Functional role of aspartic proteinase cathepsin D in insect metamorphosis
- Zhong Zheng Gui†1,
- Kwang Sik Lee†1,
- Bo Yeon Kim1,
- Yong Soo Choi1,
- Ya Dong Wei1,
- Young Moo Choo1,
- Pil Don Kang2,
- Hyung Joo Yoon2,
- Iksoo Kim3,
- Yeon Ho Je4,
- Sook Jae Seo5,
- Sang Mong Lee6,
- Xijie Guo7,
- Hung Dae Sohn1 and
- Byung Rae Jin1Email author
© Gui et al; licensee BioMed Central Ltd. 2006
Received: 08 August 2006
Accepted: 25 October 2006
Published: 25 October 2006
Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis.
Cathepsin D of the silkworm Bombyx mori (BmCatD) was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi)-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage.
Based on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis.
Insect metamorphosis is a complex, highly conserved, and strictly regulated process of developmental events. Metamorphosis is triggered by the steroid hormone ecdysone in the absence of the sesquiterpenoid juvenile hormone and is carried out by self-destructive mechanisms of programmed cell death . The developmental process of different larval tissues during metamorphic transformation showed that tissues such as the silk gland and gut are completely histolyzed [2–4], while other tissues such as fat body undergo reorganization with histolysis [5, 6], and predetermined imaginal tissues differentiate and grow into organs and external structures [4, 7].
The ecdysone-induced transcription factor Broad-Complex (BR-C) plays an important regulatory role in metamorphosis [8–14]. It is required for differentiation of adult structures as well as for the programmed death of obsolete larval organs during metamorphosis. The Bombyx BR-C RNAi disrupted the differentiation of adult compound eyes, legs and wings, and also perturbed the programmed cell death of larval silk glands .
Additionally, the Bombyx BR-C function uncovers the programmed cell death of larval fat body and larval gut during silkworm metamorphosis. It is still unclear what gene products function in the programmed cell death of larval fat body and/or larval gut. Therefore, we asked whether cathepsins are involved in the metamorphic events of silkworm because, to date, studies in insects reveal that cathepsins also participate in developmental processes [2, 15–23]. Recently, a study has shown that the temporal activity profile of an aspartic proteinase is associated with fat body histolysis during Ceratitis capitata early metamorphosis . Studies of insect cathepsins strongly implicate the involvement of activated proteinases in metamorphic events. Thus, it is of interest to know whether cathepsin has any functional roles in insect metamorphosis through a loss-of-function test.
Here, we have focused on cathepsin D, a lysosomal aspartic proteinase, as a metamorphosis-specific proteinase involved in metamorphic events. To help elucidate the molecular mechanisms of metamorphosis in the silkworm, we first cloned the Bombyx mori cathepsin D (BmCatD) gene from the silkworm. We examined the expression profile of BmCatD during development; BmCatD is induced by the steroid hormone ecdysone and baculovirus infection, and is expressed in a tissue- and developmental stage-specific pattern in the larval fat body of the final instar and in the larval gut of pupal stage. Finally, we demonstrate that loss of BmCatD function disrupts two classes of metamorphic events in Bombyx, larval-pupal transformation and programmed cell death of larval gut.
Results and discussion
A novel aspartic proteinase (BmCatD) gene cloned from the silkworm B. mori
Cathepsin D has been reported to be an N-glycosylated high mannose glycoprotein that functions as an acidic proteinase, with an optimal pH of 3.0 [6, 15, 26]. We found that recombinant BmCatD expressed in baculovirus-infected insect cells was N-linked glycosylated, but its N-linked glycosylation is not necessary for enzyme activity and that the purified recombinant BmCatD exhibited a high proteolytic activity at pH 3.0–3.5, establishing BmCatD as an aspartic proteinase (Fig. 1B,c).
BmCatD is expressed in a developmental stage- and tissue-specific manner and its expression causes programmed cell death
The prospective fates of these tissues such as fat body and gut during metamorphic transformation are different. The larval gut is completely histolyzed during the pupal stage [2–4]; on the other hand, the fat body undergoes reorganization with histolysis during larval-pupal transformation [5, 6]. It seems logical that the histolysis of larval fat body and larval gut represents a programmed cell death response. Therefore, we assayed whether the histolysis of larval fat body and larval gut is accompanied by internucleosomal DNA fragmentation that is a rapid and accurate indicator of the involvement of apoptosis in cell death [6, 9]. To determine whether the BmCatD expression correlates with the histolysis of larval fat body and larval gut during metamorphosis, we analyzed the induction of programmed cell death in the fat body and gut tissues. Figure 2 also shows internucleosomal DNA fragments, seen as programmed cell death-specific laddering on agarose gel electrophoresis, for the fat body and gut during metamorphosis. The DNA fragmentation by histolysis of larval fat body was observed from day 3, peaked on day 7 in the fifth instar, and dramatically reduced on day 2 in the spinning stage (lower panel of Fig. 2B). In the larval gut, DNA fragmentation initiated on day 1 of the pupal stage and thereafter gradually increased until adult eclosion (lower panel of Fig. 2C). These results suggest that in fat body and gut, BmCatD expression was accompanied with DNA fragmentation. In addition, the DNA fragmentation rapidly and severely occurred in larval gut in the pupal stage. The result suggests that the developmental profiles of BmCatD expression, as judged by DNA fragmentation, differed between the larval gut, which undergoes programmed cell death during the pupal stage, and larval fat body, which survives into the adult phase but undergoes reorganization during larval-pupal transformation.
BmCatD is induced by ecdysone and viral infection
Host insect infection with baculovirus induces metamorphosis [32, 33]. This raises the possibility that BmCatD may be involved in the induction of metamorphosis of host insects infected with baculovirus. To examine this possibility, we carried out transcriptional induction analysis of BmCatD in baculovirus-infected silkworm larvae. When BmNPV was injected into larvae on day 1 of the fifth instar, BmCatD mRNA was detected in the fat body on day 2 of the fifth larval instar (Fig. 3A-d), although no BmCatD expression was observed in the controls (Fig. 3A-a). Consistent with this, the induced programmed cell death of larval fat body by viral infection was clearly observed on day 2 of the fifth larval instar (Fig. 3C). Furthermore, the level of BmCatD expression was found to be higher in virus-infected larvae than in uninfected controls (Fig. 3B). This result indicates that viral infection induced the BmCatD expression, which resulted in the induced programmed cell death in larval fat body, and suggests that BmCatD was involved in the induction of metamorphosis in the host insect infected with baculovirus. During virus replication, the prothoracic gland of host insects was observed to maintain characteristics indicative of ecdysone biosynthetic activities . The haemolymph ecdysteroid level and prothoracic gland activity in baculovirus-infected larvae were higher than in controls, which continued until the late stages of viral infection, even during the time that controls had ceased to secrete ecdysone after molting [32, 33]. Thus, these observations indicate that viral infection resulted in the up-regulation of BmCatD, as shown in 20E treatment, and suggest that BmCatD up-regulation is the result of alteration of host's hormonal system by viral infection.
To further understand the correlation between hormonal system and viral infection, we injected larvae on day 1 of the fifth instar with Bombyx mori nucleopolyhedrovirus (BmNPV) just after JHA treatment, which down-regulates BmCatD expression. Interestingly, in both JHA and BmNPV treatments, BmCatD expression was up-regulated in a viral infection-dependent manner (Fig. 3A-e), thus indicating that viral infection plays a role in the regulation of BmCatD expression, regardless of the presence or absence of JHA. The host's hormonal system, altered by viral infection, induces metamorphosis [32, 33], which suggests that metamorphosis induction due to virus infection functions as a viral defense system of the host insect. In contrast, baculoviruses are known to contain the unique p35 gene, which blocks virus-induced apoptosis . Therefore, the inhibition of DNA fragmentation at later stages of viral infection (Fig. 3C) was likely due to P35 of BmNPV. This result was consistent with previous observations indicating that baculovirus infection blocks the progression of fat body degradation .
Loss of BmCatD function disrupts metamorphic events in the silkworm
In BmCatD RNAi-mediated silkworm larvae, larval-pupal metamorphosis was strongly affected by BmCatD RNAi. Compared to controls, all of which underwent regular larval-pupal transformation, three-fourths of BmCatD RNAi on day 3 fifth instar larvae arrested during larval-pupal metamorphosis (Fig. 4B). In this case, a large portion of BmCatD RNAi-mediated silkworm larvae was nonpupated (58.3%) and 16.7% of larvae were abnormally pupated. Similar effects were respectively seen in 55% and 48% of BmCatD RNAi on day 4 and 5 fifth instar larvae, and the abnormal pupation rate was relatively high in these cases (Fig. 4B). The arrest of larval-pupal transformation was observed in BmCatD RNAi-mediated silkworm larvae and indicated that BmCatD is necessary for the larval-pupal metamorphosis in the silkworm. It is formally conceivable that BmCatD might have additional functions in other developmental processes, and reduction in BmCatD via RNAi in these processes might directly or indirectly contribute to developmental arrest.
As judged from the observed effect of BmCatD RNAi during metamorphosis, all observations in this study provide strong evidence that BmCatD was involved in the histolysis of larval fat body and larval gut, demonstrating a functional involvement as a metamorphosis-specific lysosomal proteinase. Recently, most studies of molecular mechanisms of metamorphosis in silkworm have focused on the metamorphosis-specific transcriptional factor BR-C [4, 38–40]. It has been shown that the Bombyx BR-C is expressed in an ecdysone-induced manner and is required for programmed cell death of larval silk glands, as well as for the differentiation of adult structures including compound eyes, legs, and wings . By focusing our findings on the BmCatD, we have been able to explain metamorphosis-specific functions of BmCatD.
The work provided here demonstrates the involvement of cathepsin D as a metamorphosis-specific proteinase in metamorphic events. This finding is important in that it sheds new light on the functional role of cathepsin D in silkworm metamorphosis. The metamorphic defects seen in the BmCatD RNAi-mediated silkworm, such as larval-pupal transformation arrest and programmed cell death inhibition, highlight an important functional role of BmCatD in metamorphic processes and provide a foundation for a better understanding of the molecular mechanisms of insect metamorphosis.
Larvae of the silkworm, Bombyx mori, used in this study were F1 hybrid Baekok-Jam supplied by Department of Agricultural Biology, The National Institute of Agricultural Science and Technology, Korea. Silkworms were reared on fresh mulberry leaves at 25°C, 65 ± % relative humidity, and 12 h light: 12 h dark photoperiod. Spinning (wandering) occurred on day 7 of the fifth instar, and pre-pupation and pupation occurred 2 days and 3 days thereafter. The first days corresponding to the developmental stages of the fifth larval ecdysis, spinning, and pupation were designated as day 1 of the fifth larval instar, spinning, and pupal stage, respectively.
The BmCatD cDNA was cloned from a cDNA library using whole bodies of B. mori larvae . The sequences were compared using the DNASIS and BLAST programs provided by the NCBI . MacVector (ver. 6.5, Oxford Molecular Ltd) was used to align the amino acid sequences of CatD. Genomic DNA, extracted from the fat body of single B. mori larva using a Wizard Genomic DNA Purification Kit (Promega), was used for PCR amplification with oligonucleotide primers designed from the BmCatD cDNA sequences. The nucleotide sequence was determined as described previously .
A baculovirus expression vector system , using the Autographa californica nucleopolyhedrovirus (AcNPV) and an insect cell line Sf9, was employed for the production of recombinant BmCatD protein. Recombinant BmCatD purification, antibody preparation, and Western blot analysis were performed as described previously . The loading volume of protein samples in all Western blot analyses was 5 μg/lane. Tunicamycin treatment was performed as described previously . Aspartic proteinase activity of BmCatD was measured as described previously .
Total RNA was isolated as described . Northern blot and its image analysis were performed as described previously . The loading volume of total RNA in all Northern blot analyses was 5 μg/lane.
DNA fragmentation assay
DNA fragmentation from larval fat body and larval gut was assayed using an Apoptotic DNA-Ladder Kit (Roche Applied Science, Germany) according to the manufacturer's protocols. DNA was analyzed on a 1.0% agarose gel and visualized by ethidium bromide staining.
Hormonal treatment and viral injection
Twenty-hydroxyecdysone (20E, Sigma) was dissolved in distilled water and stored at -20°C until used. Twenty micrograms of 20E dissolved in 20 μl of distilled water was injected into B. mori larvae on day 1 of the fifth instar. Fifty nanograms of a juvenile hormone analogue, fenoxycarb (Sankyo, Japan), dissolved in 20 μl of acetone were applied topically to larvae with a micropipette along the dorsal midline. For viral infection, BmNPV [44, 46] was suspended in TC100 medium. B. mori larvae on day 1 of the fifth instar were injected with 50 μl of a viral suspension (1.0 × 105 PFU/larva). The fat body from all treated larvae was collected at 1-day intervals post-treatment and washed twice with PBS. Total RNA and genomic DNA were isolated from the tissues as described above. For injection in experiments, larvae of B. mori were injected with a sample solution between the first and second abdominal segments with a microsyringe. Each assay was replicated three times based on three independent tissue preparations. For comparison of relative BmCatD mRNA levels, statistical analysis of images of Northern blots was performed with Tukey's pairwise comparison test. Results are shown as mean ± SE of three animals per group. Significant P values were obtained by Tukey's test.
The MEGAscript RNAi kit (Ambion) was used to generate double-stranded RNA (dsRNA) corresponding to nucleotides 226 to 741 of the BmCatD cDNA. T7 promoter sites were added to the PCR primers BmCatD-Fi (5'-GCGTAATACGACTCACTATAGGGAGACCGCAGTCGTTCAAGGTGGTA-3') and BmCatD-Ri (5'-GCGTAATACGACTCACTATAGGGAGAGAACTCCCAGTACGTGTCCCG-3'). PCR reactions were conducted to generate complementary templates with a single T7 promoter site. T7 RNA polymerase was used to transcribe single-stranded RNA (ssRNA) from each DNA template over 4 h at 37°C. BmCatD dsRNA was produced by mixing solutions containing equal amounts of complementary ssRNA, incubating at 75°C for 5 min, and allowing the solution to cool down to room temperature. DNA and ssRNA were removed from the solution by digestion with DNase I and RNase at 37°C for 1 h. The dsRNA was purified using purification cartridges provided in the kit and dsRNA was eluted with two successive 50 μl washings of pre-heated (95°C) 10 mM Tris-HCl (pH 7.0) containing 1 mM EDTA. Finally, total dsRNA was quantified from the A260. BmCatD dsRNA (≈1 mg ml-1) was injected into larvae or pupae of B. mori (injection volume ≈50 μl/individual) using a sterile needle. After injection, all individuals were covered with paraffin.
This work was supported by a grant from the Biogreen 21 Program, Rural Development Administration, Republic of Korea and the Brain Korea 21 Project, the Ministry of Education, Republic of Korea.
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