Short sequence-paper

Characterization of JDP genes, an evolutionarily conservedJ domain-only protein family, from human and moths1

Jieun Lee a, Yoonsoo Hahn a, Ji Hye Yun a, Kazuei Mita b, Jae Hoon Chung a;*a Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701, South Koreab Genome Research Group, National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba 263-8555, Japan

Received 30 November 1999; accepted 10 February 2000

Abstract

We characterized evolutionarily conserved J domain containing protein (JDP) genes from human, Bombyx mori, andManduca sexta. Each of the JDP proteins contains a J domain at its N-terminus and a highly conserved C-terminal domain.Southern blot analysis revealed that the human JDP1 gene is present as a single copy in the human genome. Expression washigher in brain, heart, and testis than in kidney or stomach. Human JDP1 was mapped in silico to chromosome 10q21.1,which exhibits a conserved synteny with the central region of mouse chromosome 10. Drosophila jdp is located at 99F4^99F11 on the right arm of the third chromosome. ß 2000 Elsevier Science B.V. All rights reserved.

Keywords: J domain protein; Phylogenetic analysis ; Human; Bombyx mori ; Manduca sexta

Molecular chaperones of the Hsp70 family pro-teins perform numerous processes in protein metab-olism, such as protein folding and degradation, as-sembly and disassembly of protein complexes, andsuppression of protein aggregation, all of which areessential for cell survival under stressed as well asnormal circumstances [1]. The Hsp40 partner pro-teins directly interact with the Hsp70 proteins coop-eratively to perform the chaperone function. The re-versible capture and release of protein substrates aretightly coupled with ATP hydrolysis and dependenton conformational changes [2,3]. The Hsp40 familyproteins stimulate ATPase activity of the Hsp70 fam-

ily proteins and consequently change the conforma-tion of Hsp70s to an elevated a¤nity form for theirpeptide substrates.

DnaJ was identi¢ed as the Escherichia coli ortho-log of Hsp40. To date, several Hsp40/DnaJ familyproteins have been reported in various eukaryoticorganisms [4^10]. The E. coli DnaJ protein hasfour domains: the N-terminal J domain; a glycine/phenylalanine (G/F)-rich domain; a central repeatregion (CRR); and a weakly conserved C-terminaldomain. The J domain which is believed to mediateinteraction with Hsp70 protein contains the highlyconserved histidine-proline-aspartate (HPD) tripep-tide. The C-termini of Hsp40/DnaJ family proteinsare thought to be involved in binding to their peptidesubstrate. Other Hsp40 family proteins contain all orcombinations of these four domains, sometimes withcellular localization signals or post-translationalmodi¢cation sequences [5,9,10].

0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 0 4 7 - 6

* Corresponding author. Fax: +82-42-869-2610;E-mail : jhchung@sorak.kaist.ac.kr

1 The nucleotide sequence data have been submitted to Gen-Bank database under accession numbers AF176012^AF176015,AF192462, and AF192463.

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The J domain-only proteins refer to members of asubclass of the Hsp40/DnaJ family which possess theJ domain, but lack G/F-rich domain and CRR. Sincethe J domain is widely believed to interact directlywith the Hsp70 chaperone machinery, any proteinwith a J domain is plausibly thought to assistHsp70 protein for its function in protein folding.Several proteins containing J domain without preva-lent three domains mentioned above were identi¢ed;SV40 T-antigen [11], cysteine string proteins [12],Sec63p [13], Zuotin [14], Auxilin [15], and TID1[16]. High-resolution three-dimensional structures ofthe E. coli DnaJ [17,18] and human HDJ1 [19] Jdomains provide clues to how J domains aid Hsp70proteins [20,21]. In this study, we characterized evo-lutionarily conserved J domain proteins (JDPs) fromhuman, Bombyx mori (silkworm) and Manduca sexta(tobacco hornworm). Gene copy number and tissuedistribution of the transcript were investigated in hu-man. The chromosomal locations of the genes inhuman, mouse, and Drosophila melanogaster weredetermined.

We previously identi¢ed highly conserved JDPgenes from mouse and Drosophila [22]. Amino acidsequences of murine Jdp1 and Drosophila jdp pro-teins were used as database queries to identify or-thologous genes from various organisms. Sequencesimilarity searches were performed using BLASTprograms [23] at the NCBI server (http://www.ncbi.nlm.nih.gov/BLAST). Initial searchagainst non-redundant GenBank/EMBL/DDBJ Da-tabase yielded plenty of sequences encoding J do-main proteins. None of the known genes showedan overall homology beyond the J domain. A searchagainst the expressed sequence tags database(dbEST) detected several ESTs encoding orthologousproteins in human, rat, B. mori, and M. sexta. Twoisoforms of putative human ortholog JDP1 cDNAcontigs were generated by iterative searches againstthe dbEST and by assembly of retrieved EST sequen-ces using the CAP program [24]. Two EST clones, c-2fe02 for the longer isoform and IMAGE:1649677for the shorter isoform (GenBank accession numbersZ45047 and AI034345, respectively) were purchasedfrom Genome Systems. The two clones containingthe longest 3P-untranslated region (UTR) were chos-en for further study. The B. mori EST clone e40154(GenBank accession number AU000134) was found

in the cDNA library derived from embryos 40 h afterfertilization [25]. The M. sexta EST clone pMsmaB8(GenBank accession number AI142147) isolatedfrom male antennae was kindly provided by H.M.Robertson [26]. The full sequences of the cDNAclones were determined by a Bigdye Terminator cyclesequencing kit (Perkin-Elmer, ABI) and an autose-quencer model 373A (Perkin-Elmer, ABI).

Two isoforms of human JDP1 transcripts wereidenti¢ed by dbEST search and sequence determina-tion of cDNA clones. Both clones contained the 5P-UTR, full open reading frame (ORF), and the poly-(A) tail. The cDNA sequences and deduced aminoacids of two isoforms are shown in Fig. 1. The iso-form a (clone c-2fe02) was 1.2 kb-long and encoded apolypeptide of 198 amino acids. The isoform b (cloneIMAGE:1649677) was 0.7 kb-long and lacked 68 bpat the 5P-end with respect to the isoform a. The iso-form b contained a shorter ORF encoding a poly-peptide of 107 amino acids, which di¡ered in C-ter-minal region with respect to isoform a. The peptideof isoform b diverged from that of isoform a at theamino acid position 100. Two potential polyadenyla-tion signals and three alternative polyadenylationsites in each of two isoforms were identi¢ed bycDNA sequencing and exhaustive EST sequenceanalysis. Each polyadenylation signal preceded poly-(A) tract by 10^22 nucleotides. The sequence com-parison revealed that the isoform a was orthologousto previously identi¢ed murine Jdp1 and Drosophilajdp. The nucleotide sequences of human JDP1 cDNAclones were deposited in the GenBank databaseunder accession numbers AF176012 and AF176013.

The isoform b seemed to be originated by intronicpolyadenylation of the primary transcript. SuccessfulPCR ampli¢cation of genomic fragment encompass-ing the diverging position using a forward primerand an isoform b-speci¢c primer con¢rmed the in-tronic polyadenylation (data not shown). Interest-ingly, we found no evidence for the presence of ro-dent EST sequences corresponding to the humanisoform b, implying that this intronic polyadenyla-tion had been newly evolved after divergence be-tween rodents and primates.

To determine the copy number of human JDP1gene, Southern blot analysis was performed as de-scribed [27]. Human genomic DNA isolated fromblood was separately digested with the restriction

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enzymes, EcoRI, HindIII, BamHI, and PvuII andsubjected to Southern blot analysis using a radiola-beled partial cDNA probe. As shown in Fig. 2A,only a single hybridized band was detected in eachof the lanes for HindIII, BamHI, and PvuII, suggest-ing that the JDP1 gene is present as a single copy inthe human genome. The presence of multiple bandsin the EcoRI-digest lane indicated that additionalEcoRI sites were present in the intronic regions.The three bands in the manifold suggested that the

region used as the probe contained at least two in-trons. The corresponding region of previously char-acterized mouse Jdp1 cDNA was also intervened byat least two introns [22].

Tissue distribution of human JDP1 transcript wasinvestigated by Northern blot analysis using MultipleChoice Northern Blots (OriGene Technologies) ac-cording to the manufacturer's instruction. The iso-form a full cDNA clone c-2fe02 was used as a probe.Though two isoforms were identi¢ed by dbEST anal-

Fig. 1. Complementary DNA sequences and deduced amino acid sequences of human JDP1 transcript isoforms a and b. Two iso-forms of human JDP1 cDNA clones were purchased from Genome Systems and sequenced. (A) The isoform a was 1.2 kb-long andencoded a polypeptide of 198 amino acids. An arrowhead indicates the diverging position of isoform b from isoform a. (B) The iso-form b was 0.7 kb-long and encoded a polypeptide of 107 amino acids. The 3P-end sequence of the isoform b which di¡ers from theisoform a is presented. The position +1 of isoform b corresponds to +69 of isoform a. The numbers at the right are nucleotide andamino acid position. Amino acid residues comprising J domain, translational start codons (ATG), stop codons (TGA), and polyade-nylation signal sequences (AATAAA) are in boldface letters. Three poly(A) addition sites in each isoform are in boldface and under-lined.

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ysis, only a single transcript could be detected intissues examined (Fig. 2B). The estimated transcriptsize was about 1.3 kb, commensurable with thelength of isoform a. RT-PCR analysis using eachisoform-speci¢c primer pair revealed that the expres-sion level of isoform b is negligible compared to thatof isoform a in fetal and adult liver (data not shown).The JDP1 gene was expressed at similar levels inbrain, heart, and testis, but at reduced levels in kid-ney and stomach. Previously, we showed that murineJdp1 gene was expressed reproducibly at the highestlevel in kidney and Drosophila jdp gene transcriptwas restricted to heads [22]. The JDP proteins seemto be utilized in di¡erent ways in these organisms.

Moth jdp genes were identi¢ed from two species,B. mori and M. sexta. The nucleotide sequences anddeduced amino acid sequences of moth jdp cDNAclones were presented in Fig. 3. The moth jdpcDNA clones analyzed in this study were about 1.5

Fig. 2. Gene copy number and tissue distribution of humanJDP1 gene. (A) Human blood genomic DNA was indepen-dently digested with EcoRI, HindIII, BamHI, or PvuII and sub-jected to Southern blot analysis. The hybridization probe wasgenerated by PCR ampli¢cation of human JDP1 isoform acDNA clone c-2fe02 using a gene-speci¢c primer (5P-AGA-GAGTCGAGCCCGCTATGACCACT-3P) and a vector primer(5P-GTAAAACGACGGCCAGT-3P). The probe encompassedthe 3P part of coding region and the complete 3P-UTR and in-cluded at least one intron from which two human isoformswere diverged. Though the probe contained a recognition sitefor HindIII, the shorter fragment did not generate the hybrid-ization signal in tested condition. A single band was detected inthe lanes for HindIII, BamHI, and PvuII, indicating that theJDP1 is present as a single copy in the human genome. Thenumber of bands in the lane for EcoRI implies that the regionused as the probe is intervened by at least two introns. DNAsize markers are indicated at the right of the panel in kb. (B)Tissue distribution of human JDP1 transcript was investigatedby Northern blot analysis using Multiple Choice Northern Blots(OriGene Technologies). Each lane contained 2 Wg of poly(A)�

RNA. The probe was prepared from the isoform a full cDNAclone c-2fe02. The sizes of RNA fragments are indicated at theright of the panel in kb.6

C

Fig. 3. Complementary DNA sequences and deduced amino acid sequences of moth jdp genes. (A) The Bombyx mori jdp cDNA clonewas isolated from embryos 40 h after fertilization. (B) The Manduca sexta jdp cDNA clone was isolated from male antennae andkindly provided by H.M. Robertson. The moth jdp cDNA clones are about 1.5 kb in length and encode 170 residues proteins. The se-quence features are represented as described in Fig. 1.

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kb in length and composed of very short 5P-UTRand relatively long 3P-UTR of 0.9^1.0 kb. A poly-adenylation signal sequence (AATAAA) was foundat 12 nucleotides upstream to the poly(A) tract inboth of the moth species. Each cDNA containedthe complete ORF encoding a polypeptide of 170amino acids. The nucleotide sequences of moth jdpcDNA clones were submitted to the GenBank data-base (accession numbers AF176014 and AF176015).

Multiple sequence alignment was performed to in-fer conserved primary structures among JDP pro-teins. The deduced amino acid sequences of JDPgenes from human, mouse, Drosophila, B. mori,and M. sexta were aligned using ClustalW program[28] and shown in Fig. 4A. The J domain as de¢nedby Pfam database [29] was found at amino acid po-sition 14^79 in mammals and at 17^82 in insects. TheN-terminal halves, including J domain, of the JDP

Fig. 4. Multiple sequence alignment and phylogenetic relationship of JDP proteins. (A) Deduced amino acid sequences of JDP genesfrom human (isoform a), mouse, Bombyx mori, Manduca sexta, and Drosophila (isoform a) were aligned using ClustalW program.The N-terminal J domain and the conserved C-terminal heptapeptide are denoted by asterisks and hash marks, respectively. The ami-no acids identical in more than three sequences are highlighted against a black background. The conserved amino acids are boxed ingray. An arrow indicates the diverging position of isoform b from isoform a in both human and Drosophila. Calculated molecularmasses and identities with human JDP1 isoform a are presented after the sequences. Amino acid numbers for each protein are shownat the right. The GenBank accession numbers of the sequences are as follows: human, AF176012; mouse, AF132906; Bombyx mori,AF176014; Manduca sexta, AF176015; Drosophila, AF192462. (B) A phylogenetic tree was constructed from the ClustalW alignment.The scale bar is 0.05 substitutions per site.

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proteins were highly conserved between mammalsand insects. The major distinguishing feature is theC-terminal heptapeptide, KFRNYEI, which wasfound in all JDP proteins, suggesting that it plays acrucial role in biological function of JDP proteins,such as speci¢c substrate binding or catalytic func-tions. The diverging positions of human isoformsand Drosophila isoforms were identical, indicatingthat the corresponding intron positions were con-served in these organisms. Calculated molecularmasses of JDP proteins were within a range of 19^23 kDa. Amino acid identities with human JDP1protein isoform a were 77.3% and about 40% formouse and insect JDP proteins, respectively. Molec-ular masses were calculated using AACOMP pro-gram and identities with human JDP1 were obtainedusing ALIGN program in FASTA program package[30]. The phylogenetic tree (Fig. 4B) was constructedbased on ClustalW alignment.

A comprehensive similarity search against com-

plete genome sequences of Caenorhabditis elegansand Saccharomyces cerevisiae failed to detect obviousorthologs in these organisms. It was expected that anortholog exist at least in C. elegans since C. elegansand insects are grouped in the same clade of Ecdy-sozoa [31]. A plausible explanation is that JDP genein C. elegans was lost during evolution or highlydiverged by extremely rapid nucleotide-substitutionsfound in rhabditid nematodes.

Chromosomal location of the human JDP1 wasdetermined by similarity search of the human JDP1cDNA sequence against the sequence tagged sitesdatabase (dbSTS) [32]. The human JDP1 cDNAwas revealed to contain STS marker SHGC-33772(GenBank accession number G30458) which wasmapped between markers D10S1646 and D10S210at 10q21.1 (http://www.ncbi.nlm.nih.gov/genemap)[33]. Human JDP1 was £anked proximally byANK3 and distally by HK1. Mouse mapping datafrom the Mouse Genome Database (MGD, http://

Fig. 5. Comparative genetic map of human chromosome 10q21^q22. Homologous regions between human chromosome 10 (HSA10)and mouse chromosome 10 (MMU10) are depicted. Human JDP1 is located between markers D10S1646 and D10S210 at 10q21.1 and£anked proximally by ANK3 and distally by HK1. Murine genes located between Ank3 and Hk1 on the central region of chromosome10 belong to a conserved linkage group on human chromosome 10q21^q22, suggesting that mouse Jdp1 lies on the same region. Ori-entation of these linkage groups on human and mouse chromosome is reversed relative to centromere (¢lled circle). Genes localized inthis region but the exact position in G3 map was not determined are presented in brackets. Murine orthologs of human genes CDC2,TCF6, PCBD, PRF1, PP are Cdc2a, Tfam, Dcoh, Pfp, Pyp, respectively. The cM position refers only to the mouse chromosome.

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www.informatics.jax.org) [34] showed that murineAnk3 and Hk1 were located on the central regionof chromosome 10. Several genes located betweenAnk3 and Hk1 belong to a conserved linkage groupon human chromosome 10q21^q22, suggesting thatmouse Jdp1 lies on the same region (Fig. 5). In Dro-sophila, draft sequences of two clones from chromo-some 3R, BACR12J15 and BACR48G16 (GenBankaccession numbers AC007820 and AC008299, respec-tively), were con¢rmed to contain pieces of jdp ge-nomic sequences, placing the gene at position 99F4^99F11 on the right arm of the third chromosome.

In summary, we characterized an evolutionarilyconserved subclass of J domain-containing proteinsfrom human, B. mori, and M. sexta. The humanJDP1 gene encodes two isoforms by alternative us-age of polyadenylation sites and its transcript wasdetected in brain, heart, and testis. The primarystructure of JDP proteins is extremely conserved inmammalian and insect lineage. Human JDP1 is lo-calized to chromosome 10q21.1, which is homolo-gous to the central region of mouse chromosome10, and Drosophila jdp gene is located at the rightarm of chromosome 3.

This work was supported by a grant from KoreaScience and Engineering Foundation (KOSEF),South Korea.

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