Functional analysis of four processing products from multiple precursors encoded by a lebocin-related gene from Manduca sexta

Antimicrobial peptides (AMPs) are a crucial component of the natural immune system in insects. Five types of AMPs have been identified in the tobacco hornworm Manduca sexta, including attacin, cecropin, moricin, gloverin, and lebocin. Here we report the isolation of lebocin-related cDNA clones and antibacterial activity of their processed protein products. The 17 cDNA sequences are composed of a constant 5′ end and a variable 3′ region containing 3–16 copies of an 81-nucleotide repeat. The sequence of the corresponding gene isolated from a M. sexta genomic library and Southern blotting results indicated that the gene lacks introns and exists as a single copy in the genome. The genomic sequence contained 13 complete and one partial copy of the 81-nucleotide repeat. Northern blot analysis revealed multiple transcripts with major size differences. The mRNA level of M. sexta lebocin increased substantially in fat body after larvae had been injected with bacteria. The RXXR motifs in the protein sequences led us to postulate that the precursors are processed by an intracellular convertase to form four bioactive peptides. To test this hypothesis, we chemically synthesized the peptides and examined their antibacterial activity. Peptide 1 killed Gram-positive and Gram-negative bacteria. Peptide 2, similar in sequence to a Galleria mellonella AMP, did not affect the bacterial growth. Peptide 3 was inactive but peptide 3 with an extra Arg at the carboxyl terminus was active against Escherichia coli at a high minimum inhibitory concentration. Peptide 4, encoded by the 81-bp repeat, was inactive in the antibacterial tests. The hypothesis that posttranslational processing of the precursor proteins produces multiple bioactive peptides for defense purposes was validated by identification of peptides 1, 2, and 3 from larval hemolymph via liquid chromatography and tandem mass spectrometry. Comparison with the orthologs from other lepidopteran insects indicates that the same mechanism may be used to generate several functional products from a single precursor.