Animal cells and systems

The Korean Society for Integrative Biology (한국통합생물학회)
권호 표지

Molecular Cloning and Characterization of Lysozyme II from Artogeia rapae and its Expression in Baculovirus-infected Insect Cells

  • Bang, In-Seok (Department of Biological Science and the Research Institute for Basic Sciences, Hoseo University) ;
  • Kang, Chang-Soo (Department of Biological Science and the Research Institute for Basic Sciences, Hoseo University)
  • Published : 2007.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

The lysozyme II gene of cabbage butterfly Artogeia rapae was cloned from fat body of the larvae injected with E. coli and its nucleotide sequence was determined by the RACE-PCR. It has an open reading frame of 414 bp nucleotides corresponding to 138 amino acids including a signal sequence of 18 amino acids. The estimated molecular weight and the isoelectric point of the lysozyme II without the signal peptide were 13,649.38 Da and 9.11, respectively. The A. rapae lysozyme II (ARL II) showed the highest identity (81%) in the amino acid sequence to Manduca sexta lysozyme among other lepidopteran species. The two catalytic residues ($Glu^{32}$ and $Asp^{50}$) and the eight Cys residue motifs, which are highly conserved among other c-type lysozymes in invertebrates and vertebrates, are also completely conserved. A phylogenetic analysis based on amino acid sequences indicated that the ARL II was more closely related to M. sexta, Hyphantria cunea, Heliothis virescens, and Trichoplusia ni lysozymes. The ARL II gene was expressed in Spodoptera frugiperda 21 insect cells and the recombinant ARL II (rARL II) was purified from cell-conditioned media by cation exchange column chromatography and reverse phase FPLC. The purified rARL II was able to form a clear zone in lysoplate assay against Micrococcus luteus. The lytic activity was estimated to be 511.41 U/mg, 1.53 times higher than that of the chicken lysozyme. The optimum temperature for the lytic activity of the rARL II was $50^{\circ}C$, the temperature dependency of the absolute lytic activity of rARL II was higher than that of the chicken lysozyme at low temperatures under $65^{\circ}C$.

Keywords

References

  1. Abraham EO, Nagaraju J, Salunke D, Gupta HM and Datta RK (1995) Purification and partial characterization of an induced antibacterial protein in the silkworm, Bombyx mori. J lnvertebr Pathol 65: 17-24 https://doi.org/10.1006/jipa.1995.1003
  2. Bang IS, Son SY and Yoe SM (1997) Hinnavin, an antibacterial peptide from cabbage butterfly, Artogeia rapae. Mol Cells 7: 509-513
  3. Bang IS and Yoe SM (2005) Purification and cDNA cloning of lysozyme from cabbage butterfly, Artogeia rapae larvae. Korean J Entomol 35: 207-211 https://doi.org/10.1111/j.1748-5967.2005.tb00161.x
  4. Boman HG, Faye I, Gudmundsson GH, Lee JY and Lidholm DA (1991) Cell-free immunity in Cecropia. A model system for antibacterial proteins. Eur J Biochem 201: 23-31 https://doi.org/10.1111/j.1432-1033.1991.tb16252.x
  5. Bradford M (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  6. Bulet P, Hetru C, Dimarcq JL and Hoffmann D (1999) Antimicrobial peptides in insects; structure and function. Dev Comp Immunol 23: 329-344 https://doi.org/10.1016/S0145-305X(99)00015-4
  7. Dunn PE, Dai W, Kanost MR and Geng C (1985) Soluble peptidoglycan fragments stimulate antibacterial protein synthesis by fat body from larvae of Manduca Sexta. Dev Comp Immunol 9: 559-568 https://doi.org/10.1016/0145-305X(85)90019-9
  8. Engstrom A, Xanthopoulos KG, Boman HG and Bennich H (1985) Amino acid and cDNA sequences of lysozyme from Hyalophora cecropia. EMBO J 4: 2119-2122
  9. Engstrom Y (1999) Induction and regulation of antimicrobial peptides in Drosophila. Dev Comp Immunol 23: 345-358 https://doi.org/10.1016/S0145-305X(99)00016-6
  10. Fusimoto S, Toshimori- Tsuda I, Kishimoto K, Yamano Y and Morishima I (2001) Protein purification, cDNA cloning and gene expression of lysozyme from eri silkworm, Samia cynthia ricini. Comp Biochem Physiol 128: 709-718
  11. Ganz T, Selsted ME, Szklarek D, Harwig SS, Daher KA, Bainton DF and Lehrer RI (1985) Defensins. Natural peptide antibiotics of human neutrophils. J Clin Invest 76: 1427-1435 https://doi.org/10.1172/JCI112120
  12. Gao Y and Fallon AM (2000) Immune activation upregulate lysozyme gene expression in Aedes aegypti mosquito cell culture. Insect Mol Biol 9: 553-558 https://doi.org/10.1046/j.1365-2583.2000.00216.x
  13. Grinde B, Jolles J and Jolles P (1988) Purification and characterization of two Iysozymes from rainbow trout (Salmo gairdneri). Eur J Biochem 173: 269-273 https://doi.org/10.1111/j.1432-1033.1988.tb13994.x
  14. Higgins D, Thompson J, Gibson T, Thompson JD, Higgins DG and Gibson TJ (1994) Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673-4680 https://doi.org/10.1093/nar/22.22.4673
  15. Hoffmann JA and Reichhart JM (2002) Drosophila innate immunity: an evolutionary perspective. Nature Immunol 3: 121-126 https://doi.org/10.1038/ni0202-121
  16. Howard JB and Glazer AN (1969) Papaya lysozyme. Terminal sequence and enzymatic properties. J Biol Chem 244: 1399-1409
  17. Hultmark D (1996) Insect ysozymes. EXS 75: 87-102
  18. Imler JL and Bulet P (2005) Antimicrobial peptides in Drosophila structures, activities, and gene regulation. In: Kabelitz D, and Schroder JM (ed), Mechanisms of Epithelial Defense, Basil, Karger, pp 1-21
  19. Inouye M and Tsugita A (1968) Amino acid sequence of T2 phage lysozyme. J Mol Biol 37: 213-223 https://doi.org/10.1016/0022-2836(68)90084-3
  20. Ito Y, Nakamura M, Hotani T and Imoto T (1995) Insect lysozyme from house fly (Musca domestica) larvae: possible digestive function based on sequence and enzymatic properties. J Biochem 118: 546-551 https://doi.org/10.1093/oxfordjournals.jbchem.a124943
  21. Ito Y, Yamada H, Nakamula M, Yoshikawa A, Ueda T and Imoto T (1993) The primary structures and properties of nonstomach Iysozymes of sheep and cow, and implication for functional divergence oflysozyme. Eup J Biochem 213: 649-658 https://doi.org/10.1111/j.1432-1033.1993.tb17805.x
  22. Jain D, Nair DT, Swaminathan OJ, Abraham EG, Nagaraju J and Salunke DM (2001) Structure of the induced antibacterial protein from Tasar Silkworm, Antheraea mylitta. J Biol Chem 276: 41377-41382 https://doi.org/10.1074/jbc.M104674200
  23. Jolles P and Jolles J (1984) What's new in lysozyme research? Mol Cell Biochem 63: 165-189
  24. Jung A, Sippel AE, Grez M and Schutz G (1980) Exons encode functional and structural units of chicken lysozyme. Proc Natl Acad Sci USA 77: 5759-5763
  25. Kang D, Liu G, Gunne H and Steiner H (1996) PCR differential display of immune gene expression in Trichoplusia ni. Insect Biochem Mol Biol 26: 177-184 https://doi.org/10.1016/0965-1748(95)00080-1
  26. Kumar S, Tamura K and Nei M (1994) MEGA molecular evolutionary genetics analysis software for microcomputers. Comput Appl Biosci 10: 189-191
  27. Kylsten P, Kimbrell DA, Daffre S, Samakovlis C and Hutmark D (1992) The lysozyme locus in Drosophila melanogaster: different genes are expressed in midgut and salivary glands. Mol Gen Genet 232: 335-343
  28. Lee WJ and Brey PT (1995) Isolation and characterization of the lysozyme-encoding gene from the silkworm Bombyx mori. Gene 161: 199-203 https://doi.org/10.1016/0378-1119(95)00199-G
  29. Lehrer RI, Rosenman M, Harwig SS, Jackson R and Eisenhauer P (1991) Ultrasensitive assays for endogenous antimicrobial polypeptides. J Immunol Methods 137: 167-173 https://doi.org/10.1016/0022-1759(91)90021-7
  30. Lockey TO and Ourth DD (1996) Purification and characterization of lysozyme from hemolymph of Heliothis virescens larvae. Biochem Biophys Res Commun 220: 502-508 https://doi.org/10.1006/bbrc.1996.0434
  31. Lowenberger C (2001) Innate immune response of Aedes aegypti. Insect Biochem Mol Biol 31 : 219-229 https://doi.org/10.1016/S0965-1748(00)00141-7
  32. Mohrig W and Messner B (1968) Lysozyme as antibacterial agent in honey and bees venom. Acta Biol Med Ger 21: 85-95
  33. Morishima I, Yamada K and Ueno T (1992) Bacterial peptidoglycan as elicitor of antibacterial protein synthesis in larvae of the silkworm, Bombyx mori. Insect Biochem Mol Biol 22: 363-367 https://doi.org/10.1016/0965-1748(92)90074-O
  34. Mulnix AB and Dunn PE (1994) Structure and induction of a lysozyme gene from the tobacco homworm, Manduca sexta. Insect Biochem Mol Biol 24: 271-281 https://doi.org/10.1016/0965-1748(94)90007-8
  35. Powning RF and Davidson WJ (1976) Studies on insect bacteriolytic enzymes-. Some physical and enzymatic properties of lysozyme from haemo1ymph of Galleria mellonella. Comp Biochem Physiol B 55: 221-228 https://doi.org/10.1016/0305-0491(76)90234-0
  36. Regel R, Matioli SR and Terra WR (1998) Molecular adaptation of Drosophila melanogaster Iysozymes to a digestive function. Insect Biochem Mol Biol 28: 309-319 https://doi.org/10.1016/S0965-1748(97)00108-2
  37. Rossignol PA and Lueders AM (1986) Bacteriolytic factor in the salivary glands of Aedes aegypti. Comp Biochem Physiol B 83: 819-822 https://doi.org/10.1016/0305-0491(86)90153-7
  38. Saitou N and Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: 406-425
  39. Schager Hand von Jagow G (1987) Tricine-sodium dodecylsulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 KDa. Anal Biochem 166: 368-379 https://doi.org/10.1016/0003-2697(87)90587-2
  40. Towbin H, Staehelin T and Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nat Acad Sci USA 76: 4350-43504
  41. Ursic Bedoya RJ, Mitzey AM, Obraztsova M and Lowenberger C (2005) Molecular cloning and transcription activation of lysozyme-encoding cDNAs in the mosquito Aedes Aegypti. Insect Mol Biol 14: 89-94 https://doi.org/10.1111/j.1365-2583.2004.00534.x
  42. Weaver LH, Gmtter MG, Remington SI, Gray TM, Issacs NW and Mattews BW (1985) Comparison of goose type, chickentype, and phage-type Iysozymes illustrates the changes that occur in both amino acid sequence and three dimensional structures during evolution. J Mol Evol 21 : 97-111 https://doi.org/10.1007/BF02100084
  43. Yoe SM, Bang IS, Kang CS and Kim HI (1996) Purification and chracterization of two lysozyme from larval haemolymph of cabbage butterfly, Artogeia rapae. Mol. Cells 6: 609-614
  44. Yoe SM, Kang CS, Han SS and Bang IS (2006) Characterization and cDNA cloning of hinnavin, a cecropin family antibacterial peptide from the cabbage butterfly, Artogeia rapae. Comp Biochem Physiol B 144: 199-205 https://doi.org/10.1016/j.cbpb.2006.02.010
  45. Zachary D and Hoffmann JA (1984) Lysozyme is stored in the granules of certain hemocyte types in Locusta. J Insect Physiol 30: 405-411 https://doi.org/10.1016/0022-1910(84)90098-2