研究 D-肝素結合凝血附著素c (D-HBHAc) 胜肽之安定性及蛋白質運輸能力

Investigation on the Stability and Protein delivery of D form Heparin-Binding Haemagglutinin Adhesin c (D-HBHAc) peptide

陳瑩倫(Ying-Luen Chen)

指導教授 ： 沈麗娟

國立臺灣大學/醫學院/藥學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.00431

摘要

肺結核桿菌 (Mycobacterium tuberculosis) 中的肝素結合凝血附著素 (heparin-binding haemaglutinin adhesin , HBHA) C端區域是HBHA蛋白黏合上宿主肺部表皮細胞的重要區段，且主宰了肺結核桿菌的肺外擴散。此C端區域主要由兩個片段重覆排列而成：KKAAPA (R1) 和 KKAAAKK (R2)。過去，實驗室將兩片段依不同排列組合合成出六段胜肽：HBHAa (3R1)、HBHAb (3R1+1R2)、HBHAc (1R1+2R2)、HBHAd (2R1+2R2)、HBHAe (2R2)及HBHA (3R1+2R2)，並研究其結合、攝取及穿胞能力的不同。結果顯示，HBHAc在人類肺臟上皮癌細胞 (A549) 與人類結腸癌細胞 (Caco-2) 中，具有極佳的結合、攝取及穿胞能力之最短HBHA相關胜肽；然而，與A549細胞共同培養時，HBHA胜肽的安定性不佳，故為了克服胜肽被降解的問題，本研究將HBHAc胜肽序列上的L-form胺基酸置換成D-form，並比較L- 及D-HBHAc之間的安定性、穿胞能力、細胞內化機制和蛋白質遞送能力。
　　本研究以人類肺臟上皮癌細胞 (A549) 與人類乳腺癌細胞 (MCF-7) 作為體外研究模式。首先，在適當的蛋白質遞送濃度下，L- 及D-HBHAc並不具有細胞毒性，即使分別投予10-100倍遞送濃度的兩段胜肽，對A549的細胞存活率仍可維持80 – 90%以上；利用逆相高效能液相層析儀分析L- 及D-HBHAc胜肽於細胞培養液中的安定性，發現D-HBHAc在6、16、24小時條件培養液中的安定性，確實優於L-HBHAc，在培養2小時於16小時條件培養液中，L-HBHAc已降解至31.58%，而D-HBHAc仍維持83.55%的完整性。幸運地，D-HBHAc仍保有和L-HBHAc相當的穿胞能力；胜肽濃度對於L- 及D-HBHAc細胞攝取之間的關係，在同種細胞株中，兩段胜肽間並無顯著地不同。
　　再來，MβCD會顯著地降低L- 及D-HBHAc的細胞攝取，表示兩段胜肽的細胞內化可能與lipid-rafts相關的胞噬作用有關。然而，這兩段胜肽在不同細胞株中的表現並不完全相同。L- 及D-HBHAc胜肽在A549中為部分專一性攝取，但在MCF-7細胞中卻多為非專一性。在A549中，肝素 (heparin) 和硫酸葡聚醣 (dextran sulfate) 確實會降低L- 及D-HBHAc胜肽的細胞攝取，但這個現象在MCF-7細胞中並未觀察到。隨著培養溫度的降低 (攝氏16度及4度)，L- 及D-HBHAc胜肽在MCF-7中的細胞攝取亦隨著下降，然而，在A549細胞中，L- 及D-HBHAc胜肽於攝氏16與37度時的細胞攝取，僅有些微差異。
　　基因重組精胺酸去亞胺酶 (recombinant arginine deiminase，rADI) 雖對大部分肝癌及黑色素癌細胞具有抑制生長之作用，但仍有部分癌細胞對rADI具有抗性，如MCF-7細胞株。為了提高這些抗性癌細胞對rADI的敏感性，故將rADI遞送入細胞中，不僅清除了細胞外的精胺酸也清除了細胞內的。本研究利用rADI作為模式蛋白，研究L- 及D-HBHAc胜肽的蛋白質遞送能力。而細胞內rADI的遞送和細胞存活率分別用流式細胞儀和MTT (二甲基硫醇二苯基四唑溴) 試驗來分析。結果顯示，D-HBHAc-SPDP-rADI比L-HBHAc-SPDP-rADI將更多的rADI運送入MCF-7細胞且顯著地抑制了MCF-7細胞的生長。接著，以超高效能液相層析儀分析細胞內胺基酸的變化，相對於單獨投予rADI，L- 及D-HBHAc接上rADI後，均使得瓜胺酸與精胺酸的比值增加，但這兩種接合體之間並無顯著差異。
　　總結，本研究指出，在三種不同的條件細胞培養液中，D-HBHAc的安定性優於L-HBHAc。而L- 及D-HBHAc胜肽的細胞攝取專一性與否、與細胞膜之間存在的交互作用及受到溫度的影響，與細胞株的不同有關。利用化學性鍵結胜肽及具有活性的rADI蛋白，可以有效地被運用在蛋白質地遞送，且似乎能夠增加MCF-7對rADI的敏感性。

關鍵字

肝素結合凝血附著素 ； 細胞穿透胜肽d-胺基酸取代 ； 胞噬作用 ； 基因重組精胺酸去亞胺酶蛋白質遞送

並列摘要

The c-terminal domain of heparin-binding haemaglutinin adhesin (HBHA), a protein from Mycobacterim tuberculosis, has been considered as the crucial domain to adhere to hosts’ pulmonary epithelial cells and mediate extra-pulmonary dissemination. The c-terminal region is identified with two repeated motifs: KKAAPA (R1) and KKAAAKK (R2). In the past, we investigated the characteristics of binding, uptake, and transport in six modified sequences 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 good ability of binding, uptake and the best ability of transport in A549 and Caco-2 cell models. However, HBHA showed the instability when incubated with A549 cells. In order to overcome the degradation issue, we replaced the L-amino acids with the corresponding D-amino acids of HBHAc. And study the stability, cell-penetrating ability, internalization mechanism, and protein delivery ability between L-HBHAc and D-HBHAc.
Human lung carcinoma epithelial cell line, A549, and human breast adenocarcinoma cell line, MCF-7, were used as in vitro models. First, the stability of L- and D-HBHAc in cell culture supernatant was measured by reverse-phase high performance liquid chromatography (RP-HPLC). We found that L- and D-HBHAc showed non-cytotoxicity at protein delivery concentrations. Even when using 10 -100 fold higher concentrations, L- and D-HBHAc only slightly impaired the cell proliferation in A549. D-HBHAc showed better stability than L-HBHAc in 6, 16, 24 hr-conditional medium. After 2 hr incubation time, the intact L-HBHAc was 31.58% and the intact D-HBHAc still retained 83.55% in 16 hr-conditional medium. Fortunately, D-HBHAc kept the cell-penetrating ability of L-HBHAc. No significant differences were observed on the cellular uptake profiles between these two peptides in A549 or MCF-7.
In addition, the cellular uptake of L- and D-HBHAc was significantly decreased with MβCD treatment in A549 and MCF-7 cells. It indicated that L- and D-HBHAc internalize into cells through lipid-rafts dependent endocytosis pathway. However, L- and D-HBHAc had 20% specific uptake of total uptake in A549 cells, but not in MCF-7 cells. Moreover, heparin and dextran sulfate decreased about 50% of the internalization of L- and D-HBHAc in A549. Nevertheless, the phenomenon was not observed in MCF-7 cells. When decreasing the incubation temperature to 16 and 4℃, the cellular uptake of L- and D-HBHAc were significantly reduced in MCF-7. However, the cellular uptake of L- and D-HBHAc only slightly decreased at 16℃ compared to that at 37℃ in A549.
Some cancer cells were resistant to recombinant arginine deiminase (rADI), used as an anti-cancer treatment in clinical trials for a range of cancers. To increase the sensitivity of rADI in the resistant cancer cells, intracellular delivery of rADI has been tested by deprivation not only extracellular arginine but also intracellular arginine. We utilized rADI as a protein drug model to investigate the intracellular delivery ability between L- and D-HBHAc. We separately treated L- and D-HBHAc-SPDP-rADI conjugates with rADI-resistant MCF-7. And the intracellular rADI delivery and cell viability were measured by flow cytometry and MTT (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H- tetrazolium bromide) assay, respectively. D-HBHAc-SPDP-rADI conjugates significantly increased intracellular rADI delivery and inhibited cell proliferation compared with L-HBHAc-SPDP-rADI conjugates. Subsequently, the intracellular amino acids diversification was performed by UPLC (Ultra Performance Liquid Chromatography). The ratio of intracellular citrulline to arginine under the treatment of L- and D-HBHAc-SPDP-rADI conjugates was significant higher than the one under the treatment of rADI alone. However, there’s no significant difference in citrulline to arginine ratio between these two kinds of conjugates.
In conclusion, we demonstrated D-HBHAc showed better stability than L-HBHAc in three different conditional media. The specific uptake, cell membrane interaction, and temperature effects of L- and D-HBHAc were related to cell types. Using chemical linkage to produce peptides and bioactive rADI conjugates is successfully applied in the delivery of proteins and might restore the sensitivity of rADI in MCF-7.

並列關鍵字

heparin-binding haemaglutinin adhesin ； cell-penetrating peptides ； d-amino acids replacement ； endocytosis ； recombinant arginine deiminase ； protein delivery

參考文獻

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