Characterization and cDNA cloning of an immune-induced lysozyme from cultured Aedes albopictus mosquito cells

Vida P. Hernandez, LeeAnn Higgins, Ann M Fallon

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

Protein chemistry and cDNA sequencing were used to identify an Aedes albopictus mosquito lysozyme secreted after treatment of cultured cells with heat-killed bacteria. On acid gels, the putative lysozyme activity ran just ahead of the cecropin band. Elution of this activity yielded a single band on SDS gels, with a mass of ∼14kDa. Mass spectral analysis of the silver-stained band uncovered five tryptic peptides with masses that matched peptides from an Aedes aegypti lysozyme, which we had previously characterized from the Aag-2 mosquito cell line. Based on this tentative identification, the Ae. albopictus lysozyme cDNA was cloned using PCR-based approaches. The full length cDNA sequence was used to deduce the sequences and masses of theoretical tryptic peptides that would be detected after matrix-assisted laser desorption ionization time of flight (MALDI-TOF) and tandem mass spectrometry (MS/MS). In aggregate, this analysis uncovered seven peptides that encoded 75 of the 125 amino acids in the mature Ae. albopictus lysozyme. In a phylogenetic analysis, the Aedes lysozymes were most closely related to the Anopheles lysozymes. As a group the mosquito lysozymes were more closely related to lysozymes from various Lepidopteran species than to those from higher Diptera such as Drosophila and Musca, which have evolved a digestive function.

Original languageEnglish (US)
Pages (from-to)11-20
Number of pages10
JournalDevelopmental and Comparative Immunology
Volume27
Issue number1
DOIs
StatePublished - Jan 1 2003

Keywords

  • CDNA
  • Cell line
  • Immune response
  • Lysozyme
  • MALDI-TOF
  • MS/MS
  • Mosquito

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