title: Cloning and functional characterization of the Lymantria dispar initiator caspase dronc.
abstract: Ld652Y cells from the gypsy moth, Lymantria dispar, are extremely sensitive to various apoptotic stimuli, whereas BM-N cells from the silkworm, Bombyx mori, are relatively resistant to apoptotic stimuli. We previously cloned and characterized a B. mori homologue (bm-dronc) of Drosophila melanogaster dronc. In the present study, we cloned and characterized an L. dispar homologue of dronc (ld-dronc) comparatively with Bm-Dronc. The open reading frame of ld-dronc consisted of 1329bp that was predicted to encode a 443 amino-acid polypeptide with a molecular mass of 50,706Da and 54-57% amino acid sequence identity with Dronc homologues from other lepidopteran insects identified to date. Ld-Dronc had a long prodomain, large p20 domain, and small p10 domain, and a catalytic site composed of (308)QTCRG(312), which was distinct from the sites QACRG in Bm-Dronc and QMCRG in Dronc homologues of several other lepidopteran insects. Transiently expressed Ld-Dronc underwent proteolytic processing in the lepidopteran cell lines L. dispar Ld652Y, Spodoptera frugiperda Sf9, and B. mori BM-N, and dipteran D. melanogaster S2, but only triggered apoptosis in the lepidopteran cell lines. Endogenous Ld-Dronc underwent processing in Ld652Y cells upon infection with vAcΔp35, but not in mock-infected Ld652Y cells, supporting the involvement of Ld-Dronc in apoptosis induction. In vAcΔp35-infected apoptotic cells, Ld-Dronc underwent proteolytic processing more rapidly and extensively than Bm-Dronc. Similar results were obtained for Ld-Dronc and Bm-Dronc expressed transiently in S2, Ld652Y, Sf9, and BM-N cells. Taken together, these findings suggest that the intrinsic properties of Dronc proteinsare responsible, at least in part, for the differing sensitivity of Ld652Y and BM-N to apoptosis induction upon NPV infection.
authors.affiliation: Laboratory of Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Kitaguchi
authors.forename: Rina
authors.initials: R
authors.lastname: Hamajima
authors.forename: Hayato
authors.initials: H
authors.lastname: Yamada
authors.forename: Michihiro
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Motoko
authors.initials: M
authors.lastname: Ikeda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D012831:Bombyx / Q000166:cytology / Q000235:genetics / Q000821:virology; D053471:Caspases, Initiator / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D004331:Drosophila melanogaster / Q000166:cytology / Q000235:genetics / Q000821:virology; D054884:Host-Pathogen Interactions; D015151:Immunoblotting; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics / Q000378:metabolism; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009036:Moths / Q000166:cytology / Q000235:genetics* / Q000821:virology; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D061987:Sf9 Cells; D018411:Spodoptera / Q000166:cytology / Q000235:genetics / Q000821:virology; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D051034:Inhibitor of Apoptosis Proteins; D019476:Insect Proteins; D014764:Viral Proteins; C086430:inhibitor of apoptosis, Nucleopolyhedrovirus; D053471:Caspases, Initiator