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

• Main Authors: Jyun-wei Kang, 康竣崴
• Other Authors: Li-Juan Shen
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/80887530580094398915

碩士 === 國立臺灣大學 === 藥學研究所 === 101 === 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.