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  • 學位論文

以親合質譜術研究白細胞介素2與其受體的作用

Study of the Interaction between Interleukin-2 with Its Alpha Receptor by Nanoprobe-Based Affinity Mass Spectrometry

指導教授 : 吳文桂 陳玉如
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摘要


白細胞介素2 (interleukin-2, IL-2)是受到活化T細胞所產生的重要細胞激素,透過與其受體結合調控著細胞傳遞訊息的功能。具有高親合力的白細胞介素2受體是分別由α (CD25)、β、和γ三條長胜肽鏈所組成,曾有文獻推測白細胞介素2辨識其受體的關鍵組成是特定序列的胜肽鏈與多甘露糖(high mannose)鏈。本論文主要以親合質譜術來研究白細胞介素2與其受體結合的作用。在論文的第一部分是利用分子生物技術來大量表達重組白細胞介素2 (rIL-2)作為探針。將卵白蛋白(ovalbumin)上的多甘露糖(Man-5-Asn)以共價鍵結裝配於磁性奈米粒子表面作為探針,可分離白細胞介素2且直接以基質輔助雷射脫附游離飛行式質譜儀(MALDI-TOF MS)分析,由質譜圖可證明Man-5-Asn能專一地分離出白細胞介素2,此結果也再次確認多甘露糖在白細胞介素2辨識其受體中扮演重要的角色。接著在尋找與Man-5-Asn結合的決定位(epitope)實驗中,我們發現白細胞介素2的胺基酸序列中79到94可能參與Man-5-Asn的辨識及結合,此實驗結果也與最近文獻中X-光繞射所得到結構具有一致性,將更進一步的利用競爭實驗和醣晶片技術去確認決定位的正確性。我們證明了利用奈米探針結合質譜分析可有效的研究醣和蛋白質之間的作用,並可快速鑑定醣和蛋白質的結合位置。 最後實驗部分是將重組白細胞介素2與奈米粒子共價鍵結(MNP@rIL-2)作為探針,從CTLL-2細胞株分離出受體蛋白CD25。利用離心分離技術從CTLL-2細胞株取得膜蛋白且將膜蛋白與MNP@rIL-2反應,當CD25與重組白細胞介素2反應後,利用磁鐵可分離奈米粒子複合體且將其以西方點墨法分析之;雖然經過MNP@rIL-2萃取後並沒有成功的偵測到CD25的訊號,我們推測失敗的因素可能有下列兩點:第一,合成MNP@rIL-2的過程中需要更穩定rIL-2的生物活性。第二,分離膜蛋白的過程中視否改變了白細胞介素2受體固有的生物結構。如何維持白細胞介素2與其受體上的醣之間微弱親合力將是未來實驗修正的方向。

並列摘要


Interleukin-2 (IL-2) is an important cytokine synthesized by activated T cell and well known as a mediator of cellular signalling through interaction with its receptor, IL-2R. The high affinity IL-2R is a heterotrimer composed of α, β, and γ-polypeptide chains. Specific peptide sequence and high mannose-type oligosaccharides on IL-2Rα (CD25) play a critical role in the IL-2 recognition to IL-2R. In this thesis, we aim to develop new methodology, nanoprobe-based affinity mass spectrometry (NBAMS), to study the interaction between IL-2 and its receptor. In the first part of the thesis, rIL-2 was successfully expressed in E. coli to serve as a probe protein. Oligomannose-5-Asn glycan (Man-5-Asn) prepared from ovalbumin was covalently conjugated on magnetic nanoparticle (MNP@Man-5-Asn) to simultaneously isolate rIL-2 for the analysis of MALDI-TOF MS without an additional elution step. The clean spectrum showed the specific and efficient isolation of rIL-2 by MNP@Man-5-Asn. The results confirmed the importance of oligomannose for rIL-2 recognition by its receptor. Subsequent mapping the binding sequence of Man-5-Asn-interacting epitope revealed that peptide H79-L94 in rIL-2 potentially participates in the rIL-2 recognition of Man-5-Asn. Our results were consistent with the complex structure of IL-2—receptor that was recently determined by X-ray crystallography. In order for further validation, competition assay and carbohydrate array technology were also performed on few synthetic peptide sequences. We demonstrated the combination of functionalized nanoprobe and mass spectrometry provides efficient analysis for probing the carbohydrate-protein interaction and mapping the epitope sequences. In addition, rIL-2 encapsulated nanoparticles (MNP@rIL-2) was used to as a probe to isolate CD25 from CTLL-2 cell. Membrane fraction from CTLL-2 cells was prepared by centrifugation and incubated with MNP@rIL-2. After glycoprotein-protein interaction, nanoparticles/ bound protein complexes were isolated by magnet and analyzed by Western blotting. So far, we failed to detect CD25 signal after MNP@rIL-2 enrichment. We speculate two possibilities of failure of detection. First, the biological activity of rIL-2 on MNP@rIL-2 may require further optimization by specific immobilization of IL-2 with active conformation. Secondly, the extraction process of membrane fraction may change the native structure of CD25 and/or the β and γ-polypeptide chains. How to maintain the weak IL-2 —oligomannose receptor interaction remain future study.

參考文獻


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