驗室發現以1R1+2R2之25個胺基酸組合的胜肽，HBHAc，在人類肺臟上皮癌細胞 (A549) 與人類結腸癌細胞 (Caco-2) 具有極佳的結合、攝取與穿胞之能力。為了更進一步了解HBHAc作為蛋白質載體的能力，本研究以綠色螢光蛋白 (EGFP) 作為模式蛋白，利用EGFP的螢光活性來評估HBHAc遞送蛋白質進入細胞的能力，並進一步研究其在細胞內的分布途徑與被細胞攝取的相關機轉。
本研究利用E. coli BL21大量表現帶有histidine-tag的EGFP與HBHAc-EGFP重組蛋白後以nickel-nitrilotriactic acid (Ni-NTA) 作為親合性的材料來進行純化。以HepG2人類肝癌細胞株為本研究中體外試驗的給藥目標，首先，以MTT assay來研究EGFP或是HBHAc-EGFP重組蛋白是否會影響細胞存活率，接著，再以流式細胞儀與共軛焦螢光顯微鏡來分析並觀察HBHAc-EGFP進入細胞的效率，確認HBHAc-EGFP能有效地進入細胞後則進一步以免疫螢光分析法來觀察HBHAc-EGFP在細胞內的分布位置，並且藉由給予chloroquine來研究HBHAc-EGFP在細胞內是否會經由endolysosomal pathway，最後，利用溫度、胞吞作用抑制劑與膜作用競爭劑對HBHAc-EGFP進入細胞的影響來進一步研究HBHAc-EGFP的細胞攝取機轉。
研究結果顯示，本研究的純化方法可以有效地純化出高純度的EGFP與HBHAc-EGFP重組蛋白。首先，在有效的遞送濃度下，HBHAc-EGFP並不會對細胞的存活率造成明顯的影響；接著，在進入細胞的效率分析方面，以1 μM的給藥濃度培養1小時後，HBHAc-EGFP可進入超過95%的細胞，EGFP則是只有進入約3%左右的細胞，且投予HBHAc-EGFP的細胞平均螢光強度為投予EGFP的細胞平均螢光強度的10倍以上；另外，在1小時的給藥時間內，HBHAc-EGFP進入細胞的效率與給藥劑量成正相關性，細胞平均螢光強度亦與給藥時間成正相關性，在共軛焦螢光顯微鏡的結果中亦可觀察到此現象，但是在給藥2小時後，不論是在高濃度或是低濃度下，細胞平均螢光強度均有明顯下降的趨勢。
在細胞內的分布途徑方面，以共軛焦螢光顯微鏡可觀察到HBHAc-EGFP分布於細胞內的囊泡中，與early endosome antigen-1有明顯的交集，但未觀察到HBHAc-EGFP與caveolin-1有交集的現象；另外，chloroquine的給予可使已進入細胞內的HBHAc-EGFP在12小時後仍維持與起始時間相當的螢光強度，而未給予chloroquine的細胞平均螢光強度在12個小時後則是下降至原本的10%左右；以共軛焦螢光顯微鏡亦可觀察到給予chloroquine的組別在停藥後3小時細胞内仍有明顯的螢光訊號，並且呈現大範圍的聚集，而未給予chloroquine的組別在停藥後3小時細胞内則是幾乎無法偵測到螢光訊號。
在細胞的攝取機轉方面，在16℃和4℃的條件下，不論給藥劑量的高低，HBHAc-EGFP的相對細胞攝取量均有非常顯著的下降；不同的胞吞作用抑制劑對HBHAc-EGFP進入細胞均有不同程度的抑制效果，而其中抑制程度最高的為chlorpromazine，當給予濃度至20 μg/mL時會有40%左右的抑制效果；膜作用競爭劑 (heparin、dextran sulfate) 對HBHAc-EGFP的細胞攝取均有顯著的抑制效果，當給予濃度至30 μg/mL會有90%左右的抑制效果。
綜合以上研究結果，可推測HBHAc-EGFP會先與細胞膜上的醣蛋白產生作用而黏附於細胞膜上，主要經由clathrin-mediated endocytosis進入細胞，進入細胞後會分布於endosome中，並且經由endolysosomal pathway被降解，而此機制與HBHAc本身進入細胞的機制不同，過去的結果顯示，HBHAc可能會利用direct tranduction或是lipid-raft mediated endocytosis的途徑進入細胞。另外，膜作用競爭劑對HBHAc-EGFP進入細胞的抑制效果較HBHAc來得顯著。
HBHAc能有效地將EGFP遞送進入細胞内並維持EGFP的活性，並且在有效的遞送濃度內不會對細胞造成明顯的毒性，但由於HBHAc-EGFP主要會經由endolysosomal pathway被降解，因此，若要作為有效的蛋白質藥物遞送載體，可能必須配合適當的endosome escape策略使蛋白質藥物在細胞內得以發揮最大的效用。

關鍵字

肝素結合凝血附著素 ； 細胞穿透胜肽綠色螢光蛋白 ； 蛋白質胞內遞送 ； 胞噬作用

並列摘要

Heparin-binding hemagglutinin adhesin (HBHA) is a 28 kDa heparin-binding mycobacterial protein expressed on the surface of Mycobacterium tuberculosis. The C-terminal domain of HBHA dominates adherence to epithelial cells and mediates extra-pulmonary dissemination. The C-terminal region is identified with two repeated motifs : KKAAPA (R1) and KKAAAKK (R2) . In our previous study, one peptide with the sequence of 1R1+2R2 named HBHAc showed the good ability of cell binding, uptake and transport in A549 and Caco-2 cell line. To understandthe the ability and mechanism of HBHAc in protein delivery, enhanced green fluorescence protein (EGFP) fused with HBHAc was used in this study.
Fisrt, we used nickel-nitrilotriactic acid (Ni-NTA) to purify histidine tagged EGFP or HBHAc-EGFP overexpressed in E. coli BL21. The human hepatocellular carcinoma cell line, HepG2, was used as an in vitro model. We utilized MTT assay to examine whether the cell viability is affected by recombinant EGFP and HBHAc-EGFP, respectively. We further analyzed the uptake efficiency of HBHAc-EGFP by flow cytometry and confocal microscopy. Immunofluorescence assay was used to investigate the intracellular distribution of HBHAc-EGFP. We also applied chloroquine to investigate the escape of HBHAc-EGFP from endolysosomal pathway. The cellular uptake mechanism of HBHAc-EGFP was studied by studying the effect of different temperature, endocytosis inhibitors, and membrane interaction competitors.
Based on SDS-PAGE analysis, EGFP and HBHAc-EGFP recombinant protein was obtained with high purity after purification. No cytotoxicity of both protiens was found in HepG2 cells after 24 h incubation at concentration up to 10 μM. Based on the results of flow cytometry, HBHAc-EGFP efficienctly entered into above 95% of total cells and the mean fluorescence intensity was 25.43 a.u. after 1 h incubation at 1 μM concentration. On the contrary, EGFP only entered into 2% of total cells and the mean fluorescence intensity was 2.97 a.u. under the same condition. Based on the confocal microscopy images, after 1 h incubation at 10 μM concentration, HBHAc-EGFP treated cells showed obvious green fluorescence in the cytoplasm and barely detectable signals were observed in the EGFP treated cells. Furthermore, the transduction of HBHAc-EGFP occurred rapidly and the mean fluorescence intensity gradually intensified until about 1 h of incubation based on the results of flow cytometry and confocal microscopy. However, the mean fluorescence intensity decreased significantly after 2 h incubation.
According to the results of immunofluorescence assay, the internalized HBHAc-EGFP was co-localized with early endosome antigen-1 (EEA1) but was not co-localized with caveolin-1. In the endolyosomal blockade assay, HepG2 cells were treated with HBHAc-EGFP and chloroquine for 1 h and replaced with new medium with chloroquine for a period of time. The mean fluorescence intensity measured by flow cyometry remained until 12 h in chloroquine treated cells but dropped dramatically within 1 h in non-chloroquine treated cells. It suggests that chloroquine could effectively block the endolysosomal degradation of HBHAc-EGFP in HepG2 cells.
Furthermore, the cellular uptake of HBHAc-EGFP was significantly decreased when cells were incubated at 4℃ or 16℃. In the endocytosis inhibition assay, the entry of HBHAc-EGFP was inhibited by 40% in chlorpromazine treated cells, and methyl-β-cyclodextrin and amiloride only showed about 20% of inhibition. Besides, membrane competitors, heparin and dextran sulfate, greatly reduced the internalization of HBHAc-EGFP up to 90%.
These results suggest that the internalization of HBHAc-EGFP fusion protein in HepG2 cells is mostly via clathrin-mediated endocytosis through interaction with heparan sulfate proteoglycans on the plasma membrane. Furthermore, most internalized HBHAc-EGFP was distributed in the endosome and degraded through endolysosmal pathway. However, our previous study showed that HBHAc may utilize direct transduction or lipid-raft endocytosis. In addition, membrane interaction competitors inhibit the cellular uptake of HBHAc-EGFP more effectively than the cellular uptake of HBHAc.
HBHAc can effectively deliver EGFP into cells without disrupting the activity of EGFP and no obvious cytototoxicity was observed at the concentrations used for delivering EGFP. Due to the degradation of HBHAc-EGFP by endolysosomal pathway, appropriate endosomal escape strategies are needed for HBHAc to be used as a potiential protein carrier.

並列關鍵字

heparin-binding haemaglutinin adhesin ； cell-penetrating peptide ； green fluorescence protein ； protein delivery ； endocytosis

參考文獻

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[105] 邱予芹，研究肝素結合凝血附著素c之蛋白質運送能力，National Taiwan University (2010)

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