| Summary: | 碩士 === 國立清華大學 === 生物資訊與結構生物研究所 === 95 === 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.
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