Characterization and cDNA cloning of an immune-induced lysozyme from cultured Aedes albopictus mosquito cells
Autor(es): Hernandez, VP; Higgins, L; Fallon, AM
Resumo: 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 similar to14 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. (C) 2002 Elsevier Science Ltd. All rights reserved.
Palavras-Chave: Mosquito; Cell line; Immune response; lysozyme; cDNA; MALDI-TOF; MS/MS
Imprenta: Developmental and Comparative Immunology, v. 27, n. 1, p. 11-20, 2003
Identificador do objeto digital: 10.1016/S0145-305X(02)00065-4
Descritores: Aedes aegypti - Biochemistry ; Aedes aegypti - DNA ; Aedes aegypti - Immunology
Data de publicação: 2003
