title: Functional analysis of two lebocin-related proteins from Manduca sexta.
abstract: Insects produce a group of antimicrobial peptides (AMPs) in response to microbial infections. Most AMPs are synthesized as inactive precursors/pro-proteins and require proteolytic processing to generate small active peptides. Here we report identification and functional analysis of two lebocin-related proteins (Leb-B and Leb-C) from the tobacco hornworm, Manduca sexta. The mRNA levels of Leb-B and Leb-C increased significantly in larval fat body and hemocytes after injection of Escherichia coli, Micrococcus luteus and Saccharomyces cerevisiae. Western blotting using rabbit polyclonal antibody to Leb-B showed accumulation of large protein(s) and small peptide(s) in larval hemolymph after microbial injection. This result and the presence of RXXR motifs in the deduced amino acid sequences led to our postulation that Leb-B/C may be inactive precursors that are processed in larval hemolymph to generate short active peptides. To test this hypothesis, we expressed and purified full-length and various fragments of Leb-B and Leb-C as thioredoxin (TRX) fusion proteins. We found that fusion proteins could be cleaved by induced larval plasma, and the cleavage sites were determined by protein sequencing. Antibacterial activity of peptide fragments was also verified using synthetic peptides, and active M. sexta lebocin peptides were located at the N-termini of Leb-B/C, which are different from Bombyx mori lebocins 1-4 that are located close to the C-termini. In addition, we found that synthetic Leb-B(22-48) peptide not only had higher antibacterial activity but also caused agglutination of E. coli cells. Our results provide valuable information for studying processing of lebocin precursors in lepidopteran insects.
authors.affiliation: Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, 5007 Rockhill Road, Kansas City, MO 64110, USA.
authors.forename: Xiang-Jun
authors.initials: XJ
authors.lastname: Rao
authors.forename: Xiao-Xia
authors.initials: XX
authors.lastname: Xu
authors.forename: Xiao-Qiang
authors.initials: XQ
authors.lastname: Yu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000378:metabolism*; D005786:Gene Expression Regulation; D006458:Hemolymph / Q000378:metabolism; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D018402:Manduca / Q000235:genetics / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein
chemical_list: D023181:Antimicrobial Cationic Peptides; D019476:Insect Proteins; D011993:Recombinant Fusion Proteins