A lepidopteran pacifastin member: Cloning, gene structure, recombinant production, transcript profiling and in vitro activity

• Published in: Insect biochemistry and molecular biology 2009-07, Vol.39 (7), p.430-439
• Main Authors: Breugelmans, Bert, Simonet, Gert, van Hoef, Vincent, Van Soest, Sofie, Smagghe, Guy, Vanden Broeck, Jozef
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Arthropoda; Bombyx - chemistry; Bombyx - genetics; Bombyx - metabolism; Bombyx - microbiology; Bombyx mori; Cloning, Molecular; disease resistance; entomopathogenic fungi; enzyme inhibition; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - metabolism; Enzyme Inhibitors - pharmacology; enzymes; Escherichia coli - genetics; Escherichia coli - metabolism; Exons; Fungi - drug effects; Gene Expression; Gene structure; genes; in vitro studies; Inhibitor; insect genetics; Insect Proteins - chemistry; Insect Proteins - genetics; Insect Proteins - metabolism; Insect Proteins - pharmacology; Lepidoptera; Medicin och hälsovetenskap; metabolic inhibitors; molecular cloning; Molecular Sequence Data; Pacifastin; Peptide; peptides; Protein Structure, Tertiary; Proteins - chemistry; Proteins - genetics; Proteins - metabolism; Proteins - pharmacology; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; Sequence Alignment; sequence analysis; Serine peptidase; serine proteinases; silkworms; transcription (genetics); trypsin; trypsin inhibitors
• ISSN: 0965-1748; 1879-0240; 1879-0240
• DOI: 10.1016/j.ibmb.2009.03.005

Members of the pacifastin family have been characterized as serine peptidase inhibitors (PI), but their target enzyme(s) are unknown in insects. So far, the structural and biochemical characteristics of pacifastin-like PI have only been studied in locusts. Here we report the molecular identification and functional characterization of a pacifastin-like precursor in a lepidopteran insect, i.e. the silkworm Bombyx mori. The bmpp-1 gene contains 17 exons and codes for two pacifastin-related precursors of different length. The longest splice variant encodes 13 inhibitor domains, more than any other pacifastin-like precursor in arthropods. The second transcript lacks two exons and codes for 11 inhibitor domains. By studying the expression profile of the Bombyx pacifastin-like gene a different expression pattern for the two variants was observed suggesting functional diversification. Next, several PI domains of BMPP-1 were produced and, contrary to locust pacifastin peptides, they were found to be potent inhibitors of both bovine trypsin and chymotrypsin. Surprisingly, the same Bombyx PI are only weak inhibitors of endogenous digestive peptidases, indicating that other peptidases are the in vivo targets. Interestingly, the Bombyx PI inhibit a fungal trypsin-like cuticle degrading enzyme, suggesting a protective function for BMPP-1 against entomopathogenic fungi.