Purification, characterization and gene expression of a glycine and proline-rich antibacterial protein family from larvae of a beetle, Allomyrina dichotoma.
Two structurally related antibacterial proteins were isolated from larvae of a beetle, Allomyrina dichotoma, immunized with Escherichia coli. The two proteins were designated A. dichotoma (A. d.) coleoptericin A and B. The mature portion of A. d. coleoptericins deduced from nucleotide sequences of the cDNAs consists of seventy-two amino acids without cysteine residues and is rich in glycine (11.1%) and proline (11.1%). Comparison of the amino acid sequences of the A. d. coleoptericins revealed that these antibacterial proteins have 94%, 75%, 50% and 43% similarity to rhinocerosin, holotricin 2, coleoptericin and acaloleptin A1. Recombinant A. d. coleoptericin A and B showed strong antibacterial activity against Staphylococcus aureus, methicillin resistant S. aureus (MRSA) and Bacillus subtilis. Recombinant A. d. coleoptericin A and B were shown to not form pores through bacterial membranes of E. coli, but to hamper cell division. Results of Northern blotting showed that A. d. coleoptericin genes are inducible by bacteria and are expressed strongly in the fat bodies and haemocytes, and weakly in the Malpighian tubules. Analysis of the evolutionary relationship of amino acid sequences among A. d. coleoptericins and other antibacterial proteins suggests that A. d. coleoptericins, rhinocerosin and holotricin 2 are closely related and form a gene family.
Sagisaka
A
A
Laboratory of Biological Defense, National Institute of Sericultural and Entomological Science, Tsukuba, Ibaraki 305-8634, Japan.
Miyanoshita
A
A
Ishibashi
J
J
Yamakawa
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000276:immunology / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D001517:Coleoptera / Q000235:genetics / Q000276:immunology*; D018076:DNA, Complementary; D004926:Escherichia coli; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D005998:Glycine; D019476:Insect Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D007814:Larva; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D011392:Proline; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA
D000900:Anti-Bacterial Agents; D018076:DNA, Complementary; D019476:Insect Proteins; D008081:Liposomes; D011993:Recombinant Fusion Proteins; C437993:coleoptericin A protein, Allomyrina dichotoma; C437994:coleoptericin Bm protein, Allomyrina dichotoma; D011392:Proline; D005947:Glucose; D005998:Glycine