Elsevier

Gene

Volume 510, Issue 2, 1 December 2012, Pages 133-141
Gene

Expression analysis of inhibitor of apoptosis and related caspases in the midgut and silk gland of the greater wax moth, Galleria mellonella, during metamorphosis and under starvation

https://doi.org/10.1016/j.gene.2012.08.036Get rights and content

Abstract

We cloned a cDNA encoding inhibitor of apoptosis (IAP) from the greater wax moth, Galleria mellonella. The deduced amino acid sequence showed that GmIAP contains two baculoviral IAP repeat (BIR) motifs, followed by a RING finger. The sequence comparison showed that GmIAP had high homology to lepidopteran IAPs and baculoviral IAPs, as well as dipteran IAPs. GmIAP transcript and its protein appeared in both the midgut and the silk gland during metamorphosis and starvation where cell death was detected by TUNEL test. IAP, and capases-1, -3, -4 and ‐6 appeared as at least two peaks in the midgut and silk gland during metamorphosis. Caspase-1 transcript appeared at the highest level among caspases, while caspase-3 and caspase-6 seemed to be the most relevant caspases to IAP during metamorphosis suggesting that IAP and caspases may be involved in a core apoptosis pathway in the wax moth as in flies and mosquitoes.

Highlights

► GmIAP contains two BIR motifs and a RING finger. ► GmIAP expressed in the midgut and silk gland during metamorphosis and starvation. ► GmIAP is the most relative to caspase-3 and ‐6. ► It may represent a core death pathway in Galleria mellonella.

Introduction

Apoptosis is a genetically programmed cellular suicide process that eliminates unwanted or diseased cells; it plays important roles in embryogenesis, homeostasis, morphogenesis, metamorphosis, and immunity (Opferman and Korsmeyer, 2003, Steller and Grether, 1994, Vaux and Korsmeyer, 1999). Cells undergoing apoptosis display a series of morphological changes, including cell shrinkage, chromatin condensation, DNA fragmentation, membrane blebbing, and finally, formation of apoptotic bodies (Häcker, 2000). The molecular pathways that regulate apoptosis include positive or negative interactions of Bcl2 family containing BH3 domain to the anti-apoptosis members and leading activated caspases to induce cell death (Huang and Strasser, 2000).

Inhibitor of apoptosis (IAP), a family of potent anti-apoptotic protein, was first identified in baculoviruses (Crook et al., 1993) and cellular homologs have been identified in yeast, nematodes, flies, and higher vertebrates. All IAPs contain one to three N-terminal baculoviral IAP repeats (BIRs) and usually a C-terminal RING finger motif (Miller, 1999, Nachmias et al., 2004). The BIR domains consist of approximately 70 amino acids that contain the characteristic sequence CX2CX6WX3DX5HX6C (C, cysteine; W, tryptophan; D, aspartic acid; H, histidine; and X, any amino acid) (Deveraux et al., 1999, Hinds et al., 1999, Miller, 1999). With both hydrophobic and hydrophilic residues on its surface, the BIR core is theoretically capable of supporting protein-protein interactions. A RING finger domain (C3HC4) exists at the C-terminal region in some IAPs. It contains one zinc atom chelated to three cysteines and one histidine and another zinc atom bound to four cysteines (Wei et al., 2008).

The IAP inhibits downstream components of the caspase activation pathway in the regulation of apoptosis and plays important roles in regulating the progress of apoptosis in many species (Crook et al., 1993, Schimmer, 2004). IAPs inhibit caspases by binding their conserved BIR domain to promote the degradation of active caspases, or by sequestering the caspases away from their substrates (Tenev et al., 2005). In Drosophila melanogaster, the activities of the core death genes rpr and hid are upregulated, which in turn induces destruction of IAPs. This allows free DRONC to associate with Dark, which then activates the DrICE and DCP1 that induce apoptosis in D. melanogaster (Hawkins et al., 1999, Hawkins et al., 2000, Kaiser et al., 1998, Meier et al., 2000). The same apoptotic pathway was defined in Aedes aegypti. However, in the place of rpr, hid, DrICE, and DCP1 in D. melanogaster, A. aegypti has AeMichelob-x, AeIMP, AeCaspase7, and AeCaspase8 (Liu and Clem, 2011).

IAPs have been described in lepidopteran insects (Huang et al., 2000, Huang et al., 2001, Seshagiri et al., 1999, Vilaplana et al., 2007). Biochemical and recombination data of TnIAP from Trichoplusia ni, SfIAP from Spodoptera frugiperda, BmIAP from Bombyx mori showed that they could inhibit mammalian caspase-9 activity but not downstream caspase-3 and caspase-7 (Huang et al., 2000, Liao et al., 2002). SlIAP transcript decreased concurrently with programmed cell death that appears in the midgut of S. littoralis during metamorphosis (Vilaplana et al., 2007). In Heliothis virescens, increases in caspase-1 and ICE transcripts were found to follow downregulation of IAP transcript (Parthasarathy and Palli, 2007).

In Galleria mellonella, Uwo et al. (2002) showed that replacement of midgut during metamorphosis involved apoptosis because of the condensation of chromatin, DNA fragmentation, toluidine blue staining in dying tissue, and increased activity of caspase-3-like protease (C3P). These results were confirmed by the expression profile of caspase-1 (Khoa et al., 2012). Recently, four caspases, caspase-1, -3, -4, and ‐6, were identified from G. mellonella (Courtiade et al., 2011, Khoa et al., 2012). However, the character of inhibitor of apoptosis (IAP) and relevant caspase expressions have not been described. Therefore, in this study, we continue the identification and characterization of the expression of an IAP and related caspases in the midgut and silk gland of G. mellonella during metamorphosis and under starvation.

Section snippets

Insects

Insects were reared in the laboratory under LD 16:8 at 30 °C on an artificial diet used by Burges (1976) and Uwo et al. (2002). The midgut transformation was tentatively divided into 15 stages during metamorphosis. Six larval stages were determined based on the degree of withdrawal of pigments from stemmata, according to Kühn and Piepho (1936) and Uwo et al. (2002): stages 0, I, II, III, IV, and V. Pupal stages were designated as follows: Stage VI, a “white pupa” just after pupation; stages VII,

Isolation of cDNA encoding GmIAP

Rapid amplification of cDNA ends PCR (RACE-PCR) was performed with gene-specific primers designed from partial sequence and adaptor primer as reported by Khoa et al. (2012). The first sequence, containing N-terminus sequence, was identified using 5′-RACE cDNA with GSP and adaptor. The second sequence, containing carboxyl-terminus, was identified using 3′-RACE cDNA with GSP and adaptor primer. Use of GENETYX-WIN (Software Development, Tokyo, Japan) to align the first and second sequences

Discussion

In this study, we retrieved cDNA encoding inhibitor of apoptosis (IAP) from G. mellonella. GmIAP contains the characteristic domains and structures of most IAP proteins with two BIR motifs and one RING finger. Sequence alignment and phylogenetic analyses revealed that GmIAP has a high similarity to lepidopteran IAPs and baculoviruses (Fig. 1, Fig. 2), which were proven to suppress apoptosis by inhibiting caspase (Huang et al., 2000, Seshagiri and Miller, 1997). Moreover, among the known

Acknowledgment

We would like to thank to Dr. Hiroshi Kitakawa, Laboratory of Animal Histophysiology, Graduate School of Agricultural Science, Kobe University for providing us with the X-IAP antibody.

References (44)

  • R. Parthasarathy et al.

    Developmental and hormonal regulation of midgut remodeling in a lepidopteran insect, Heliothis virescens

    Mech. Dev.

    (2007)
  • H. Richardson et al.

    Death to flies: Drosophila as a model system to study programmed cell death

    J. Immunol. Methods

    (2002)
  • S. Seshagiri et al.

    Baculovirus-based genetic screen for antiapoptotic genes identifies a novel IAP

    J. Biol. Chem.

    (1999)
  • H. Steller et al.

    Programmed cell death in Drosophila

    Neuron

    (1994)
  • M. Tufail et al.

    Molecular cloning and developmental expression pattern of the vitellogenin receptor from the cockroach, Leucophaea maderae

    Insect Biochem. Mol. Biol.

    (2007)
  • D.L. Vaux et al.

    Cell death in development

    Cell

    (1999)
  • L. Vilaplana et al.

    Molecular characterization of an inhibitor of apoptosis in the Egyptian armyworm, Spodoptera littoralis, and midgut cell death during metamorphosis

    Insect Biochem. Mol. Biol.

    (2007)
  • S.L. Wang et al.

    The Drosophila caspase inhibitor DIAP1 is essential for cell survival and is negatively regulated by HID

    Cell

    (1999)
  • D.D. Burges

    Techniques for the bioassay of Bacillus thuringiensis with Galleria mellonella

    Entomol. Exp. Appl.

    (1976)
  • J. Courtiade et al.

    A comprehensive characterization of the caspase gene family in insects from the order Lepidoptera

    BMC Genomics

    (2011)
  • N.E. Crook et al.

    An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif

    J. Virol.

    (1993)
  • Q.L. Deveraux et al.

    Endogenous inhibitors of caspases

    J. Clin. Immunol.

    (1999)
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