Investigation on the Characteristic of Heparin-Binding Haemagglutinin Adhesin (HBHA)-Related Peptides and their Transcytosis

肝素結合凝血附著素（heparin-binding haemagglutinin adhesin，HBHA）是來自結核菌 Mycobacterium tuberculosis 或 Mycobacterium bovis的表面抗原蛋白（antigenic protein），分子量約為28 kDa，已被認為是結核菌黏附於表皮細胞或在肺外散佈（extrapulmonary dissemination）的主要媒介。其C-terminal端區段主要含有lysine、alanine和proline三種胺基酸，並由兩個小片段（motif）：KKAAPA（R1）和KKAAAKK（R2）重複排列而成。由於此序列負責結核菌與細胞膜的交互作用，所以我們藉由研究胜肽進入細胞的能力來評估各motif所扮演的角色與重要性。經由合成的六段HBHA相關胜肽序列：胜肽A（3R1）、胜肽B（3R1+1R2）、胜肽C（1R1+2R2）、胜肽D（2R1+2R2）、胜肽E（2R2）和全長HBHA（3R1+2R2），純度經LC-MS檢測均符合95％以上。在安定性試驗中，我們以HPLC分析來探討胜肽在細胞共存下的完整性，一小時內胜肽仍維持其完整性，而在細胞共存六小時後胜肽會被代謝到只剩約40％，加入蛋白酶抑制劑後可減緩胜肽的降解。本研究中，以人類肺部癌細胞A549與人類結腸腺癌細胞Caco-2作為肺部與腸道細胞吸收的體外研究模式。我們利用I-125來標記各胜肽並以sephadex G10 層析法來純化I-125標記胜肽並計算濃度。將表皮細胞培養於24孔盤中，待其生長至八分滿後，給予不同濃度的I-125標記胜肽，分別培養於4℃與37℃下一小時來進行結合與攝取試驗。結果顯示全長HBHA在兩細胞株中均具有最高的結合與攝取能力，胜肽D和C次之，而胜肽A、B和E的量則相當低，並且只有胜肽C、D和HBHA的結合與攝取曲線在濃度約為3 μM時有呈現飽和的趨勢。此外，利用20倍過量的cold peptides和hot peptides同時治療細胞來進行專一性/非專一性試驗，發現胜肽C、D、HBHA均具有細胞攝取專一性，但其他胜肽均已失去了對Caco-2細胞的專一性。再者，我們以pulse-chase assay來測試胜肽C、D和HBHA，發現胜肽C的chase/pulse百分比在Caco-2細胞中能高達67.23％，多於其他胜肽，但在A549細胞中此三段胜肽的結果均相似，約為25-30％。此結果顯示在chase之後胜肽C留於Caco-2細胞中的量高於其他胜肽，且兩細胞間有不同的結果。為了進一步了解胜肽穿胞性質，我們將I-125標定胜肽置於Caco-2 cell monolayer transwell model的頂端，並於六小時之間在底端偵測胜肽含量。結果證實胜肽C具有最好的穿胞能力，在6小時的穿胞含量約為58.61 ± 3.57 pmole（9.76 ± 2.06％），高於胜肽D和HBHA且具有統計上差異。綜觀以上試驗結果，胜肽C為具有專一性結合、攝取與穿胞性質之最短HBHA相關胜肽序列。經過這些體外試驗篩選之後，我們選擇胜肽C作為後續研究攝取機制與結合接受器的主要胜肽。將胜肽C與fluorescein isothiocyanate (FITC) 鍵結後，經純化分離出FITC-胜肽C，並以confocal進行影像分析。在4℃下胜肽C會少量結合於A549細胞膜，然而在37℃下發現胜肽C會明顯地進入細胞並分布於細胞質中。此結果顯示胜肽C應是經由energy-dependent的途徑來進入細胞。另外，我們也利用了胞噬抑制劑，如：amiloride （鈉氫交換抑制劑）、methyl-β-cyclodextrin (MβCD) (cholesterol depleting agent) 和chloropromazine (CPZ) (clathrin-mediated endocytosis inhibitor)，與胜肽C同時給予細胞進行觀察。結果顯示MβCD抑制胜肽C攝取進入細胞最為顯著，而amiloride和CPZ僅少量減少其攝取的量。此外，heparin和sulfate dextran也會降低胜肽C約50-60％的細胞攝取作用。從這些實驗結果來看，我們推論胜肽C可和表皮細胞膜上的硫化醣類（sulfated carbohydrate）相似物產生交互作用，並經由energy- 和 lipid-rafts dependent 的胞噬途徑來進入細胞。因合成精胺酸去亞胺酶（recombinant arginine deiminase，rADI）能將環境中的精胺酸（arginine）催化水解產生瓜氨酸（citrulline）和氨（ammonia），並能抑制某些癌細胞的生長。我們利用其作為蛋白質模式藥物（protein model drug）來測試rADI-胜肽C結合體在細胞內運輸與穿胞遞輸的能力與效果。我們將rADI-胜肽C直接治療對rADI具有抗藥性的乳癌細胞株 MCF-7 （rADI-resistant cell line），發現其在治療四天後能抑制約30％的細胞生長。然而，我們以Caco-2/A375 co-culture系統來測試穿胞遞輸能力，但rADI-胜肽C結合體在給予transwell頂端後，並沒有顯著抑制底部A375細胞（rADI-sensitive cell line）的生長作用。納本研究結果，我們致力於分析HBHA相關胜肽對於表皮細胞的結合、攝取機制、以及細胞內和穿胞運輸能力，並利用體外細胞模式證實胜肽C（1R1+2R2）為具有專一結合、攝取與穿胞作用的最短序列胜肽，並能經由energy- 和lipid-rafts dependent 胞噬作用進入細胞。由此可知一個R1 motif和兩個R2 motifs在表皮細胞膜表面的交互作用與內化過程中扮演很重要的角色。同時我們也期望胜肽C能繼續研究其穿胞性質並發展成為未來大分子藥物傳遞的有效載體。Heparin-binding haemagglutinin adhesin (HBHA), a 28-kDa protein from Mycobacterium tuberculosis or Mycobacterium bovis, predominantly adheres to pulmonary epithelial cells and mediates extra-pulmonary dissemination. Its 36-mer C-terminal domain contains lysine, alanine, and proline, and is identified with two different repeats, KKAAPA motif (R1) and KKAAAKK motif (R2). Internalizated competence of HBHA was evaluated to understand the roles of motifs interacting with epithelial cells. Six modified sequences of HBHA-related peptides, peptide A (3R1), peptide B (3R1+1R2), peptide C (R1+2R2), peptide D (2R1+2R2), peptide E (2R2), and HBHA (3R1+2R2) were applied to the binding, uptake and transport studies. The peptides were synthesized with purity greater than 95% at LC-MS level. Subsequently, the peptides were radio-labeled with I125 by chloramine-T reaction and separated from free I125 by chromatography G10 in our laboratory. ulmonary carcinoma cell line, A549, and colorectal adenocarcinoma cell line, Caco-2, were used as in vitro models. After both cells reached 80% of confluence on 24-well plates, cells were incubated with each I125-labed peptide at 4℃ and 37℃ for an hr, for the binding and uptake studies, respectively. Full-length HBHA had the highest binding and uptake amounts in both cell lines, but peptide A, B, and E were quite a few of amounts. The binding and uptake curves of peptide C, D, and HBHA were saturated at concentration of about 3 μM. Additionally, we utilized excess cold peptides to compete with hot peptides in the specific/nonspecific assay, and found out peptide C, D, and HBHA had specific uptake, but others didn’t. Furthermore, we also used pulse-chase experiment to investigate whether they would stay inside the cells after entering. The chase/pulse percentage of peptide C was the highest one at about 67.23% to compare with peptide D and HBHA in Caco-2 cells, but they were similar at 25-30％ in A549 cells. In order to examination of transcytosis, we tested peptides with transport assay by Caco-2 cell monolayer on transwell model. The result exhibited peptide C had the supreme efficiency for six hrs at the amount of 58.61 ± 3.57 pmole （9.76 ± 2.06％） to cross through the Caco-2 cells. According to these results, peptide C was the shortest seguence of HBHA-related peptides with specific binding, uptake, and transcytosis capacity in epithelium cells. fter the selection of HBHA-related peptides, peptide C was chosen to be labled with fluorescein isothiocyanate (FITC) for further investigation. FITC-peptide C was puried by Sephadex G10 size exclusion column, and the fluorescein to peptide molar ratio (F/P) was about 4.1. The confocal image of FITC- peptide C at 4℃ was located on the A549 cell membrane, but it was strongly presented within the cytoplasma at 37℃. It was intimated that peptide C would internalize into cells through energy-dependent pathway. Moreover, we co-treated cells with peptide C and endocytosis inhibitors, such as amiloride (Na+-H+ exchanger inhibitor), methyl-β-cyclodextrin (MβCD) (cholesterol depleting agent), and chloropromazine (CPZ) (clathrin-mediated endocytosis inhibitor), and the uptake amount of peptide C was reduced more obviously with MβCD to compare with other treatments. In addition, heparin and sulfate dextran also decreased about 50-60％ of the internalization of peptide C. Above all, we suggested that peptide C could interact with sulfated carbohydrate analogs on the cell membrane, and internalize into cells through energy- and lipid-rafts dependent endocytosis pathway.ecombinant arginine deiminase (rADI) is a mycoplasma enzyme that catalyzes the imine hydrolysis of arginine to produce citrulline and ammonia, and has been studied as a potential anti-cancer drug for arginine-auxotrophic tumors. It was utilized as a protein model drug to research the ability of intracellular and transcellular delivery of peptide C. rADI was generated at our lab and conjugated with peptide C by disulfide bond. We treated rADI-peptide C with MCF-7 cell line which is resistant to rADI-treatment, could decrease cell proliferation to about 30％. However, when rADI-peptide C was also treated with Caco-2/A375 cells co-culture system as transcellular delivery model, it had no better effect by conjugation with peptide C. n summary, six HBHA-related peptides were investigated on their functionalities of interaction with cell membrane, and demonstrated that peptide C was the shortest sequence that still had binding, uptake, and transcytosis efficiency through lipid-rafts dependent endocytosis pathway. It indicates that the sequence which consists of one-unit R1 and two-unit R2 motifs plays an important role in cell surface binding and internalization. The further studies are required to evaluate the possibility of using peptide C as a peptide-based delivery system for therapeutic applications.圖目錄 iii寫表 v文摘要 vi一章 緒論 1.1. 生物製劑的發展與藥物遞輸 1.2. 細胞穿透胜肽 (cell-penetrating peptide，CPPs) 3.2.1. 背景和分類 3.2.2. 細胞內化過程（cell internalization and intracellular processing） 4.2.3. 製藥技術的應用（pharmaceutical application） 6.2.4. 藥物遞輸上的限制（limitation） 6.3. Heparin-binding haemagglutinin adhesin（HBHA） 7.3.1. 結核桿菌（Mycobacteria）的HBHA 蛋白 7.3.2. HBHA與細胞的交互作用（interaction） 8.3.3. HBHA 的extrapulmonary dissemination特性 10.3.4. HBHA胜肽片段的發現與辨識（identification） 10.3.5. HBHA 目前的發展與應用 11.3.6. 基因合成精胺酸去亞胺酶 11二章 實驗目的 13三章 實驗材料與方法 15.1. 實驗材料 15.2. 細胞培養 15.3. 胜肽合成（參考表 1） 16.4. 胜肽安定性試驗 16.5. I-125同位素標記胜肽 17.6. 表皮細胞的結合與攝取試驗 18.7. 穿胞運輸試驗（transport assay） 18.8. Pulse-chase assay 19.9. 專一性/非專一性試驗（specific/nonspecific assay） 19.10. 細胞毒性試驗（MTT assay） 20.11. 螢光素標記法（fluorescein labeling method） 20.12. 共軛焦螢光影像分析法 20.13. 胞噬作用試驗（endocytosis inhibitor assay） 21.14. 醣類競爭試驗（carbohydrate competing assay） 21.15. 原二色光譜（circular dichroism） 21.16. rADI-胜肽C結合體在MCF-7細胞的細胞內運輸 22.17. Caco-2/A375系統（co-culture）的細胞穿胞運輸 22.18. 其他試劑和緩衝液 23.19. 資料統計分析 23四章 實驗結果 24.1. HBHA相關胜肽的安定性 24.2. 在Caco-2和A549細胞中的結合與攝取試驗 24.3. 專一性/非專一性結合與攝取試驗 25.4. 動力學試驗 26.5. Pulse-chase assay和Caco-2細胞的穿胞運輸試驗 27.6. 細胞毒性試驗 28.7. 胜肽C的結合與攝取螢光影像圖 28.8. HBHA和胜肽C的胞噬作用試驗與醣類競爭試驗 30.9. 胜肽C的原二色光譜 31.10. rADI-胜肽C結合體的細胞內運輸和穿胞運輸試驗 31五章 討論 33.1. HBHA相關胜肽的安定性 33.2. HBHA相關胜肽與表皮細胞結合、攝取和專一性的篩選 34.3. HBHA相關胜肽的動力學與穿胞運輸性質 36.4. 胜肽C的內化機制與細胞膜的作用 38.5. HBHA相關胜肽在Caco-2細胞與A549細胞間的差異 40.6. rADI-胜肽C結合體的細胞內運輸和穿胞運輸 41.7. HBHA相關胜肽作為細胞內藥物遞輸載體之發展潛力 43.8. 未來研究方向 43.8.1. 確立胜肽C穿胞運輸的能力 43.8.2. 胜肽C對於肺部細胞的選擇性（selectivity） 44.8.3. 胜肽C細胞內的target胞器 44.9. 本研究之實驗限制 45六章 結論 46七章 參考文獻 9