Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

A Monoclonal Antibody That Specifically Binds Chitosan In Vitro and In Situ on Fungal Cell Walls

  • Schubert, Max (Fraunhofer Institute for Molecular Biology and Applied Ecology (IME)) ;
  • Agdour, Siham (Fraunhofer Institute for Molecular Biology and Applied Ecology (IME)) ;
  • Fischer, Rainer (Fraunhofer Institute for Molecular Biology and Applied Ecology (IME)) ;
  • Olbrich, Yvonne (RWTH Aachen University, Institute for Molecular Biotechnology) ;
  • Schinkel, Helga (Fraunhofer Institute for Molecular Biology and Applied Ecology (IME)) ;
  • Schillberg, Stefan (Fraunhofer Institute for Molecular Biology and Applied Ecology (IME))
  • Received : 2010.01.05
  • Accepted : 2010.05.07
  • Published : 2010.08.28
Download PDF
⟨ Previous Next ⟩

Abstract

We report the generation of the first monoclonal antibody that specifically binds to the polysaccharide chitosan. Mice were immunized with a mixture of chitosans, and hybridoma clones were screened for specific binders, resulting in the isolation of a single clone secreting a chitosan-specific IgM, mAbG7. In ELISAs, the antibody could bind to chitosans of varying composition, but demonstrated the highest affinity for chitosans with lower degrees of acetylation (DA) and very poor binding to chitin. We tested the ability of the antibody to bind to chitosan in situ, using preparations of fungal cell walls. Immunofluorescence microscopy confirmed that the antibody bound strongly to the cell walls of fungi with high levels of chitosan, whereas poor staining was observed in those species with cell walls of predominantly chitin or cellulose. The potential use of this antibody for the detection of fungal contamination and the protection of plants against fungal pathogens is discussed.

Keywords

References

  1. Adorini, L. 1991. Peptide interactions with MHC class II molecules. Br. J. Rheumatol. 30 Suppl 2: 10-13. https://doi.org/10.1093/rheumatology/30.1.10
  2. Bakkers, J., J. W. Kijne, and H. P. Spaink. 1999. Function of chitin oligosaccharides in plant and animal development. EXS 87: 71-83.
  3. Bartnicki-Garcia, S. and W. J. Nickerson. 1962. Isolation, composition, and structure of cell walls of filamentous and yeast-like forms of Mucor rouxii. Biochim. Biophys. Acta 58: 102-119. https://doi.org/10.1016/0006-3002(62)90822-3
  4. Briza, P., A. Ellinger, G. Winkler, and M. Breitenbach. 1988. Chemical composition of the yeast ascospore wall. The second outer layer consists of chitosan. J. Biol. Chem. 263: 11569-11574.
  5. Cherif, M., N. Benhamou, and R. R. Belanger. 1993. Occurrence of cellulose and chitin in the hyphal walls of Pythium ultimum - a comparative study with other plant pathogenic fungi. Can. J. Microbiol. 39: 213-222. https://doi.org/10.1139/m93-030
  6. Cohen-Kupiec, R. and I. Chet. 1998. The molecular biology of chitin digestion. Curr. Opin. Biotechnol. 9: 270-277. https://doi.org/10.1016/S0958-1669(98)80058-X
  7. Coligan, J. E., A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strobel. 2000. Current Protocols in Immunology. Wiley, NY.
  8. Comer, F. I., K. Vosseller, L. Wells, M. A. Accavitti, and G. W. Hart. 2001. Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine. Anal. Biochem. 293: 169-177. https://doi.org/10.1006/abio.2001.5132
  9. Duvick, J. P., T. Rood, A. G. Rao, and D. R. Marshak. 1992. Purification and characterization of a novel antimicrobial peptide from maize (Zea mays L.) kernels. J. Biol. Chem. 267: 18814-18820.
  10. El Gueddari, N. E., U. Rauchhaus, B. M. Moerschbacher, and H. B. Deising. 2002. Developmentally regulated conversion of surface-exposed chitin to chitosan in cell walls of plant pathogenic fungi. New Phytol. 156: 103-112. https://doi.org/10.1046/j.1469-8137.2002.00487.x
  11. Erwin, D. C. and O. K. Ribeiro. 1996. Phytophthora Diseases Worldwide. American Phytopathological Society., St. Paul, MN, U.S.A.
  12. Hadwiger, L. A., J. M. Beckman, and M. J. Adams. 1981. Localization of fungal components in the pea-Fusarium interaction detected immunochemically with anti-chitosan and anti-fungal cell-wall antisera. Plant Physiol. 67: 170-175. https://doi.org/10.1104/pp.67.1.170
  13. Hammerling, G. J. and U. Hämmerling. 1981. Production of antibody-producing hybridomas in the rodent system. Res Monographs Immunol. 3: 563-587.
  14. Harrington, B. J. and G. J. Hageage. 2003. Calcofluor white: A review of its uses and applications in clinical mycology and parasitology. Lab. Med. 34: 361-367. https://doi.org/10.1309/EPH2TDT8335GH0R3
  15. Kasaai, M. R. 2009. Various methods for determination of the degree of N-acetylation of chitin and chitosan: A review. J. Agric. Food Chem. 57: 1667-1676. https://doi.org/10.1021/jf803001m
  16. Khalaf, S. A. 2004. Production and characterization of fungal chitosan under solid-state fermentation conditions. Int. J. Agric. Biol. 6: 1033-1036.
  17. Khor, E. and L. Y. Lim. 2003. Implantable applications of chitin and chitosan. Biomaterials 24: 2339-2349. https://doi.org/10.1016/S0142-9612(03)00026-7
  18. Kim, S. Y., D. H. Shon, and K. H. Lee. 2000. Enzyme-linked immunosorbent assay for detection of chitooligosaccharides. Biosci. Biotechnol. Biochem. 64: 696-701. https://doi.org/10.1271/bbb.64.696
  19. Knorr, D. 1991. Recovery and utilization of chitin and chitosan in food-processing waste management. Food Technol. 45: 114-123.
  20. Kumar, M. N. V. R. 2000. A review of chitin and chitosan applications. React. Funct. Polym. 46: 1-27. https://doi.org/10.1016/S1381-5148(00)00038-9
  21. Kwak, B. Y., B. J. Kwon, C. H. Kweon, and D. H. Shon. 2004. Detection of Aspergillus, Penicillium, and Fusarium species by sandwich enzyme-linked immunosorbent assay using mixed monoclonal antibodies. J. Microbiol. Biotechnol. 14: 385-389.
  22. Lamarque, G., J. M. Lucas, C. Viton, and A. Domard. 2005. Physicochemical behavior of homogeneous series of acetylated chitosans in aqueous solution: Role of various structural parameters. Biomacromolecules 6: 131-142. https://doi.org/10.1021/bm0496357
  23. Latge, J. P. 2007. The cell wall: A carbohydrate armour for the fungal cell. Mol. Microbiol. 66: 279-290. https://doi.org/10.1111/j.1365-2958.2007.05872.x
  24. Li, H. P., J. B. Zhang, R. P. Shi, T. Huang, R. Fischer, and Y. C. Liao. 2008. Engineering Fusarium head blight resistance in wheat by expression of a fusion protein containing a Fusariumspecific antibody and an antifungal peptide. Mol. Plant Microbe Interact. 21: 1242-1248. https://doi.org/10.1094/MPMI-21-9-1242
  25. Liao, Y. C., F. Kreuzaler, R. Fischer, H. J. Reisener, and R. Tiburzy. 1994. Characterization of a wheat class Ib chitinase gene differentially induced in isogenic lines by infection with Puccinia graminis. Plant Sci. 103: 177-187. https://doi.org/10.1016/0168-9452(94)90206-2
  26. Merzendorfer, H. and L. Zimoch. 2003. Chitin metabolism in insects: Structure, function and regulation of chitin synthases and chitinases. J. Exp. Biol. 206: 4393-4412. https://doi.org/10.1242/jeb.00709
  27. Monzavi-Karbassi, B., G. Cunto-Amesty, P. Luo, and T. Kieber- Emmons. 2002. Peptide mimotopes as surrogate antigens of carbohydrates in vaccine discovery. Trends Biotechnol. 20: 207-214. https://doi.org/10.1016/S0167-7799(02)01940-6
  28. Nicholas, R. O., D. W. Williams, and P. A. Hunter. 1994. Investigation of the value of beta-glucan-specific fluorochromes for predicting the beta-glucan content of the cell-walls of zoopathogenic fungi. Mycol. Res. 98: 694-698. https://doi.org/10.1016/S0953-7562(09)80419-X
  29. Peschen, D., H. P. Li, R. Fischer, F. Kreuzaler, and Y. C. Liao. 2004. Fusion proteins comprising a Fusarium-specific antibody linked to antifungal peptides protect plants against a fungal pathogen. Nat. Biotechnol. 22: 732-738. https://doi.org/10.1038/nbt970
  30. Peter, M. G. 2005. Chitin and chitosan in fungi, pp. 123-147. In A. Steinbüchel, S. De Baets, and E. J. Vandamme (eds.). Biopolymers. Wiley-Vch, Weinheim.
  31. Pochanavanich, P. and W. Suntornsuk. 2002. Fungal chitosan production and its characterization. Lett. Appl. Microbiol. 35: 17-21. https://doi.org/10.1046/j.1472-765X.2002.01118.x
  32. Ryan, G. B., W. T. Jones, R. E. Mitchell, and V. Mett. 2001. Polyclonal antibody production against chito-oligosaccharides. Food Agric. Immunol. 13: 127-130. https://doi.org/10.1080/09540100120055600
  33. Snow, C. M., A. Senior, and L. Gerace. 1987. Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J. Cell Biol. 104: 1143-1156. https://doi.org/10.1083/jcb.104.5.1143
  34. Sorlier, P., D. J. Hartmann, A. Denuziere, C. Viton, and A. Domard. 2003. Preparation and development of anti-chitosan antibodies. J. Biomed. Mater. Res. A 67: 766-774.
  35. Tharanathan, R. N. and F. S. Kittur. 2003. Chitin - The undisputed biomolecule of great potential. Crit. Rev. Food Sci. Nutr. 43: 61-87. https://doi.org/10.1080/10408690390826455
  36. Walker, A. N., R. E. Garner, and M. N. Horst. 1990. Immunocytochemical detection of chitin in Pneumocystis carinii. Infect. Immun. 58: 412-415.
  37. Westerwoudt, R. J. 1985. Improved fusion methods. IV. Technical aspects. J. Immunol. Methods 77: 181-196. https://doi.org/10.1016/0022-1759(85)90031-6
  38. White, S. A., P. R. Farina, and I. Fulton. 1979. Production and isolation of chitosan from Mucor rouxii. Appl. Environ. Microbiol. 38: 323-328.
  39. Wu, T., S. Zivanovic, F. A. Draughon, W. S. Conway, and C. E. Sams. 2005. Physicochemical properties and bioactivity of fungal chitin and chitosan. J. Agric. Food Chem. 53: 3888-3894. https://doi.org/10.1021/jf048202s

Cited by

  1. Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of ChitosanIn VitroandIn Situ vol.78, pp.9, 2010, https://doi.org/10.1128/aem.07506-11
  2. Thanatin confers partial resistance against aflatoxigenic fungi in maize (Zea mays) vol.24, pp.5, 2015, https://doi.org/10.1007/s11248-015-9888-2
  3. Chitosan Mediates Germling Adhesion in Magnaporthe oryzae and Is Required for Surface Sensing and Germling Morphogenesis vol.12, pp.6, 2016, https://doi.org/10.1371/journal.ppat.1005703
  4. Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae vol.19, pp.9, 2010, https://doi.org/10.1111/cmi.12743
  5. Aspergillus-specific antibodies – Targets and applications vol.36, pp.4, 2018, https://doi.org/10.1016/j.biotechadv.2018.03.016
  6. In-vitro Detection of Phytopathogenic Fungal Cell Wall by Polyclonal Sera Raised Against Trimethyl Chitosan Nanoparticles vol.14, pp.None, 2010, https://doi.org/10.2147/ijn.s220488