大腸桿菌和霍亂弧菌的忌熱腸毒素(LT)在結構上屬於AB5毒素，是由一個A次單元和一個五聚體B次單元組成。有許多研究表明忌熱腸毒素具有強大的佐劑活性。第二型b型忌熱腸毒素的B次單元(LT-IIb-B5)具有與Toll-like receptor (TLR-2/1)和GD1a神經節苷脂結合的能力，藉此誘導NF- ĸB的反應途徑並能夠增強先天免疫反應。基於該機制LT-IIb-B5顯示出誘導保護性粘膜免疫的潛力。先前的研究已經指出LT-IIb-B5表面上的特定GD1a結合位點，我們推測藉由與GD1a結合力的調節可以改變誘導的免疫反應性。因此我們設計LT-IIb-B5環狀排列重組蛋白(CP)來調節LT-IIb-B5與 GD1a反應的親和力。我們希望能透過這種方式使新的蛋白質具有減弱的GD1a結合力和足夠的TLR-2/1親和力，以降低細胞毒性和維持免疫反應。本篇研究透過圓偏光二色光譜、核磁共振光譜和X射線晶體學來評估候選物的結構特性。此研究可以滿足開發一種新型、高效、安全黏膜佐劑的迫切需求。我們的結果可能有助於未來流感和其他呼吸道疾病的粘膜疫苗開發。

The heat-labile enterotoxins (LTs) of Escherichia coli and Vibrio cholerae structurally belong to AB5-toxin that consists of an A-subunit and a pentameric B-subunit. LTs have been comprehensively reported for their strong adjuvant activities. Among the two components, the B-subunit of type II LT (named LT-IIb-B5) shows the ability in recognizing Toll-like receptors (TLR-2/1) and GD1a ganglioside, which are involved in the activation of the NF-ĸB pathway and with the ability to boost innate immune response. Based on the mechanism, LT-IIb-B5 showed the potential to induce protective mucosal immunity. Previous research revealed the specific GD1a binding sites on the LT-IIb-B5 surface. We expect that the modulation of GD1a binding could change the induced immunoreactivity. Therefore, we designed LT-IIb-B5 circular permutants (CPs) to modulate the binding effect. We hope to have a new protein with decreased GD1a binding ability and sufficient TLR-2/1 affinity to reduce cell toxicity and maintain immune response. Several CP candidates with good structural stability were characterized in the study. Circular dichroism, NMR, and X-Ray crystallography are employed to evaluate the structural property of the candidates. The study might satisfy the urgent need for the development of a novel, efficient and safe mucosal adjuvant for influenza and other respiratory diseases in the future.

中文摘要 I
Abstract II
Acknowledgement III
Abbreviations IV
Content V
1. Introduction 1
1.1 The general function and structure of Escherichia coli heat-labile enterotoxins 1
1.2 Adjuvant activities of LT-IIb-B5 2
1.3 Mucosal adjuvant and the side effect derived by heat-labile enterotoxin 2
1.4 GD1a binding site in LT-IIb-B5 3
1.5 Aim of the study 4
2. Materials and methods 5
2.1 Construct design of LT-IIb-B5 circular permutations 5
2.2 Expression and purification of LT-IIb-B5 CPs 5
2.3 15N labeled sample for NMR HSQC spectroscopy 6
2.4 Circular dichroism 7
2.5 X-ray data collection, processing, and determination 7
2.6 Ellman‘s Assay 8
3. Results 9
3.1 Circular permutation of LT-IIb-B5 9
3.2 Purification and conformation of LT-IIb-B5 10
3.3 The structural stability comparison of CP constructs 12
3.4 Crystal structure 14
3.5 The binding of GD1a to CP constructs 16
4. Discussion 17
References 21
Figures 24
Figure 1. Model display differential on NF-ĸB activation by LT-IIb holotoxin and its B pentamer 24
Figure 2. Neu5Ac-nLT binding region of LT-IIb-B5 25
Figure 3. Close view for Neu5Ac-nLT primary and secondary binding site 26
Figure 4. Prediction of potential circular permutation sites by CPred 27
Figure 5. SDS-PAGE and FPLC size exclusion chromatography of LT-IIb-B5 28
Figure 6. FPLC size exclusion chromatography comparison of LT-IIb-B5 and circular permutants 29
Figure 7. SDS-PAGE of LT-IIb-B5 and CP construct samples in the presence and absence of β-ME 29
Figure 8. NMR HSQC spectra of LT-IIb-B5 and CP constructs 30
Figure 9. Circular dichroism spectroscopy of LT-IIb-B5 and circular permutants at different temperatures 31
Figure 10. Crystals of CP constructs 32
Figure 11. Pentameric structure and partial electron density of CP13-14 33
Figure 12. Structural comparison of CP13-14 and native LT-IIb-B5 34
Figure 13. Close-up view of Trp92 from each monomer 35
Figure 14. Pentameric structure and partial electron density of CP52-53 36
Figure 15. Structural Comparison of CP52-53 and native LT-IIb-B5 37
Figure 16. HSQC spectra of CP13-14 titrating with GD1a 38
Figure 17. HSQC spectra of CP52-53 titrating with GD1a 39
Figure 18. Structural comparison of CP constructs and LT-IIb-B5 complexed with Neu5Ac-nLT 40
Tables 41
Table 1. The sequences of LT-IIb-B5 and CP constructs. 41
Table 2. Ellman’s assay of LT-IIb-B5 and CP constructs. 42
Appendix 43
Appendix 1. The temperature dependence of the absorbance at 208 nm from LT-IIb-B5 and CP constructs 43
Appendix 2. Data collection and refinement statistics 44

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