Cloning and analysis of DnaJ family members in the silkworm, Bombyx mori

    Cloning and analysis of DnaJ family members in the silkworm, Bombyx mori Yin¨u Li, Cuiyu Bu, Tiantian Li, Shibao Wang, Feng Jiang, Yongzhu Yi, Huipeng Yang, Zhifang Zhang PII: DOI: Reference:

S0378-1119(15)01188-9 doi: 10.1016/j.gene.2015.09.079 GENE 40897

To appear in:

Gene

Received date: Revised date: Accepted date:

15 June 2015 24 September 2015 28 September 2015

Please cite this article as: Li, Yin¨ u, Bu, Cuiyu, Li, Tiantian, Wang, Shibao, Jiang, Feng, Yi, Yongzhu, Yang, Huipeng, Zhang, Zhifang, Cloning and analysis of DnaJ family members in the silkworm, Bombyx mori, Gene (2015), doi: 10.1016/j.gene.2015.09.079

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ACCEPTED MANUSCRIPT Cloning and Analysis of DnaJ Family Members in the Silkworm, Bombyx

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Yinü Li1, Cuiyu Bu1, Tiantian Li1, Shibao Wang1, Feng Jiang1, Yongzhu Yi2, Huipeng Yang1, Zhifang Zhang1*

Biotechnology Research Institute, Chinese Academy of Agricultural Sciences,

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Beijing, China

The Sericultural Research Institute, Chinese Academy of Agricultural Sciences,

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Zhenjiang, China

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E-mail: Yinü Li, [email protected]

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Cuiyu Bu, [email protected] Tiantian Li, [email protected]

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Shibao Wang, [email protected] Feng Jiang, [email protected] Yongzhu Yi, [email protected] Huipeng Yang, [email protected] Zhifang Zhang, [email protected] Tel: +86-10-82105495 Fax: +86-10-82103006 *Corresponding author

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ACCEPTED MANUSCRIPT Abstract: Heat shock proteins (Hsps) are involved in a variety of critical biological functions,

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including protein folding, degradation, and translocation and macromolecule assembly,

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act as molecular chaperones during periods of stress by binding to other proteins. Using expressed sequence tag (EST) and silkworm (Bombyx mori) transcriptome databases, we identified 27 cDNA sequences encoding the conserved J domain, which

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is found in DnaJ-type Hsps. Of the 27 J domain-containing sequences, 25 were

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complete cDNA sequences. We divided them into three types according to the number and presence of conserved domains. By analyzing the gene structures, intron numbers,

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and conserved domains and constructing a phylogenetic tree, we found that the DnaJ

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family had undergone convergent evolution, obtaining new domains to expand the

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diversity of its family members. The acquisition of the new DnaJ domains most likely occurred prior to the evolutionary divergence of prokaryotes and eukaryotes. The

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expression of DnaJ genes in the silkworm was generally higher in the fat body. The tissue distribution of DnaJ1 proteins was detected by western blotting, demonstrating that in the fifth-instar larvae, the DnaJ1 proteins were expressed at their highest levels in hemocytes, followed by the fat body and head. We also found that the DnaJ1 transcripts were likely differentially translated in different tissues. Using immunofluorescence cytochemistry, we revealed that in the blood cells, DnaJ1 was mainly localized in the cytoplasm.

Keywords: silkworm; DnaJ; convergent evolution; tissue expression profile 2

ACCEPTED MANUSCRIPT 1. Introduction Heat shock proteins (Hsps) are highly conserved, act as molecular chaperones

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during periods of stress by binding to other proteins. These proteins are involved in a

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variety of important biological functions, including protein folding, degradation, and translocation and macromolecule assembly (Lindquist 1986). Hsps are divided into five major families based on their molecular weights as follows: Hsp40, Hsp60,

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Hsp70, Hsp90, and Hsp100. DnaJ is the Escherichia coli homolog of the eukaryotic

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Hsp40 family and is an important molecular chaperone. DnaJ was first identified in E. coli as a 41-kDa heat shock protein (Georgopoulos et al. 1980). Its homologs have

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been identified in yeast, plants, animals, and humans. These homologs are

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characterized by a highly conserved J domain, which is typically a 70-amino-acid

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N-terminal consensus sequence that facilitates interactions with Hsp70 family members (Kelley 1999; Greene et al. 1998). DnaJ proteins possess at least four

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conserved domains: the J domain, a glycine- and phenylalanine-rich domain (termed G/F domain), a cysteine-rich zinc-finger domain (zf domain), and a less well-conserved C-terminal domain (C domain) that is thought to be involved in substrate binding. Although they are moderately conserved, the DnaJ homologs do vary in structure. DnaJ proteins can be divided into three subtypes (Qiu et al. 2006; Cheetham and Caplan 1998). Type I DnaJ proteins contain the three conserved domains, the J domain, G/F domain, and zf domain. Type II proteins contain the J domain and the G/F domain. Type III proteins only possess the J domain. For example, human HDJ-2 (Chellaiah et al. 1993) and yeast YDJ-1 (Caplan and Douglas 1991) 3

ACCEPTED MANUSCRIPT contain the J, G/F, and zf domains; human HDJ-1 (Freeman et al. 1995) and yeast Sis1p (Lu and Cyr 1998) contain the J and G/F domains; and human DNAJB6

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(Menezes et al. 2012) and zuotin, which is a DnaJ molecular chaperone in yeast (Lu

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and Cyr 1998), contain only the J domain. The J domain is typically located at the N terminus of the protein, but there are family members, such as yeast zuotin (Zhang et al. 1992), yeast Sec63 (Sadler et al. 1989), and human auxilin (Ahle and Ungewickell

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1990), in which it is found in the middle of the protein or at the C terminus.

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DnaJ proteins are cofactors for DnaK/Hsp70. By stimulating ATPase activity via the J domain, DnaJ is able to change the conformation of DnaK/Hsp70, leading to the

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folding of substrate polypeptides and the binding to Hsp70 (Kelley 1999). Through its

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interaction with Hsp70, DnaJ/Hsp40 is involved in DNA transcription, viral

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replication, protein transmembrane transport, cell proliferation, signal transduction, the suppression of apoptosis, and other biological processes (Kelley 1998).

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In E. coli, DnaK and DnaJ are the two main types of molecular chaperones. DnaJ was originally shown to be involved in DNA transcription initiation and phage replication. J-domain mutations were shown to cause functional defects in DNA and RNA synthesis, cell division, and protein degradation (Wickner et al. 1991; Yochem et al. 1978). In addition, ABBP-2, a mammalian DnaJ homolog, has been shown to be involved in editing the RNA of apolipoprotein B through its interaction with Hsp70 (Lau et al. 2001). It is now known that DnaJ proteins also stimulate the ATPase activity of chaperone proteins, making them important in many biological functions. For example, auxilin participates in the uncoating and endocytosis of clathrin-coated 4

ACCEPTED MANUSCRIPT vesicles by stimulating the ATPase activity of Hsc73, which is an Hsp70 family member (Ungewickell et al. 1995). The stimulation of ATPase activity requires the

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conserved J domain of DnaJ. Furthermore, in yeast, Ydj-1 regulates polypeptide

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translocation across the endoplasmic reticulum and the mitochondrial membrane (Becker et al. 1996). In addition, ERdjl/MTJ1 (Chevalier et al. 2000), ERdj2/hSec63 (Scidmore et al. 1993), ERdj3/HEDJ (Yu et al. 2000), ERdj4/MDG1 (Kurisu et al.

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2003), and Erdj5 (Cunnea et al. 2003) are J domain-containing Hsp40 proteins that

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are located in the endoplasmic reticulum and regulate protein transport from the endoplasmic reticulum to the cytoplasm via interactions with the Hsp70-like

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cellular functions.

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chaperone, Bip. Thus, these chaperones have been shown to play multiple roles in

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Hsp40 may aid in the regulation of the homeostatic balance of eukaryotic cell survival and apoptosis. For example, the Hsp70-DnaJ molecular chaperone complex

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can protect cells from apoptosis induced by heat shock, nitric oxide, and ischemic stress (Gotoh et al. 2004). In addition, the large T antigen of the SV40 virus also contains a J domain, which may act as a proto-oncogene via its interaction with the product of the retinoblastoma gene, Rb (Srinivasan et al. 1997; Stubdal et al. 1997). Furthermore, the human protein Tidl, which is a homolog of the Drosophila tumor suppressor Tid56 (Kurzik-Dumke et al. 1995), can reduce the degree of tumor malignancy and promote the apoptosis of cancer cells (Trentin et al. 2004; Kim et al. 2004). Finally, the DnaJ protein P58PIK is a protein kinase inhibitor that can promote protein synthesis by inhibiting double-stranded RNA-activated serine/threonine 5

ACCEPTED MANUSCRIPT kinases and may also function as a proto-oncogene (Melville et al. 2000; Tan et al. 1998). Thus, the Hsp40 family has a wide array of functions as well as differing

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expression profiles and subcellular localizations.

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At present, there are few reports related to the DnaJ gene family in insects. Sirigineedi et al. (Sirigineedi et al. 2014) conducted phylogenetic analyses of 13 DnaJ gene sequences obtained by BLAST using the Bombyx mori genomic DNA database.

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They studied the expression profiles of DnaJ homologs in diapause-induced eggs at

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different time intervals. The phylogenetic analysis of the DnaJ family members found in this insect and those found in other species confirmed the complicated evolutionary

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history of this protein.

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In this study, we cloned and analyzed 27 members of the silkworm DnaJ gene

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family. Thirteen of the cDNA sequences with a conserved DnaJ J domain were identified using EST databases and random amplification of cDNA ends (RACE)

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technology; these sequences were named DnaJ1-DnaJ13. Additionally, 14 cDNA sequences with a conserved J domain were found through transcriptome data retrieval and were named DnaJ14-DnaJ27. Using quantitative RT-PCR, we analyzed the expression differences among DnaJ1-DnaJ13 in 10 different tissues in day 3 fifth-instar larvae as well as differences among DnaJ1-DnaJ27 in the fat bodies of the larvae. The tissue distribution and location of DnaJ1 in the blood cells were analyzed by western blotting and immunofluorescence cytochemistry.

2. Materials and Methods 2.1. Materials 6

ACCEPTED MANUSCRIPT The silkworm strain JY-1 was kept at the Sericultural Research Institute, Chinese Academy of Agricultural Sciences. The silkworms were fed fresh mulberry leaves in

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an environment maintained at 25°C with 70-80% relative humidity (Lu 1991).

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2.2. Transcriptome database retrieval and sequence assembly We used the conserved J domain of the DnaJ protein to search the GenBank EST

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database, limiting the search to the organism Bombyx mori. Furthermore, using the conserved J domain sequence, we performed a BLAST search of the silkworm

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transcriptome database (SilkTransDB) (Li et al. 2012), which was built by our

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laboratory. The sample used to establish the SilkTransDB was pooled from over 100

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samples from different tissues at various stages of silkworm development; therefore, this database represents a comprehensive overview of the silkworm transcriptome.

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The EST sequences are listed in Table 1. We identified the silkworm DnaJ cDNA sequences by assembling homologous EST sequences using the DNASTAR software

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package (http://www.dnastar.com).

2.3. RNA extraction, cDNA synthesis, and RACE Total RNA was extracted from a variety of tissues (head, epidermis, midgut, wing disc, silk gland, tracheal cluster, Malpighian tubule, hemocyte, ovary, testis, and fat body) from day 3 fifth-instar larvae using a Trizol kit (Invitrogen). Total RNA (1 g) was used to perform RACE amplification of the 3′ and 5′ ends using the Clontech (CA, USA) RACE kit. The primers used are listed in Table 2. The PCR products were separated using 1% agarose gel electrophoresis and purified using the Axygen DNA Gel Extraction Kit. The products were then ligated to the pMD18-T vector (TaKaRa), 7

ACCEPTED MANUSCRIPT and the positive clones were selected and sequenced.

2.4. DNA sequence analysis, protein sequence alignment, and

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phylogenetic analysis

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The genomic sequences were identified based on cDNA sequences, using the silkworm genomic database (SilkDB) (http://silkworm.genomics.org.cn/) (Xia et al. 2004; Wang et al. 2005). The protein sequences were analyzed by ClustalX

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(Thompson et al. 2002) and GeneDoc. The cladogram was constructed according to

analyzed

using

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these data by MEGA4 (Tamura et al. 2007). The conserved protein domains were the

NCBI

conserved

domain

database

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(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (Marchler-Bauer et al. 2011).

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2.5. RT-PCR and data processing

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RT-PCR was conducted according to the user manual for Toyobo SYBR Green RT-PCR Master Mix, using BmActinA3 as a reference gene. Three independent

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samples were measured to evaluate the expression levels. The primers used for the RT-PCR are listed in Table 2. The relative expression level was measured by the equation 2–ΔCt (ΔCt = Cttarget gene – CtActinA3).

2.6. BmDnaJ1 prokaryotic expression and antibody preparation A 3’-terminal partial fragment of the DnaJ1 gene (561 bp) was cloned into the prokaryotic expression plasmid pET-28a (Novagen), using the primers J1F and J1R listed in Table 2, to construct the recombinant pET28a-BmDnaJ1 plasmid. The fusion protein His-BmDnaJ1F was expressed in E. coli and purified by nickel affinity column chromatography, using the Ni-NTA Purification System (Invitrogen). The 8

ACCEPTED MANUSCRIPT expression of the BmDnaJ1 protein fragment was confirmed by liquid chromatography–mass spectrometry. The purified protein was used to immunize New

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Zealand white rabbits for the production of polyclonal antibodies. The DnaJ1

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antibody preparation was carried out at the Animal Experiment Center of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

2.7. DnaJ1 tissue distribution and subcellular localization

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Proteins were extracted with the Total Protein Extraction Kit (BestBio) from a

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variety of tissues, including the epidermis, testis, ovary, wing disc, silk gland, fat body, midgut, hemolymph, and head, in silkworm day 3 fifth-instar larvae. Western blotting

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was conducted using the DnaJ1 antibody. The subcellular localization in the blood

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cells of day 3 fifth-instar silkworm larvae was determined using immunofluorescence

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cytochemistry(Li et al. 2006). Samples were observed with the TCS SP2 confocal microscope (Leica, Germany).

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3. Results

3.1. Sequence analysis of the silkworm DnaJ family A BLAST search was performed of the GenBank EST database using the J domain, and the search was limited to the organism Bombyx mori. As a result, several EST sequences were identified (Table 1) and compiled with homologous sequences. The first DnaJ cDNA sequence that was obtained contained a 1227-bp open reading frame (ORF) encoding a complete DnaJ protein, which was then named DnaJ1. We searched both the GenBank EST database and the SilkTransDB using the J domain of DnaJ1. We obtained a series of homologous silkworm EST sequences, which were 9

ACCEPTED MANUSCRIPT classified and assembled by the DNASTAR software package. Thirteen gene sequences, each of which encoded a conserved region of DnaJ, were identified and

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named BmDnaJ1-BmDnaJ13. Of these, 10 sequences contained complete ORFs.

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BmDnaJ8, BmDnaJ10, and BmDnaJ11 had incomplete ORFs, so we used RACE technology to obtain intact ORFs for each of these three genes. We submitted the 13 sequences to GenBank (accession numbers: FJ592074-FJ592086).

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Using the J domain to identify coding regions of interest, we found 27 cDNA

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sequences containing DnaJ domains in the silkworm transcriptome database. In addition to the 13 J domain-containing proteins described above, we found and named

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the additional 14 (BmDnaJ14-BmDnaJ27), of which two (BmDnaJ26 and BmDnaJ27)

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had incomplete ORFs. We were unable to obtain the complete ORFs for these two

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cDNAs using RACE. We submitted the additional 14 sequences to GenBank (accession numbers: JN872888–JN872902). Detailed information is listed in Table 1.

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The members of the DnaJ family that were identified all contained a J domain, which is most often located at the N terminus. BmDnaJ1, BmDnaJ2, and BmDnaJ14 contained the J domain, G/F domain, zf domain and C domain. DnaJ3 and DnaJ5 contained the J domain, G/F domain and C domain, and the remaining members contained only the J domain. Thus, DnaJ1, DnaJ2, and DnaJ14 were classified as type I proteins, DnaJ3 and DnaJ5 as type II proteins, and the remaining members as type III proteins. Using genomic sequence alignment, we analyzed the gene structures of the silkworm DnaJ family members, as shown in Fig. 1.

3.2. Multiple sequence alignment and evolutionary analysis of the 10

ACCEPTED MANUSCRIPT silkworm DnaJ protein Sequence alignments of silkworm DnaJ family members were generated and

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analyzed by ClustalX and GeneDoc (Fig. 2). The conserved J domains were

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consistently located near the N termini, and the remainder of the sequences was not highly conserved. A cladogram was constructed of the 25 complete silkworm DnaJ proteins and other DnaJ family members from E. coli, Saccharomyces cerevisiae,

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Drosophila melanogaster, Tribolium castaneum (Herbst), and humans (at least three

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proteins from each species) (Fig. 3). The DnaJ/Hsp40 homologs were divided broadly into two major clades. Clade I contained 15 members, including eight type I DnaJ

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proteins, six type II DnaJ proteins and one type III DnaJ protein. All three type I

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silkworm DnaJ proteins (BmDnaJ1, BmDnaJ2, and BmDnaJ14), both type II

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silkworm DnaJ proteins (BmDnaJ3 and BmDnaJ5) and one type III silkworm DnaJ protein (BmDnaJ8) belonged to this clade. In this clade, nine DnaJ proteins formed a

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sub-clade. This sub-clade contained all eight type I DnaJ proteins and one type II DnaJ (BmDnaJ3) protein. Clade II contained the other 25 type III DnaJ proteins with the exception of the type III DnaJ protein BmDnaJ8. A total of 19 type III silkworm DnaJ proteins belonged to this clade.

3.3. Expression patterns of DnaJ family in day 3 fifth-instar larvae The tissue-specific expression profiles of DnaJ1-DnaJ13 in day 3 fifth-instar larvae were analyzed by quantitative RT-PCR (qRT-PCR, Fig. 4). The expression levels were generally higher in the wing discs and silk glands and lower in the hemocytes, Malpighian tubules, tracheal clusters, and ovaries. Overall, BmDnaJ1, 11

ACCEPTED MANUSCRIPT BmDnaJ2, BmDnaJ3, and BmDnaJ6 were expressed at higher levels, whereas BmDnaJ8, BmDnaJ11, and BmDnaJ13 showed lower levels of expression. BmDnaJ2

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was not detected in the tracheal clusters, BmDnaJ4 was not detected in the epidermis

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or the tracheal clusters, and BmDnaJ9 was not detected in the wing discs. The expression patterns of BmDnaJ1-BmDnaJ27 in the fat bodies isolated from day 3 fifth-instar larvae were analyzed by qRT-PCR (Fig. 5). The expression levels of

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and BmDnaJ26 levels were undetectable.

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BmDnaJ14-BmDnaJ27 were lower than those of BmDnaJ1–BmDnaJ13. BmDnaJ2

3.4. BmDnaJ1 tissue distribution and localization in blood cells

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Among the BmDnaJ family members, the mRNA levels of BmDnaJ1 were

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relatively high in several of the silkworm tissue types. Thus, we chose DnaJ1 for

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further study and proceeded to prepare polyclonal antibodies for the detection of this protein. The polyclonal antibody quality was tested by enzyme-linked immunosorbent

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assay (ELISA) and was deemed appropriate for further tissue distribution experiments. Based on the western blotting of proteins isolated from the tissues of day 3 fifth-instar larvae, BmDnaJ1 showed the highest expression levels in the hemocyte, followed by the fat body and the head. The epidermis, testis, and ovary expressed BmDnaJ1 at low levels, but this protein was not detectable in the midgut and wing discs (Fig. 6). As shown in Fig. 6, two hybridization bands of different molecular weights were observed. The molecular weights of form I and form II were 45 kDa and 50 kDa respectively. The 45 kDa form was present in the epidermis, hemocyte, and head, whereas the 50 kDa DnaJ protein was present in the silk gland. The fat body, testis, 12

ACCEPTED MANUSCRIPT and ovary contained two forms of this protein. BmDnaJ1 protein expression was analyzed by immunofluorescence labeling of

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the blood cells using the DnaJ1 antibody. Serum from a pre-immunized rabbit served

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as a negative control (Fig. 7). BmDnaJ1 protein was mainly distributed in the cytoplasm, with a small amount visible in the cell nucleus (Fig. 7).

4. Discussion

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The cDNA sequence and genomic structure analyses of the silkworm DnaJ family

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members with intact ORFs showed that the ORF lengths, intron numbers, and conserved domains varied widely, demonstrating that the evolutionary history of the

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DnaJ family is complex. Despite the diversity and complexity of the silkworm DnaJ

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family members, as well as the highly repetitive sequences found within their introns,

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the DnaJ gene has been highly conserved across phyla and even across kingdoms. DnaJ genes from prokaryotes, fungi, insects, and mammals were found within the

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same cluster. This indicates that many features of this family are highly conserved, although some motifs have been lost in select homologs. Future studies of the specific functions of each family member may elucidate the phylogenetic relationships among the family members. Although BmDnaJ1, BmDnaJ2, and BmDnaJ14 contained three structural domains, they were located on different chromosomes (4, 9, and 14, respectively). BmDnaJ3 and BmDnaJ5 contained J and G/F domains and were located on chromosomes 4 and 5, respectively. The five BmDnaJ members formed a separate clade with the DnaJ genes from other species. The evolutionary relationships among 13

ACCEPTED MANUSCRIPT the DnaJ family members could be considered as relatively disparate. However, Sirigineedi et al. (Sirigineedi et al. 2014) have suggested that the DnaJ gene family

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evolved from a common ancestor and that variation occurred over time.

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Type III DnaJ members contain only one DnaJ structural domain. From a purely evolutionary standpoint, the evolutionary distance between type III DnaJ proteins of different species is far, and there is a large degree of variation. The type I and type II

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DnaJ members, which all contain more than one conserved domain, formed one

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cluster with their corresponding members in other species, and even the prokaryote (Ec-DnaJ) and eukaryote (BmDnaJ14) proteins were present on a small branch in very

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close proximity. The variation is thus smaller, and the convergent evolution of the

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gene structure is more obvious in this case. We suggest that type III DnaJ may have

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been the progenitor protein type and that it gradually obtained additional conserved structural domains to form the type I and type II DnaJ members through different

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forms of duplication and/or transposition, thereby expanding the DnaJ family function. Type III BmDnaJ8 has the closest phylogenetic relationship with the corresponding type I and type II DnaJ proteins among the type III members of the BmDnaJ family. Thus, we speculate that BmDnaJ8 may be an ancestor gene of the silkworm type I and type II DnaJ genes. In addition, type II BmDnaJ3 may be an ancestor gene of other silkworm type I DnaJ genes. Furthermore, type I and type III DnaJ family members formed the closest branches in both prokaryotes and eukaryotes, indicating that the replication and/or transposition of the DnaJ gene most likely occurred prior to the evolutionary divergence of prokaryotes and eukaryotes; therefore, DnaJ appears to be 14

ACCEPTED MANUSCRIPT an ancient gene. Each of the 27 members contained a conserved J domain that interacts with

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Hsp70 (DnaK) and stimulates its ATPase activity, thereby promoting the correct

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folding of peptide substrates. This J domain is conserved across different members and different organisms, demonstrating the physiological function of DnaJ. As described in Fig. 4, BmDnaJ family members have different mRNA levels in

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different tissues. A few DnaJ mRNAs were not detected in several tissues, which was

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likely associated with the functional differences of the different DnaJ family members.

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The fat body is an important tissue in silkworm because it stores nutrients and is

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responsible for metabolism. Protein synthesis in the fat body increases in both speed

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and variety in the early fifth-instar stage of silkworm development (Price 1973). As shown in Fig. 5, the expression levels of BmDnaJ14-BmDnaJ27 were far lower than

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those of BmDnaJ1-BmDnaJ13, which explains why BmDnaJ14-DnaJ27 were not found in our previous EST analysis of DnaJ gene splicing. DnaJ1 was one of the most highly expressed family members and a major DnaJ in silkworm; therefore, its tissue distribution and localization within blood cells were analyzed by western blotting and immunofluorescence cytochemistry. Fig. 6 shows that DnaJ1 was expressed at the highest levels in the hemocytes of the silkworm day 3 fifth-instar larvae. However, in contrast to the mRNA expression results, this protein was not detected in the wing discs. The midgut and wing discs showed higher DnaJ1 mRNA levels relative to those in other tissues, and only low levels were observed in 15

ACCEPTED MANUSCRIPT the hemocytes. DnaJ1 mRNA levels do not correlate with its protein levels in different tissues. It is possible that its transcripts are differentially translated or DnaJ1 proteins

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are involved in post-translational translocation. In addition, using western blotting, we

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detected two DnaJ1 proteins with different molecular weights. These proteins may have undergone different post-translational modifications in particular silkworm tissues. In addition, the DnaJ1 gene fragment that we selected for the antibody

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preparation could have been similar to another DnaJ gene, resulting in a

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cross-reaction. Further studies should be conducted to identify whether both forms are active and to ascertain whether there is a functional difference between the two

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45-kDa and 50-kDa proteins.

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The function of a protein is closely related to its subcellular localization,

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especially in eukaryotic cells (Kumar et al. 2002). The same protein located in different cellular compartments may have different functions. According to our

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immunolabeling experiment, BmDnaJ1 was mainly found in the cytoplasm of blood cells, providing a basis for further study of the specific role of BmDnaJ1 in hemocytes.

We found that there were a minimum of 27 DnaJ members in silkworm (Bombyx mori), of which there were three type I DnaJ members and two type II members, and the remainder were classified as type III. The phylogeny evolution analysis revealed that the type I and type II DnaJ proteins may have evolved from the type III proteins through the acquisition of the G/F and zf domains. Therefore, some co-evolutionary interactions may exist among the structures and functions of the three types of DnaJ 16

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5. Conclusion

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27 members of DnaJ family in silkworm were identified and divided into three

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types according to the number and presence of conserved domains. The phylogenetic tree of 25 complete silkworm DnaJ proteins and other DnaJ family members from E. coli, Saccharomyces cerevisiae, Drosophila melanogaster, Tribolium castaneum

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(Herbst), and humans (at least three proteins from each species) showed that the DnaJ

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family had undergone convergent evolution, obtaining new domains to expand the diversity of its family members. The acquisition of the new DnaJ domains most likely

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occurred prior to the evolutionary divergence of prokaryotes and eukaryotes. The

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mRNA levels of BmDnaJ family members were different in different tissues, which

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was likely associated with the functional differences of the different DnaJ family members. The tissue distribution of DnaJ1 proteins was detected by western blotting,

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demonstrating that in the fifth-instar larvae, the DnaJ1 proteins were expressed at their highest levels in hemocytes, followed by the fat body and head. We also found that the DnaJ1 transcripts were likely differentially translated in different tissues. Using immunofluorescence cytochemistry, we revealed that in the blood cells, DnaJ1 was mainly localized in the cytoplasm. It is possible that DnaJ1 is the major chaperone workhorse in silkworm. Acknowledgments This

work

was

supported

http://www.863.gov.cn),

the

by

the

National

“863”

Basic 17

Project

Research

(No.2011AA100603, Program

of

China

ACCEPTED MANUSCRIPT (2012CB114600,

http://www.973.gov.cn)

and

the

National

Natural

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Foundation of China (No. 31200275, http://www.nsfc.gov.cn).

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Science

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Becker J, Walter W, Yan W, Craig EA (1996) Functional interaction of cytosolic hsp70 and a DnaJ-related protein, Ydj1p, in protein translocation in vivo. Molecular and cellular biology 16

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Caplan AJ, Douglas MG (1991) Characterization of YDJ1: a yeast homologue of the bacterial dnaJ

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protein. The Journal of cell biology 114 (4):609-621

Cheetham ME, Caplan AJ (1998) Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function. Cell stress & chaperones 3 (1):28-36 Chellaiah A, Davis A, Mohanakumar T (1993) Cloning of a unique human homologue of the Escherichia

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coli DNAJ heat shock protein. Biochimica et biophysica acta 1174 (1):111-113 Chevalier M, Rhee H, Elguindi EC, Blond SY (2000) Interaction of murine BiP/GRP78 with the DnaJ homologue

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ACCEPTED MANUSCRIPT Figure Legends Fig. 1. Structural analysis of the silkworm (Bombyx mori) DnaJ gene family.

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The majority of DnaJ gene family members have complete ORFs, except for

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BmDnaJ8, BmDnaJ10, BmDnaJ11, BmDnaJ26 and BmDnaJ27. BmDnaJ22, which

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consists of 846 amino acid residues, has the longest length, with the exception of the incomplete BmDnaJ26 and BmDnaJ27 proteins. BmDnaJ12 has the shortest length,

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containing only 170 amino acid residues. This figure shows that among the members identified from the genome sequence, BmDnaJ19 has the largest number of introns

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(11 introns), whereas BmDnaJ8 has none (unknown region: sequence without high

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identities in NCBI database).

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Fig. 2. Multiple sequence alignment of the DnaJ family members. The dark gray shading indicates >90% identity, and the light gray shading indicates

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40-60% identity.

Fig. 3. Evolutionary analysis of the DnaJ family.

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The cladogram was constructed by MEGA4. The GenBank accession numbers for DnaJ1–25 are shown in Table 1. The other sequence accession numbers are as follows: Dm-DnaJL2A, NP_650283; Dm-mrjA, NP_725541; DmDNAJ1, AAP31279; Tc-Hsp40, XP_966855; Tc-DnaJA2, XP_970724; Tc-TcasGA2, EFA10292; Sc-YDJ1, CAA39910; Sc-Sis1p, GAA26088; Sc-zuotin, CAA45156; Hs-hDj2, P31689; Hs-DnaJB1, NP_006136; Hs-HSJ1a, AAA09034.1; Ec-DnaJ, EGB62553; Ec-Hsc20, ACA76817.1; and Ec-djlA, EFF03059.1. Dm = Drosophila melanogaster, Tc = Tribolium castaneum, Sc = Saccharomyces cerevisiae, Hs = Homo sapiens, Ec = Escherichia coli. 22

ACCEPTED MANUSCRIPT Fig. 4. DnaJ1–DnaJ13 expression profiles in various tissues from day 3 fifth-instar larvae.

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Individual tissues were collected, and qRT-PCR was used to quantify the expression

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levels of the individual DnaJ genes.

Key: H, head; E, epidermis; WD, wing disc; SG, silk gland; He, hemocyte; MG, midgut; MT, Malpighian tubule; TC, tracheal cluster; O, ovary; and T, testis.

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Fig. 5. DnaJ1–DnaJ27 expression profiles in the fat bodies of day 3 fifth-instar

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larvae. Fat body tissue was collected, and qRT-PCR was used to quantify the expression levels of the individual DnaJ genes.

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Fig. 6. Tissue-specific distribution of BmDnaJ1 in day 3 fifth-instar larvae.

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Tissues were collected from day 3 fifth-instar larvae, and the purified proteins were

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then analyzed by western blotting with the DnaJ1 antibody. Key: E, epidermis; T, testis; O, ovary; WD, wing disc; SG, silk gland; FB, fat body;

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MG, midgut; He, hemocyte; and H, head. Fig. 7. DnaJ1 localization within the blood cells (Scale bars, 50μm). Immunofluorescent staining of BmDnaJ1 with pre-immune serum (negative control, A) and antibody against DnaJ1 (B).

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Figure 1

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Figure 2

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Figure 3

26

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Figure 4

27

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Figure 5

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Figure 6

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BP120274, BP120813, BP124909, BP181039, BP183135, BP183711, BP184291, CK525852, CK496195, CK500081, CK538632, CK538720, CK542922, CK539570, CK543848, CK488186, CK489441, CK492479, CK493676, CK515310, CK559960, CK560127, CK560152, CK563337, CK503582, BY922108, BB990679, BB988581, BJ984389, BJ983658, BB983021

silk gland; ovary; whole body; pheromone gland; midgut; eye; fat body; maxilla; blood

BmDnaJ2

AU005384, CK545739, CK485086, CK493585, CK517857, CK561914, CK563927, DY231295, DQ311436, BW999057,

whole body; silk gland; ovary; uncharacterized tissue

1

IP

1806 (28–1254)

CR

BmDnaJ1

J

G/F

ZF

C

GenBank accession no.

Location

408

7–56

74-107

141–196

112–338

FJ592074

Chr. 4

401

6–57

75-100

150–204

109–335

FJ592075

Chr. 9

US

Origin of mRNAs

cDNA length (ORF)

MA N

Related ESTs

Conserved domain (and amino acid positions)

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Gene

Number of introns

Protein length (amino acids)

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Table 1.The gene family encoding DnaJ proteins in Silkworm, Bombyx mori

≥7

2389 (177–1382)

31

ACCEPTED MANUSCRIPT

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BB991794, NM_001046827 BP182016, CK485689, CK530190, CN211993, BW997748, BY927645, BY928617, BY920199

testis; whole body; ovary; silk gland; pheromone gland

4

1202 (93–1154)

353

25–79

BmDnaJ4

BP128110, BP128225, BP181517

testis; ovary

4

2380 (30–740)

236

3–58

BmDnaJ5

BP179953, BP183588, CK518329, AB206400, CN374958, CN374959, BW997424, BY916124, NM_001043525

pheromone gland; silk gland; ovary; Malpighian tubule

2

1885 (117–1172)

351

4–57

BmDnaJ6

BP128301, BP183667, CK527417, CK487909, CK486036, CK490992, CK562770, CK563960, CN379443, BY924089, BB993975, BB989143

silk gland; blood; testis; whole body; pheromone gland; Verson's gland

4

1970 (132–1181)

349

BmDnaJ7

BP177673, CK542314, CK485183, BY916941

Malpighian tubule; ovary; silk gland; prothoracic gland

1

1597 (177–785)

BmDnaJ8

CK489761, CK560898, CK561341, BB987831

silk gland

0

BmDnaJ9

AU002994, AV402116, AV402549, BP121428, BP125436, BP125599,

silk gland; midgut; blood; ovary; fat body; eye;

7

98-119

130–326

FJ592076

Chr. 4

FJ592077

Chr. 10

FJ592078

Chr. 5

106–159

FJ592079

–

202

14–66

FJ592080

Chr. 15

1252 (146–1162)

338

36–>68

FJ592081

Chr. 23

2596 (158–1642)

494

377–436

FJ592082

Chr. 4

74-120

173–337

AC

CE P

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CR

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BmDnaJ3

32

uncharacterized tissue

BmDnaJ10

BP120578, BP121090, BP121472, CK520483, CK484976, CK489491, CK534758, CK516806, CK518288, CK558930, CK559634, CK564707, CK560511, CK562467, CK564185

testis; eye; silk gland; blood

BmDnaJ11

AV399485, AV399696, CK529304, CK529374, CK529434, CK529216, CK494316, CK533803, CK537929, CK562933

blood; ovary; testis; midgut; whole body

BmDnaJ12

AU000134, BP121668, BP182669, BP183414,

silk gland; fat body; pheromone gland;

MA N

US

CR

IP

BP181984, CK520631, CK527384, CK494359, CK538545, CK486078, CK487945, CK536077, CK514931, CK516529, CK517702, CK559412, CK560189, DQ311264, BY925833, BY917505, BY938190, BY938514, BY939058, BB982930, NM_001046720

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ACCEPTED MANUSCRIPT

269

15–71

FJ592083

Chr. 9

4

979 (79–720)

213

27–79

FJ592084

Chr. 22

2

1460 (27–539)

170

17–71

FJ592085

Chr. 12

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1397 (188–997)

AC

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4

33

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ovary; testis; whole body; prothoracic gland

≥5

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DQ311158, NM_00104665, CK539477, CK536227, CK560568, BY936032

9

1025 (104–811)

235

26–78

2311 (14–1627)

537

74–128

146-176

227–287

290–407

FJ592086

Chr. 22

JN872889

Chr. 14

BmDnaJ15

AC

BmDnaJ14

whole body; Verson's gland; eye; ovary; uncharacterized tissue; antenna; maxilla

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BmDnaJ13

BP183483, CK508422, CK541687, CK490096, CK491405, CK491784, CK546979, CK551100, CK550326, AF176014, CN375953, CN375954, CN375955, BY933441, BY941499, BY924859, BY916797, BY925704, BY934504, BY914108, BB991836, BB986697, BJ985416, BB983655, NM_001043551

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2

1100 (61–930)

289

10–64

JN872890

Chr. 18

BmDnaJ16

6

2135 (227–2053)

608

3–58

JN872891

Chr. 23

BmDnaJ17

12

2010 (68–1789)

573

13–71

JN872892

Chr. 1

BmDnaJ18

5

1365 (71–1147)

358

286–341

JN872893

Chr. 1

34

ACCEPTED MANUSCRIPT

11

2463 (114–2408)

764

BmDnaJ20

10

1732 (116–1585)

489

BmDnaJ21

≥5

899 (77–814)

245

BmDnaJ22

≥8

2848 (128–2668)

BmDnaJ23

≥1

BmDnaJ24

3

BmDnaJ25

≥10

Chr. 24

389–445

JN872895

Chr. 15

50–105

JN872896

Chr. 9

846

606–655

JN872897

Chr. 22

2090 (174–2003)

609

79–139

JN872898

Chr. 19

1343 (19–1143)

374

38–90

JN872899

Chr. 8

2668 (220–2580)

786

32–85

JN872900

Chr. 10

≥4

786 (87–?)

–

35–88

JN872901

Chr. 10

≥2

378 (?–?)

–

<1–40

JN872902

Chr. 12

IP

CR

US

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BmDnaJ27

JN872894

AC

BmDnaJ26

105–156

T

BmDnaJ19

Footnote: The silkworm genome sequences are incomplete, so we could not define the intron numbers of some silkworm DnaJ genes and the“≥” mark are used to show the intron numbers. The intron numbers of BmDnaJ26 and BmDnaJ2 are not determined, because of the incomplete cDNA sequences。“–”indicates that data were not determined (ND). “Chr.” is the abbreviation of “chromosome”.

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Bm-DnaJ1 Bm-DnaJ2 Bm-DnaJ3 Bm-DnaJ4 Bm-DnaJ5 Bm-DnaJ6

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Bm-DnaJ5

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3’ RACE: nested universal primer cDNA synthesis 3’ RACE

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Bm-DnaJ4

SP1: CAACGGCCTACGAGATACTACGT SP2: GTATTACAGACGTCGTATGGCACCT SP1: ACTGGACAGTCTACTGGAGGCAT SP2: GCTAGAACTGAACATGAACCG SP1: CCAGAACCAGAACCTACAGATC SP2: GGTCCAAACACCATTATTCAGATG F: ACTGTGCTAACAAGGGCTGAATTG R: CACTTGCATTTCAGCGCTGTCATG F: GCTCGACCTAGGGTTGAAATACC R: ACATATCAGGTCCACCGAAAATCC F: TCACTACAGGATGCTCTGACTGG R: GCAAAGCCTCTTTCTCAGCATCTG F: GCTGAAATCAATGGTCACGGAC R: CTCGTCCGTTCTCTGTGACTTTC F: GAAAGCAACGTTCGCGCAGTTC R: CGAGGTACCATCTTGCTGTATTAC F: GCGCCGCTTCCGATAACTATAC R: CGTTGTAGATGTCTATGGGCTTC

AC

Bm-DnaJ3

Application cDNA synthesis 5’ RACE: nested universal primer 5’ RACE

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Table 2: Primers Gene Primer sequence (5’ to 3’) 5'-CDS AAGCAGTGGTATCAACGCAGAGTACGCDCCCCCCCCC 5'-NUP AAGCAGTGGTATCAACGCAGAGTACGC Bm-DnaJ1 SP1: CTTGACCGAGACAGGATGGACAGCG SP2: TATCTTCGCCACGGACTGGACCACG 3'-NUP AAGCAGTGGTATCAACGCAGAGT 3'-CDS AAGCAGTGGTATCAACGCAGAGTAC(T)30VN

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3’ RACE 3’ RACE Real-time PCR Real-time PCR Real-time PCR Real-time PCR Real-time PCR Real-time PCR

36

ACCEPTED MANUSCRIPT

Bm-DnaJ11 Bm-DnaJ12 Bm-DnaJ13 Bm-DnaJ14 Bm-DnaJ15 Bm-DnaJ16 Bm-DnaJ17 Bm-DnaJ18 Bm-DnaJ19 Bm-DnaJ20

T IP

CR

US

Real-time PCR Real-time PCR

MA N

Bm-DnaJ10

Real-time PCR

Real-time PCR Real-time PCR

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Bm-DnaJ9

Real-time PCR

CE P

Bm-DnaJ8

F: TGAACACAGGAGCGAGTGTACC R: CTACCAAGATCCCGAATGTGATG F: GCGTGGTCAAAGTATGACACTGC R: GGTGTACTTCCAGATCCATCTCAG F: GACAAGAGTGCTTCAACAGACGAC R: GTCGATATCACTCATTCCTGCGC F: CTGGTTCACAAGAAGAGAAGGATG R: CTTCAGCTTCTTTCGCCTCACGG F: CCCGGACAAATCTAAGGATGAAGC R: CATGGTGGGTGTGATATTGCGAG F: CAGAAGTTGAAGGAAGCCAAGGAG R: CCTCAGAAGCCCGACGTGTAG F: AAACTCATGTGCCTTATAATCTACG R: GAGTTCATCAGCAAGTTCCATTGAC F: ATCAATTGCTCAAGCTGTGCTTG R: CTGTCAGTGTCTTTGGTACCTG F: GATCCAAGTTTGAATAGAATTAAGA R: AAGGGTTGTCAGGTAGGCCTTT F: GACTCTGGTAAAGTTAAGCCTCTTC R: GATTCTCAGTAGATGTCTGTGACTG F: CTCAAACTCAAGTGGACATGTTCAT R: CTTTCAAAATTAGCTTCCATAGAACG F: ACAAAACGCATACAAGGTGCTCG R: CGAGTTATCTTTCTTATTGCGTCG F: CGAGGACTCTGACGTGGGTAGTT R: CCAGTACTCCTGCTTCTCCTG F: GACGCGCTCAAAGAAATCAGAGAAT

AC

Bm-DnaJ7

Real-time PCR Real-time PCR Real-time PCR Real-time PCR Real-time PCR Real-time PCR Real-time PCR Real-time PCR 37

IP

Real-time PCR

CR

Real-time PCR

US

Real-time PCR

MA N

Real-time PCR

TE D

Real-time PCR

AC

CE P

R: CTTCACCGCACAAGAGCAGAG Bm-DnaJ21 F: CGAACAGATCATATGATAGAACAGA R: TTGAGTCTCCTGTTCAATTTCAGC Bm-DnaJ22 F: CAAGCGCGTGCAGACGGACC R: CTGCCTCAAGTTCTTCTTCTGACA Bm-DnaJ23 F: TGCCAATGAAGAGGCCTTTGACC R: CACCGCTCTGAAGTGTTTACTGG Bm-DnaJ24 F: CTGAACGAGGTTCTTATCAACAATG R: ATCCAAATGGAACTTCTGCTTGTC Bm-DnaJ25 F: AAGCCGCACAAGCGCATGAGTC R: CCAGTAGTTGATGTAGTAACGGTG Bm-DnaJ26 F: AGAAGAGGTACACGGCAGAGC R: CGCTTAGCTTCTTCTTCTTTCTC Bm-DnaJ27 F: ATGAAACCTTGAAGGATCCAGAG R: GCTAACTTCTTCCAATGATCTGC Bm-ActinA3 F: CGCCGTGTTCCCCTCGATCGT R: TCTGGGTCATCTTCTCTCTGTTG Bm-DnaJ1 J1F: CGCGGATCCATGAGAGACAACCAA J1R: CCGAAGCTTCTATTGGTGAGCACAT Footnote: N = A, C, G, or T; V = A, G, or C

T

ACCEPTED MANUSCRIPT

Real-time PCR Real-time PCR Real-time PCR Plasmid construction: pET28a-BmDnaJ1F

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ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Abbreviations HSPs, Hot shock proteins; EST, expressed sequence tag; G/F domain, glycine- and phenylalanine-rich domain; zf domain, a cysteine-rich zinc-finger domain; C domain, C-terminal domain; RACE, random amplification of cDNA ends; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; dnaJ, gene encoding E. coli chaperonin; CAJ1, DnaK-like protein of yeast

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ACCEPTED MANUSCRIPT

T

IP SC R NU MA D TE CE P

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AC

 

Highlights We identified 27 cDNA sequences of DnaJ family in silkworm. DnaJ family has undergone convergent evolution, obtaining new domains to expand the diversity of its family members. DnaJ1 proteins were expressed at different levels in different tissues. DnaJ1 was mainly localized in the cytoplasm of blood cells.

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