title: Chymotrypsin-like peptidases from Tribolium castaneum: a role in molting revealed by RNA interference.
abstract: Chymotrypsin-like peptidases (CTLPs) of insects are primarily secreted into the gut lumen where they act as digestive enzymes. We studied the gene family encoding CTLPs in the genome of the red flour beetle, Tribolium castaneum. Using an extended search pattern, we identified 14 TcCTLP genes that encode peptidases with S1 specificity pocket residues typically found in chymotrypsin-like enzymes. We further analyzed the expression patterns of seven TcCTLP genes at various developmental stages. While some TcCTLP genes were exclusively expressed in feeding larval and adult stages (TcCTLP-5A/B, TcCTLP-6A), others were also detected in non-feeding embryonic (TcCTLP-5C, TcCTLP-6D) and pupal stages (TcCTLP-5C, TcCTLP-6C/D/E). TcCTLP genes were expressed predominantly in the midgut, where they presumably function in digestion. However, TcCTLP-6C and TcCTLP-5C also showed considerable expression in the carcass. The latter two genes might therefore encode peptidases that act as molting fluid enzymes. To test this hypothesis, we performed western blots using protein extracts from larval exuviae. The extracts reacted with antibodies to TcCTLP-5C and TcCTLP-6E suggesting that the corresponding peptidases are secreted into the molting fluid. Finally, we performed systemic RNAi experiments. While injections of five TcCTLP-dsRNAs into penultimate larvae did not affect growth or development, injection of dsRNA for TcCTLP-5C and TcCTLP-6C resulted in severe molting defects.
authors.affiliation: Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, Barbarastr. 11, 49069 Osnabrück, Germany.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Broehan
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Arakane
authors.forename: Richard W
authors.initials: RW
authors.lastname: Beeman
authors.forename: Karl J
authors.initials: KJ
authors.lastname: Kramer
authors.forename: Subbaratnam
authors.initials: S
authors.lastname: Muthukrishnan
authors.forename: Hans
authors.initials: H
authors.lastname: Merzendorfer
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002918:Chymotrypsin; D041981:Gastrointestinal Tract / Q000201:enzymology; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D017344:Genes, Insect; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D007814:Larva / Q000201:enzymology / Q000254:growth & development; D008969:Molecular Sequence Data; D018978:Molting; D005810:Multigene Family; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D017422:Sequence Analysis, DNA; D014231:Tribolium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
chemical_list: D019476:Insect Proteins; D010447:Peptide Hydrolases; D002918:Chymotrypsin