Cloning and expression of three cecropin cDNAs from a mosquito cell line

We have characterized full-length cDNAs encoding three isoforms of the antibiotic cecropin secreted by the C7-10 cell line from the mosquito, Aedes albopictus. The existence of two cecropin isoforms that differed from the previously described AalCecA was predicted by mass spectrometry and amino acid sequence analysis of peptides that eluted from reversed phase high performance liquid chromatography as a single peak just behind the previously described cecropin, AalCecA. Based on the amino acid sequence of the mature AalCecA peptide, we designed primers that amplified partial cDNAs encoding three different A. albopictus cecropins in reverse transcriptase polymerase chain reactions. Rapid amplification of cDNA ends was then used to complete the cDNA sequences of AalCecA, AalCecB and AalCecC, respectively. Each cDNA encoded a translation product containing a signal peptide, a pro region, and a mature cecropin peptide consistent with amino acid sequence data from chymotryptic digests. Although the mosquito cecropins shared 70-86% identity among each other, they shared only approximately 40% identity to cecropins from Drosophila melanogaster. Each of the cecropins was expressed within 2 to 4 h after induction, and transcripts measuring 0.3 to 0.5 kb continued to accumulate over 24 h. The three cecropins were secreted in roughly equimolar proportions, and 30 to 90% of AalCecB was amidated at the terminal glycine residue. In contrast, amidated forms of AalCecA and AalCecC constituted a smaller proportion of these isoforms.