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.
Bibliographical noteFunding Information:
This work was supported by National Institutes of Health grant AI 36258 and by the University of Minnesota Agricultural Experiment Station, St Paul, MN. The authors recognize the Mass Spectrometry Consortium for the Life Sciences at the University of Minnesota and various supporting agencies, including the National Science Foundation for Major Research Instrumentation grant 9871237, used to purchase the instruments described in this study.
- Cell line
- Immune response