Characterization and cDNA cloning of an immune-induced lysozyme from cultured Aedes albopictus mosquito cells.
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 approximately 14 kDa. 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.
Hernandez
Vida P
VP
Department of Entomology, University of Minnesota, 1980 Folwell Avenue, St Paul, MN 55108, USA.
Higgins
LeeAnn
L
Fallon
Ann M
AM
D000330:Aedes / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000096:biosynthesis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009113:Muramidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010802:Phylogeny; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018076:DNA, Complementary; D010446:Peptide Fragments; D009113:Muramidase