Tectonin螺旋蛋白具有抵禦病原菌、寄生物及掠食者的能力，在先天免疫系統扮演重要的角色。分離自東方鱟(Tachypleus tridentatus)的血漿凝集素1 (TPL1) 為Tectonin螺旋蛋白家族的一員，具結合病原菌特有分子構造(pathogen associated molecular pattern)及細菌之結合能力。TPL1之胺基酸序列與圓尾鱟 (Carcinoscorpius rotundicaudata)半乳糖結合蛋白 (CrGBP) 相似程度高達96%，但預測之二級與三級結構及二者對於PAMP結合能力卻不盡相同。本研究以大腸桿菌表達重組TPL1，發現餵食含重組TPL1基因之大腸桿菌可使近60%線蟲生長發育遲滯並產生異常運動。此外鈣離子與半乳糖可提升大腸桿菌表現重組蛋白對壁脂酸(lipoteichoic acid)、脂多糖(lipopolysaccharide)及細菌的結合能力，更進一步抑制細菌生長。本論文首次證明重組東方鱟血漿凝集素1具有Tectonin蛋白家族特性，探討TPL1與醣類結合特性有助科學界瞭解東方鱟之抗菌機制及新型抗菌蛋白設計。

Tectonin is a group of beta-propeller lectin played important roles in innate immunity against pathogens, parasites and predators. Tachypleus plasma lectin 1 (TPL1) derived from Taiwanese Tachypleus tridentatus that has pathogen associated molecular pattern (PAMP) and bacteria binding activities. TPL1 shared 96% amino acid sequence identity with Carcinoscorpius rotundicaudata galactose binding protein (CrGBP) but they have distinct PAMP binding activities. In silico tertiary structure prediction showed that TPL1 and CrGBP revealed beta-propeller structure. In vivo nematotoxicity assay showed that TPL1 has Tectonin property such as inhibiting approximately 60% Caenorhabditis elegans larva development leading abnormally wriggled worm movement. In addition, recombinant TPL1 expressed in Escherichia coli enhanced lipoteichoic acid (LTA), lipopolysaccharide (LPS) and bacteria binding activities, as well as anti-bacterial activity upon addition of calcium ion and galactose. Taken together, recombinant TPL1 was firstly proven in vivo Tectonin property through in silico prediction and in vitro assay. Characterization of carbohydrate binding property of TPL1 may facilitate deciphering antimicrobial mechanism of horseshoe crab and engineering of novel proteins for antimicrobial application.

1. Rudkin, D.M., G.A. Young, and G.S. Nowlan, The oldest horseshoe crab: A new xiphosurid from Late Ordovician Konservat-Lagerstatten deposits, Manitoba, Canada. Palaeontology, 2008. 51: p. 1-9.
2. Walls, E.A., J. Berkson, and S.A. Smith, The horseshoe crab, Limulus polyphemus: 200 million years of existence, 100 years of study. Reviews in Fisheries Science, 2002. 10(1): p. 39-73.
3. Kawabata, S., T. Koshiba, and T. Shibata, The lipopolysaccharide-activated innate immune response network of the horseshoe crab. Isj-Invertebrate Survival Journal, 2009. 6(1): p. 59-77.
4. Iwanaga, S., S. Kawabata, and T. Muta, New types of clotting factors and defense molecules found in horseshoe crab hemolymph: Their structures and functions. Journal of Biochemistry, 1998. 123(1): p. 1-15.
5. Li, Y., et al., Optimising the use of commercial LAL assays for the analysis of endotoxin contamination in metal colloids and metal oxide nanoparticles. Nanotoxicology, 2015. 9(4): p. 462-73.
6. Kuo, T.H., et al., Ligand specificities and structural requirements of two Tachypleus plasma lectins for bacterial trapping. Biochem J, 2006. 393(Pt 3): p. 757-66.
7. Capaldi, S., et al., Three-dimensional structure and ligand-binding site of carp fishelectin (FEL). Acta Crystallogr D Biol Crystallogr, 2015. 71(Pt 5): p. 1123-35.
8. Gupta, G.S., Animal Lectins: Form, Function and Clinical Applications. 2012, Springer Science & Business Media.
9. Rabinovich, G.A., Y. van Kooyk, and B.A. Cobb, Glycobiology of immune responses. Ann N Y Acad Sci, 2012. 1253: p. 1-15.
10. Zhu, Y., et al., Diversity in lectins enables immune recognition and differentiation of wide spectrum of pathogens. Int Immunol, 2006. 18(12): p. 1671-80.
11. Kawabata, S. and T. Muta, Sadaaki Iwanaga: Discovery of the lipopolysaccharide- and beta-1,3-D-glucan-mediated proteolytic cascade and unique proteins in invertebrate immunity. J Biochem, 2010. 147(5): p. 611-8.
12. Saito, T., et al., A novel type of limulus lectin-L6. Purification, primary structure, and antibacterial activity. J Biol Chem, 1995. 270(24): p. 14493-9.
13. Corbett, K.D., R.K. Shultzaberger, and J.M. Berger, The C-terminal domain of DNA gyrase A adopts a DNA-bending beta-pinwheel fold. Proc Natl Acad Sci U S A, 2004. 101(19): p. 7293-8.
14. Yadid, I., et al., Metamorphic proteins mediate evolutionary transitions of structure. Proc Natl Acad Sci U S A, 2010. 107(16): p. 7287-92.
15. Saito, T., et al., A newly identified horseshoe crab lectin with binding specificity to O-antigen of bacterial lipopolysaccharides. J Biol Chem, 1997. 272(49): p. 30703-8.
16. Gokudan, S., et al., Horseshoe crab acetyl group-recognizing lectins involved in innate immunity are structurally related to fibrinogen. Proc Natl Acad Sci U S A, 1999. 96(18): p. 10086-91.
17. Low, D.H., et al., Molecular interfaces of the galactose-binding protein Tectonin domains in host-pathogen interaction. J Biol Chem, 2010. 285(13): p. 9898-907.
18. Chen, S.C., et al., Biochemical properties and cDNa cloning of two new lectins from the plasma of Tachypleus tridentatus: Tachypleus plasma lectin 1 and 2+. J Biol Chem, 2001. 276(13): p. 9631-9.
19. Ng, S.K., et al., A recombinant horseshoe crab plasma lectin recognizes specific pathogen-associated molecular patterns of bacteria through rhamnose. PLoS One, 2014. 9(12): p. e115296.
20. Chen, C.K., N.L. Chan, and A.H. Wang, The many blades of the beta-propeller proteins: conserved but versatile. Trends Biochem Sci, 2011. 36(10): p. 553-61.
21. Arnaud, J., et al., Membrane deformation by neolectins with engineered glycolipid binding sites. Angew Chem Int Ed Engl, 2014. 53(35): p. 9267-70.
22. Chaudhuri, I., J. Soding, and A.N. Lupas, Evolution of the beta-propeller fold. Proteins, 2008. 71(2): p. 795-803.
23. Low, D.H., Molecular interactions of tectonin proteins in host and pathogen recognition. 2009.
24. Fulop, V. and D.T. Jones, Beta propellers: structural rigidity and functional diversity. Curr Opin Struct Biol, 1999. 9(6): p. 715-21.
25. Huh, C.G., et al., Cloning and characterization of Physarum polycephalum tectonins. Homologues of Limulus lectin L-6. J Biol Chem, 1998. 273(11): p. 6565-74.
26. Wohlschlager, T., et al., Methylated glycans as conserved targets of animal and fungal innate defense. Proc Natl Acad Sci U S A, 2014. 111(27): p. E2787-96.
27. Low, D.H., et al., A novel human tectonin protein with multivalent beta-propeller folds interacts with ficolin and binds bacterial LPS. PLoS One, 2009. 4(7): p. e6260.
28. Nagai, T., et al., Purification, characterization, and amino acid sequence of an embryonic lectin in perivitelline fluid of the horseshoe crab. J Biol Chem, 1999. 274(53): p. 37673-8.
29. Kunzler, M., et al., Biotoxicity Assays for Fruiting Body Lectins and Other Cytoplasmic Proteins. Methods in Enzymology, Vol 480: Glycobiology, 2010. 480: p. 141-150.
30. Chiou, S.T., et al., Isolation and characterization of proteins that bind to galactose, lipopolysaccharide of Escherichia coli, and protein A of Staphylococcus aureus from the hemolymph of Tachypleus tridentatus. J Biol Chem, 2000. 275(3): p. 1630-4.
31. Petersen, T.N., et al., SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods, 2011. 8(10): p. 785-6.
32. Kelley, L.A. and M.J. Sternberg, Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc, 2009. 4(3): p. 363-71.
33. Chen, C.C., J.K. Hwang, and J.M. Yang, (PS)2-v2: template-based protein structure prediction server. BMC Bioinformatics, 2009. 10: p. 366.
34. Wu, S. and Y. Zhang, MUSTER: Improving protein sequence profile-profile alignments by using multiple sources of structure information. Proteins, 2008. 72(2): p. 547-56.
35. Yang, J., A. Roy, and Y. Zhang, Protein-ligand binding site recognition using complementary binding-specific substructure comparison and sequence profile alignment. Bioinformatics, 2013. 29(20): p. 2588-95.
36. San-Miguel, T., P. Perez-Bermudez, and I. Gavidia, Production of soluble eukaryotic recombinant proteins in E. coli is favoured in early log-phase cultures induced at low temperature. Springerplus, 2013. 2(1): p. 89.
37. Wellnitz, O., E.T. Arnold, and R.M. Bruckmaier, Lipopolysaccharide and lipoteichoic acid induce different immune responses in the bovine mammary gland. J Dairy Sci, 2011. 94(11): p. 5405-12.
38. Chang, C.F., et al., Rapid characterization of sugar-binding specificity by in-solution proximity binding with photosensitizers. Glycobiology, 2011. 21(7): p. 895-902.
39. Masilamani, M., S. Commins, and W. Shreffler, Determinants of food allergy. Immunol Allergy Clin North Am, 2012. 32(1): p. 11-33.
40. Marth, J.D. and P.K. Grewal, Mammalian glycosylation in immunity. Nat Rev Immunol, 2008. 8(11): p. 874-87.
41. Hsieh, C.L., Effects of protein glycosylation on structural variation, thermal stability and ligand specificities of two Tachypleus plasma lectins, in Institute of Biochemical Sciences. 2009, National Taiwan University.
42. Chauhan, V.M., et al., Mapping the pharyngeal and intestinal pH of Caenorhabditis elegans and real-time luminal pH oscillations using extended dynamic range pH-sensitive nanosensors. ACS Nano, 2013. 7(6): p. 5577-87.
43. Staudacher, E., Methylation--an uncommon modification of glycans. Biol Chem, 2012. 393(8): p. 675-85.
44. Hsieh, C.L., Effects of protein glycosylation on structural variation, thermal stability and ligand specificities of two Tachypleus plasma lectins. 2009, National Taiwan University.
45. Mukherjee, S., et al., Antibacterial membrane attack by a pore-forming intestinal C-type lectin. Nature, 2014. 505(7481): p. 103-7.
46. Lo, Y.H., Development of a novel bacterial detection system by engineering of starch binding domain and plasma lectin, in Institute of Molecular and Cellular Biology. 2008, National Tsing Hua University.
47. Low, E.L., Characterization of bacteria and ligand binding activities of recombinant Tachypleus plasma lectin 2, in Institute of Molecular and Cellular Biology. 2010, National Tsing Hua University.
48. T., H.Y., Functional Caracterization of Bacteria and Ligand Binding Activities of Recombinant Tachypleus Plasma Lectin, in Institute of Molecular and Cellular Biology. 2013, National Tsing Hua University.
49. Chen, Y.E., In silico identification and in vitro characterization of bacteria/ligand binding residues governing antibacterial function of recombinant horseshoe crab plasma lectin, in Institute of Molecular and Cellular Biology. 2015, National Tsing Hua University.