Isolation, cDNA cloning, and structure-based functional characterization of oryctin, a hemolymph protein from the coconut rhinoceros beetle, Oryctes rhinoceros, as a novel serine protease inhibitor.
We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant (13)C,(15)N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with K(i) values of 3.9 × 10(-10) m, 6.2 × 10(-10) m, 1.4 × 10(-9) m, and 1.2 × 10(-8) m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections.
Horita
Shoichiro
S
From the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Ishibashi
Jun
J
Nagata
Koji
K
Miyakawa
Takuya
T
Yamakawa
Minoru
M
Tanokura
Masaru
M
D000818:Animals; D003001:Cloning, Molecular; D001517:Coleoptera / Q000737:chemistry / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D006458:Hemolymph / Q000737:chemistry; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D057057:Serine Proteases / Q000737:chemistry; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013329:Structure-Activity Relationship
D018076:DNA, Complementary; D019476:Insect Proteins; D015842:Serine Proteinase Inhibitors; D057057:Serine Proteases