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Biologia 68/3: 559—564, 2013 Section Cellular and Molecular Biology DOI: 10.2478/s11756-013-0182-6 Molecular cloning and expression analysis of glucose-regulated protein 78 (GRP78) gene in silkworm Bombyx mori Xing-Zi Xi1 & Ke-Shi Ma2* 1 Department of Education Sciences, Xinxiang University, Xinxiang 453003, People’s Republic of China; e-mail: [email protected] 2 College of Life Sciences, Zhoukou Normal University, Zhoukou 466001, People’s Republic of China Abstract: GRP78 (78 kDa glucose-regulated protein), also known as BiP (immunoglobulin heavy-chain-binding protein), is an essential regulator of endoplasmic reticulum (ER) homeostasis because of its multiple functions in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. In this report, we cloned the full-length cDNA of GRP78 from silkworm Bombyx mori (BmGRP78). It is 2645 bp, including an open reading frame (ORF) of 1977 bp encoding a polypeptide of 658 amino acids. We sequenced the ORF sequence from genomic DNA, and found only one intron (104 bp) existed in Bmgrp78 gene structure. The deduced amino acid sequence of Bmgrp78 carries the ER retention signal KDEL at its C-terminus and is highly homologous to GRP78 of Fenneropenaeus chinensis (83.59%) and Homo sapiens (80.27%). RT-PCR analysis shows that Bmgrp78 is ubiquitously expressed in tissues of silkworm. Heat shock over 35 ◦C notably enhanced the expression of Bmgrp78 in head tissues. In response to starvation, the transcript level of Bmgrp78 in head tissues was 2.8-fold (at WS stage) and 3.2-fold (at SD1 stage) higher than the control level, but it remained stable in the midgut tissues. After silkworms were challenged by bacteria, the relative expression level of Bmgrp78 in midgut was up-regulated and reached maximal level at 24 hour after injection, and remained higher level than that of controls at about 48 hour post challenge. We infer that BmGRP78 may play important roles in chaperoning, protein folding and immune function of silkworm. Key words: silkworm; GRP78; heat shock; starvation; bacterial challenge. Abbreviations: ER, endoplasmic reticulum; EST, expressed sequence tag; GRP78, glucose-regulated protein 78; HSP70, heat shock protein 70; ORF, open reading frame; RACE, rapid amplification of cDNA ends. Introduction Endoplasmic reticulum (ER) is a multifunctional organelle which controls important cellular processes, including protein synthesis, protein trafficking, Ca2+ homoeostasis and apoptosis (Ma & Hendershot 2004; Schröder & Kaufman 2005). Disruption of any of these processes causes ER stress, and prolonged ER stress leads to cell death. One characteristic of the ER stress is the induction of the ER resident stress proteins, such as glucose-regulated proteins (GRPs). One of the best characterized GRPs in mammal, GRP78 (78 kDa GRP), also known as BiP (immunoglobulin heavy-chain-binding protein), is thought to function in ER stress, oxidative damage and Ca2+ depletion (Rao et al. 2002; Qian et al. 2005; Kitamura & Hiramatsu 2010). Additionally, it is an antiapoptosis protein. Overexpression in vitro showed that GRP78 could protect cells against cell death caused by disturbances of ER homoeostasis (Rao et al. 2002; Reddy et al. 2003). Interestingly, recent works have manifested the role of GRP78 in the de- fence system against foreign pathogens (Luan et al. 2009). Currently, there is no more information of GRP78 in silkworm, compared with numerous works on mammalian GRP78s. In the present paper, we cloned the full-length cDNA of GRP78 from silkworm Bombyx mori (Bmgrp78), and studied the expression profiles of Bmgrp78 in response to thermal stress, starvation and bacterial challenge. The data could be very helpful to understand the physiological and immune roles of GRP78 in silkworm. Material and methods Animals and treatments Silkworms of the normal strain (DaZao) were reared routinely on mulberry leaves at 25±0.5 ◦C under a 12L:12D photoperiod and 70% relative humidity. After animals had ecdysed to the last larval stage (5th instar), they were staged and synchronized according to previously established method (Kiguchi & Agui 1981). For starvation treatment, starting from L5D3 (5th instar, Day 3), silkworm larvae were * Corresponding author c 2013 Institute of Molecular Biology, Slovak Academy of Sciences Unauthenticated Download Date | 6/12/17 7:51 AM X.-Z. Xi & K.-S. Ma 560 subjected to complete food withdrawal. Six larvae were randomly selected at five time points, corresponding to L5D3, L5D4, L5D5, WS (wandering stage), and SD1 (Spinning phase, Day 1) (Casati et al. 2012). The head and midgut of larvae were dissected out and stored in liquid nitrogen for RNA extractions. For thermal stress, larvae (L5D3, 20 animals/group) were cultured at 30 ◦C for 24 hours, followed by 35 ◦C for 24 hours, and 40 ◦C for 24 hours. Six larvae were randomly selected from each group, and their heads were preserved in liquid nitrogen immediately for RNA extraction. For the bacterial challenge experiment, the larval (5th instar, Day 3) hemocoels were injected with 10 µL/larva of live E. coli DH5α (resuspended in phosphate buffered saline, OD600=0.8). The non-injected larvae and larvae received injections of 10 µL autoclaved phosphate buffered saline were used as blank group and control group, respectively. The midgut tissues were collected at 4, 8, 12, 16, 24 and 48 hours post-injection. RNA extraction and Bmgrp78 cDNA cloning Three or four of silkworm larvae (5th instar, Day 3) were collected and grinded in liquid nitrogen to powders and stored in Trizol reagent (Invitrogen, USA). Total RNA was extracted and treated with DNase I (Takara, Japan) for removal of DNA contamination. The RNA was quantified by the ultra-violet spectrophotometer, and then 2 µg RNA were used for reverse transcription. A 640 bp expressed sequence tag (EST) fragment for grp78 gene was previously obtained by differential-display RT-PCR in our laboratory. Based on the known EST of Bmgrp78 cDNA, Bmgrp78 3’RACE specific primer (5’-GAT GAC CAA ACT CAT TCC TCG TAA CAC-3’) and Bmgrp78 5’-RACE specific primer (5’-GCA CGG TTG TCT TTT CTG ATG TCC TTG3’) were designed for rapid amplification of cDNA ends (RACE). Both 5’-RACE and 3’-RACE were carried out using a TaKaRa RACE cDNA amplification kit according to the manufacturer’s instructions. The PCR products were gel-purified, ligated into the pUCm-T vector, and submitted for sequencing. The resulting sequences were verified and subjected to cluster analysis. Amplification of Bmgrp78 ORF sequences from genomic DNA Genomic DNA was extracted using the Takara genomic DNA extraction kit Ver.3.0 according to the manufacturer’s instructions. Based on Bmgrp78 cDNA sequence, we designed a pair of specific primers (forward: 5’-ATG GTC AAA ATG CGG TGG AGT ATG TTC-3’; reverse: 5’-GAT TCA GTC TGA TTA CAA CTC GT CC-3’) to amplify Bmgrp78 open reading frame (ORF) sequences from genomic DNA. The PCR program was carried out at 94 ◦C for 5 min, followed by 30 cycles of 94 ◦C for 30 s, 62 ◦C for 30 s, 72 ◦C for 3 min and a final extension step at 72 ◦C for 10 min. The PCR products were sequenced. Homology analysis The similarity analysis of nucleotide and amino acid sequences was carried out using BLASTn and BLASTp tools at the National Center of Biotechnology Information website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The deduced amino acid sequence and protein motif features were analyzed with the Expert Protein Analysis System (http://www.expasy.org/). Multiple alignments of the GRP78 protein sequences were performed using the software Dnaman 6.0 (http://www.lynnon.com/pc/framepc.html). A phylogenetic tree was constructed using the programs Clustal X 1.83 (http://www.clustal.org/) and Mega 3.1 (http://www.megasoftware.net/) based on the amino acid sequences of BmGRP78 and its homologues. Bootstrap analysis was used with 1000 replicates to test the relative support for the branches produced by neighbour-joining analysis. Detecting the tissue distribution of Bmgrp78 gene by RTPCR For spatial expression analysis of Bmgrp78 gene, 7 main tissues, head, muscle, midgut, silk gland, fat body, Malpighian tubule and gonads, were dissected from Day 3 of the 5th instar larvae, and also immediately frozen in liquid nitrogen, respectively. Every tissue sample was collected from three or four larvae. cDNA was synthesized as described above. A pair of specific primers (forward: 5’-TGC TGT TGT TAC TGT GCC TGC TT-3’; reverse: 5’-ATC AAA GGT TCC ACC ACC CAA AT-3’) were designed to amplify Bmgrp78 cDNA fragment of 193 bp. Two specific primers, BmActinF (5’-ACC CAT CTA CGA AGG TTA CGC-3’) and BmActinR (5’-ACG AAC GAT TTC CCT CTC AGC-3’) were used for controls. The PCR program was carried out at 94 ◦C for 5 min, followed by 24 cycles of 94 ◦C for 30 s, 58 ◦C for 30 s, 72 ◦C for 30 s and a final extension step at 72 ◦C for 5 min. Changes of expression pattern of Bmgrp78 in response to different stressors In order to detect the relatively accurate expression pattern of Bmgrp78 in response to different stressors, the quantitative RT-PCR was performed using Real-Time PCR Detection System with a SYBR Premix Ex Taq kit (Takara, Japan). The PCR was carried out as follows: 30 s at 95 ◦C, followed by 40 cycles of 5 s at 95 ◦C, 1 min at 60 ◦C. The primers of the Bmgrp78 gene and the actin gene for realtime PCR analysis were the same as those in the RT-PCR analysis. The determination of the expression ratios was carried out using the method 2−∆∆CT , described by Livak & Schmittgen (2001). The data obtained from quantitative RT-PCR analysis for the expression of Djgrp78 were subjected to one-way analysis of variance (one-way ANOVA). Differences were considered significant at P < 0.05. Results cDNA cloning and gene structure of silkworm Bmgrp78 gene Based on the Bmgrp78 EST sequence, we designed two specific primers to clone the full-length cDNA sequence of Bmgrp78 gene. A cDNA fragment of approximately 1200 bp was amplified using a Bmgrp78 3’RACE specific primer and a TaKaRa 3’-RACE inner primer, which contains stop codon TAA and canonical polyadenylation signal sequence AATAAA. A cDNA fragment of approximately 1000 bp containing a start codon was amplified using a Bmgrp78 5’-RACE specific primer and a TaKaRa 5’-RACE inner primer. A 2645 bp nucleotide sequence representing the complete cDNA sequence of Bmgrp78 gene was obtained by cluster analysis of the two above fragments. In addition, we sequenced the ORF sequences from genomic DNA, and found only one intron (104 bp) existed in the Bmgrp78 gene structure. The intron interrupts the codon AAG at the position of 587 and the codon GTT at the position of 588. The 5’- and 3’-putative splicing site follows the typical “GT-AG” rule (data not shown). Unauthenticated Download Date | 6/12/17 7:51 AM Glucose-regulated protein 78 in silkworm 561 Fig. 1. Complete BmGRP78 nucleotide and deduced amino acid sequence from silkworm Bombyx mori. The signal peptide is signified in bold letters and the C-terminal KDEL in bold and italics represents the ER retention sequence. Three HSP70 protein family signatures are underlined. The ATP/GTP-binding site motif A (P-loop) is double-underlined. The start codon, stop codon and the polyadenylation signal (AATAAA) are boxed. Characterization of BmGRP78 The cDNA sequence of the Bmgrp78 gene was deposited in GenBank (Benson et al. 2012) under the accession No. JX948747. The full-length cDNA of Bmgrp78 was of 2645 bp, including a 5’-terminal untranslated region of 134 bp, a 3’-terminal untranslated region of 534 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail (Fig. 1), and an ORF of 1977 bp encoding a polypeptide of 658 amino acids with a predicted molecular mass of 73.13 kDa and theoretical isoelectric point of 5.03. The Signal P software analysis indicated that BmGRP78 contains a signal peptide of 20 amino acids (MVKMRWSMFALVLVVLAVCA). SMART analysis showed that BmGRP78 displays three conserved heat shock proteins 70 (HSP70) family signatures: IDLGTTYS at the position of 36-43, VFDLGGGTFDVSLL at 224-237, and IVLVGGSTRIPKVQQ at 361-375. The ATP/GTPbinding site motif A (P-loop), AEAYLGKK, was located at amino acid residues 159-166 (Techel et al. 1998; Luan et al. 2009). The C terminal of BmGRP78 contains the putative ER targeting tetrapeptide KDEL (residues 655-658) (Fig. 1). Homology analysis of BmGRP78 A sequence homology search using BLASTn and BLASTp tools revealed that BmGRP78 is highly homologous with other GRP78s from invertebrates and vertebrates. It displayed a high degree of sequence similarity to GRP78 of Fenneropenaeus chinensis (83.59%, EF032561), Dugesia japonica (75.45%, JN968463), Danio rerio (79.21%, NP 998223) and Homo sapiens (80.27%, NP 005338). In addition, many GRP78 homologues from different species of insects were collected from the GenBank (Benson et al. 2012). The complete alignment was done upon these sequences, and showed that all of these homologues contain the ER retrieval signal KDEL or RDEL. These tetrapeptides are responsible for the retention of proteins in the ER lumen (Munro & Pelham 1987; Persson et al. 2005; Byun et al. 2007), which suggested that BmGRP78 is an ERlocalized protein. Based on the sequences of BmGRP78 and its homologues in insects, a phylogenetic tree was constructed (Fig. 2). The phylogenetic tree shows that animals from Lepidoptera, Hymenoptera, Diptera and Orthoptera are clustered together, respectively. Interestingly, all of the complete metamorphosis insects are clustered together, and form a sister branch with that of incomplete metamorphosis insects. This phenomenon suggests that GRP78s in insects display highly species specificity. Unauthenticated Download Date | 6/12/17 7:51 AM X.-Z. Xi & K.-S. Ma 562 Fig. 2. A phylogenetic tree of GRP78 family members in insects constructed with the neighbour-joining method. The GenBank accession numbers are in brackets. Numbers at each branch indicate the percentage of times a node is supported in 1000 bootstraps pseudoreplication by neighbour joining. Fig. 3. RT-PCR analysis of the tissue distribution of Bmgrp78 mRNA in Bombyx mori. Lanes 1-7, head, fat body, muscle, silk gland, midgut, gonads and Malpighian tubule, respectively. Bmactin was used as the control to normalize the amount of templates. Analysis of the tissue distribution of Bmgrp78 gene in silkworm RT-PCR showed that the transcript of Bmgrp78 gene could be detected in all seven tissues as described above (Fig. 3). High expression was observed in head, midgut and gonads, in contrast to the weak signals in muscle, fat body, silk gland and Malpighian tubule. Expression pattern of Bmgrp78 in response to different stressors Expression profile of Bmgrp78 after heat shock treatment is shown in Figure 4A. Silkworms were transferred from 25 ◦C culture condition to 30 ◦C for 24 hours, Bmgrp78 expression level had no notable change in contrast to the normal level. After elevating the culture temperature to 35 ◦C for 24 hours, Bmgrp78 transcription level was 1.7-fold higher than the control point. After elevating the culture temperature to 40 ◦C for 24 hours, silkworms could still survive, with Bmgrp78 mRNA level 2.2-fold higher than the control level. Previous studies have revealed that GRP78 could be induced by starvation (Lee et al. 2002; Mamady & Storey 2006). Therefore, we were greatly interested in understanding whether starvation influences GRP78 expression in silkworms. We examined the Bmgrp78 mRNA in head and midgut at different time points after the initiation of starvation. In the head of silkworms, the transcriptional level of Bmgrp78 remained unchanged after 24 hours starvation (L5D4), increased slightly (1.3-fold) after 48 hours starvation (L5D5), and reached a higher level at WS (2.8-fold) and SD1 (3.2fold) stage (Fig. 4B). Unexpectedly, we did not observe the up-regulation of Bmgrp78 expression in the midgut of the starved larvae during the period of starvation (Fig. 4B). In addition, we investigated the bacterial challenge on the effects of Bmgrp78 expression. At 8 hours postinjection, the mRNA level of Bmgrp78 began to rise (1.2-fold higher than that of blank), and reached the maximum (2.5-fold higher than blank group) at 24 hours post-injection (Fig. 4C). At 48 hours post injection, the transcriptional level of Bmgrp78 in midgut of bacteria-challenged group remained higher than that of controls. The phosphate buffered saline challenged group was used as the control, and there was no significant difference from 0 to 48 hours (Fig. 4C). Discussion In this paper, the complete cDNA sequence of GRP78 gene was successfully cloned from silkworm Bombyx mori. Conserved sequences and characteristic motifs, such as HSP70 family signatures, as well as ATP/GTPbinding motif, were found in the deduced BmGRP78 amino acid sequence. The signal peptide at the Nterminus and the tetrapeptide KDEL at the extreme C-terminus of the BmGRP78, ensure its residency in ER. KDEL is a retrieval motif essential for the preUnauthenticated Download Date | 6/12/17 7:51 AM Glucose-regulated protein 78 in silkworm Fig. 4. Expression of Bmgrp78 in response to different stressors by real-time PCR. (A) Heat shock treatment. 25 ◦C indicates animals cultured in 25 ◦C as control; 30 ◦C indicates animals cultured in 30 ◦C for 24 hours; 35 ◦C indicates animals cultured in 30 ◦C for 24 hours, followed by 35 ◦C for 24 hours; 40 ◦C indicates animals cultured in 30 ◦C for 24 hours, followed by 35 ◦C for 24 hours and 40 ◦C for 24 hours. (B) Starvation treatment starting from L5D3 up to SD1 stage; L5D3 as control group. (C) The larvae were challenged by bacteria. Asterisk indicates significant differences comparing to that of control samples (*P < 0.05, **P < 0.01). Values are expressed as mean ± S.D. (n = 6) of three independent PCR amplification. cise sorting of ER resident proteins along the secretory pathway. The KDEL proteins, such as GRP78, calreticulin and protein disulfide isomerase, perform essential functions in the ER related to protein folding as well as 563 assembly (Munro & Pelham 1986). Our work revealed that mRNA of Bmgrp78 is ubiquitously distributed in all tested tissues in silkworm, especially in head, midgut and gonads displaying intense signals. The molecular characteristic of BmGRP78 and the non-induced level of expression indicated that it might play multiple functions in silkworm as its homologues reported in other living organisms. GRP78, the ER member of the HSP70 family, is regulated by heat and/or other stresses (Shiu et al. 1977; Sun et al. 2006; Quinones et al. 2008). In this paper, we verified that thermal stress and starvation can induce the up-regulation of Bmgrp78 expression in silkworms, especially in head tissues. However, we have not observed the notable changes of Bmgrp78 expression in silkworm midgut when deprived of food. This phenomenon may due to the flexibility of the gastrointestinal tract of animals, which can conserve energy through self-digestion in response to food deprivation (Chediack et al. 2012; Zeng et al. 2012). Interestingly, other studies reported that GRP78 protein level was higher in brain than in other tissues during animal hibernation (Lee et al. 2002; Mamady & Storey 2006). The head with the central nervous system is more sensitive to ER stress. GRP78 accumulation in head tissues may have a neuroprotective role in response to ER stress. This hypothesis has been supported by the findings that overexpression of GRP78/BiP diminishes α-synuclein neurotoxicity and the induction of GRP78 expression protects hippocampal neurons against excitotoxicity and apoptosis (Yu et al. 1999; Nakka et al. 2010). Recently, more and more evidences indicate that GRP78 plays diverse roles beyond the ER (Quinones et al. 2008; Morito & Nagata 2012). Studies have shown that GRP78 is expressed on the cell surface in many tissue types, and involved in transducing signals from ligands as disparate as activated α2-macroglobulin and antibodies (Gonzalez-Gronow et al. 2009; Zhang et al. 2010). In the present study, the mRNA transcripts in midgut increased and reached the maximum at 24 hours and maintained high level at 48 hours following the bacteria injection. Luan et al. (2009) reported that white spot syndrome can induce GRP78 expression in Fenneropenaeus chinensis. Our results indicate that BmGRP78 is potentially involved in silkworm immune response to bacterial infection. These data would be helpful to understand the significance of GRP78 in silkworm immune defence. In this study, we successfully cloned a full-length grp78 cDNA from silkworm Bombyx mori for the first time, and analyzed its expression pattern in response to thermal stress, starvation, and bacterial challenge. Our data indicate that Bmgrp78 may be a multifunctional gene in silkworm. Acknowledgements We thank Dr. Oliver Clarke and Youzhong Guo from Unauthenticated Download Date | 6/12/17 7:51 AM 564 Columbia University for critical reading of this manuscript. This work was supported by the Young and Key Teacher in Colleges and Universities of Henan Province (2011GGJS163), and the Natural Science Research Program of Education Department of Henan Province (13A180107). References Benson D.A., Karsch-Mizrachi I., Clark K., Lipman D.J., Ostell J. & Sayers E.W. 2012. GenBank. Nucleic Acids Res. 40 (Database Issue): D48–D53. Byun M., Wang X., Pak M., Hansen T.H. & Yokoyama W.M. 2007. Cowpox virus exploits the endoplasmic reticulum retention pathway to inhibit MHC class I transport to the cell surface. Cell Host Microbe 2: 306–315. Casati B., Terova G., Cattaneo A.G., Rimoldi S., Franzetti E., de Eguileor M. & Tettamanti G. 2012. 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Chem. 285: 15065–15075. Received November 3, 2012 Accepted March 17, 2013 Unauthenticated Download Date | 6/12/17 7:51 AM