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Rabbit ubiquitin-activating enzyme E1: cDNA cloning, sequence and expression
Gene 196 ( 1997) 19–23
Rabbit ubiquitin-activating enzyme E1: cDNA cloning, sequence and expression Binggang Sun, Kandiah Jeyaseelan, Maxey C.M. Chung, Tian-Seng Teo * Department of Biochemistry, Faculty of Medicine, National University of Singapore, Singapore 119260, Singapore Received 8 November 1996; accepted 8 February 1997; Received by T. Sekiya
Abstract A cDNA clone encoding ubiquitin-activating enzyme E1 has been isolated from a rabbit heart cDNA library and sequenced. The 3.485 kb cDNA contains an open reading frame of 1058 amino acid residues which predicts a protein of approx. 118 kDa. The deduced protein sequence exhibits a very high homology to other ubiquitin-activating enzymes identified in a variety of organisms. Northern blot analysis reveals a single transcript of apporx. 3.5 kb in all the rabbit tissues examined. The entire coding region of the rabbit E1 cDNA has been expressed as a his-tagged protein. The recombinant protein has been verified by its ability to cross-react with anti-human E1 antibodies. Ubiquitin thiolester assay shows that the recombinant rabbit E1 protein is functional. © 1997 Elsevier Science B.V. Keywords: Ubiquitination; Protein degradation; Tissue distribution
1. Introduction Protein ubiquitination plays an important role in a number of cellular processes (Ciechanover, 1994). The initial reaction of protein ubiquitination involves the activation of ubiquitin by E1 in the presence of ATP. This results in the formation of a ubiquitin adenylate intermediate from which the ubiquitin moiety is then transferred to a thiol group of an internal cysteine residue of E1 ( Haas and Rose, 1982). The activated ubiquitin is then transferred to a cysteine residue of E2 which then ligates ubiquitin to the target protein either directly or with the participation of E3 (Ciechanover and Schwartz, 1989). E1 plays a key role in the ubiquitination process as it * Corresponding author. Tel: +65 7723246; Fax: +65 7791453; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pairs (s); CDC, cell division cycle; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin protein ligase; GAPDH, glyceraldehyde3-phosphate dehydrogenase; IPTG, isopropyl b--thiogalactopyranoside; kb, kilobase (s) or 1000 bp; kDa, kilodalton(s); PVDF, polyvinylidine difluoride; RT–PCR, reverse transcription–polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SSC, 0.15 M NaCl/0.015 M Na Ωcitrate (pH 7.6 ); UTR, 3 untranslated region(s). 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0 3 78 - 11 19 ( 9 7 ) 00 15 4 -6
catalyses the first step of the ubiquitin pathway. The gene encoding yeast ubiquitin-activating enzyme UBA1, was cloned and shown to be essential for cell viability (McGrath et al., 1991). Characterization of several mammalian temperature-sensitive mutants of E1 showed that cells defective in E1 may exhibit a variety of abnormalities including cell cycle arrest, inhibition in the degradation of short-lived and abnormal proteins and defects in a number of other cellular functions (Finely et al., 1984; Kula et al., 1988; Ayusawa et al., 1992; Mori et al., 1993 ). Genes encoding E1 from mammals and plants have also been cloned and characterized (Handley et al., 1991; Imai et al., 1992; Hatfield and Vierstra, 1992). All of these genes encode 110–126 kDa proteins which are highly similar in sequence. UBA1 is the only gene encoding E1 in yeast whereas multiform of genes and proteins of E1 were identified in wheat (McGrath et al., 1991; Hatfield and Vierstra, 1992 ). A mouse E1 gene essential for spermatogenesis was identified and shown to be testis-specific ( Kay et al., 1991; Mitchell et al., 1991 ). More recently, it was suggested that unique E1s may be required to activate and transfer ubiquitin-like proteins (Narasimhan et al., 1996 ). The significance of E1 heterogeneity remains unclear. In this paper we report the isolation and expression of a cDNA clone for rabbit E1.
20
B. Sun et al. / Gene 196 (1997) 19–23
Fig. 1. Nucleotide sequence and the deduced amino acid sequence of rabbit E1 cDNA. Sequence of the 3.485 kb cDNA clone was determined by subcloning and sequencing of the overlapping cDNA fragments generated by restriction enzyme digestion of the E1 cDNA. The potential nuclear localization signal is double underlined. The stop codon is given by an asterisk (*). The positions of degenerate primers used to amplify the 560 bp E1 cDNA probe are underlined. The putative polyadenylation site is dotted underlined. This sequence has been deposited in EMBL/GeneBank Data Libraries under accession No. U58653.
B. Sun et al. / Gene 196 (1997) 19–23
2. Experimental and discussion 2.1. Cloning and sequence analysis of the full-length E1 clone
21
based on the existence of the conserved internal start site (Met-41) and a polybasic amino acid sequence KKRR as a potential nuclear localization sequence. 2.2. Tissue distribution of E1
A rabbit heart cDNA library (Stratagene) was screened using a 560 bp rabbit E1 cDNA probe which was obtained by RT–PCR using degenerate primers designed based on the peptide sequence of purified rabbit reticulocyte E1 (positions of the primers are indicated in Fig. 1 ). Approximately 1×106 plaques were screened and 12 positive clones were obtained. The cDNA clone with the largest insert of approx. 3.5 kb was sequenced completely. As shown in Fig. 1, the 3.485 kb cDNA clone contains an uninterrupted open reading frame of 3174 bp, a complete 3∞ UTR with a 50 bp poly(A) and a 68 bp 5∞ UTR. A polyadenylation site (AATAAA) is present at position 3417–3422, 13 bp upstream of the poly(A) tail. Comparison of the deduced amino acid sequence of rabbit E1 with those from other organisms using ClusterW multiple sequence alignment reveals a high homology of 96.7 and 94% to human E1 and mouse E1, respectively. Although the identity between rabbit E1 and yeast E1 is only about 50%, the similarity increases to about 70% with the conservative amino acid substitutions included. To determine the translation initiation site, we compared the 5∞ end of this E1 cDNA clone to that of human and mouse E1 cDNAs. The first ATG of the rabbit E1 cDNA cannot be the translation start site because of the existence of multiple stop codons in this reading frame. The second ATG of the rabbit cDNA clone starts a long open reading frame of 3174 bp which has an overall high homology to human and mouse E1 cDNAs both at the nucleotide and amino acid sequence levels. Sequences outside this reading frame exhibit no homology at all. The corresponding ATG codons (Met-1 ) in human and mouse E1 cDNAs were designated as the translation start sites with an open reading frame encoding a protein of approx. 118 kDa in size ( Handley et al., 1991; Imai et al., 1992 ). It has been shown, however, that human E1 protein exists as two isoforms (116/111 kDa), and translation initiation at the downstream ATG codon (Met-41) of human E1 cDNA could be responsible for the smaller E1 isoform. The larger form contains a nuclear localization sequence KKRR (amino acids 8–11 ) required for the localization of the enzyme to nuclei, and has been shown to have a dierent cellular localization pattern compared to the smaller form. Therefore, both ATG codons (Met-1 and Met-41) in human E1 cDNA represent potential translation start sites, and the two isoforms probably result from alternative translation start sites ( Handley et al., 1994 ). The same assignment of translation start sites may also be applied to rabbit and mouse E1 cDNAs
Northern analysis of total RNA from six rabbit tissues was carried out using the same 560 bp probe as the one used for library screening. A unique transcript with an approximate size of 3.5 kb was identified in all tissues examined ( Fig. 2). Ubiquitous expression of a single 3.5 kb transcript for human E1 was reported previously in a number of human tissues and cell lines ( Handley et al., 1991). These results are consistent with the observation that E1 plays important roles in cell cycle progression as well as in a number of other important cellular processes. Our results also suggest that the 3485 bp cDNA that we have isolated appears to be the intact E1 cDNA. 2.3. Expression, Western blot analysis and activity assay of recombinant E1 The entire E1 cDNA coding region was cloned into the pQE30 vector and transformed into Escherichia coli strain M15 (pREP4) (Qiagen). The resulting construct should permit the expression of a protein with a calculated molecular mass of about 118 kDa excluding the N-terminal 6 histidine tag and six other amino acid
Fig. 2. Northern blot analysis of rabbit E1 mRNA. Total RNA was extracted by the guanidium isothiocyanate/CsCl method (Chirgwin et al., 1979). Total RNA (15 mg) from each tissue was separated on a 1.2% agarose gel containing 2.2 M formaldehyde, transferred to a nitrocellulose membrane and hybridized with 32P-labelled probe as described (Sambrook et al., 1989). The probe used was a 560 bp E1 cDNA fragment labelled with 32P to a specific activity of 1×109 cpm/mg DNA. The positions of 28S and 18S rRNA are indicated. The blot was also stripped o the E1 cDNA probe and reprobed with a human GAPDH probe to normalize the loading of total RNA.
22
B. Sun et al. / Gene 196 (1997) 19–23
residues. The first attempt to purify the recombinant protein by induction with IPTG (0.02–0.5 mM ) at 37°C of E. coli harbouring the construct failed, as most of the protein expressed in this way formed inclusion bodies. Conditions were optimized so as to increase the solubility of the expressed his-tagged E1 protein. As shown in Fig. 3A, a significant amount of protein was expressed in a soluble form at 30°C when cells were induced at a very low IPTG concentration ( 7.5 mM ). The protein was partially purified by Talon metal anity
resin (Clontech) under native conditions with an estimated yield of 50–100 mg per litre of culture. Fig. 3B shows that the recombinant E1 protein could cross-react with antibodies raised against native human E1. Bands which are slightly larger than the 116 kDa molecular mass marker were specifically detected in the induced and partially purified protein samples but not in the uninduced sample. The lower molecular mass bands other than the 116 kDa E1 could be caused by nonspecific binding of antibodies to bacterial proteins. It is
Fig. 3. Expression, immunodetection and activity assay of the recombinant protein. A, recombinant E1 protein expression and SDS–PAGE analysis. An aliquot of 15 ml of overnight culture of a single transformant was diluted into 100 ml of LB containing 100 mg/ml ampicillin and 25 mg/ml kanamycin and incubated at 37°C for 1.5 h. Cells were induced with 7.5 mM IPTG for 2 h at 30°C before harvesting. Cells were collected, lysed by sonication and clarified by centrifugation. Recombinant E1 protein was purified by Talon metal anity resin using a protocol provided by the manufacturer. Protein samples were analysed by 8% SDS–PAGE and stained with Coomassie blue. Lane 1, uninduced; lane 2, induced, total protein; lane 3, induced, soluble protein; lane 4, protein purified. B, Western blot analysis of expressed E1 protein. The protein samples were separated by 8% SDS–PAGE and transferred to PDVF membrane. Blots were blocked with 5% non-fat milk, 0.1% sodium azide in TBST (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20) and probed with rabbit anti-human E1 antibodies. Secondary antibodies used were goat antirabbit IgG labelled with alkaline phosphatase. Blots were visualized using the alkaline phosphatase substrate BCIP/NBT. Lane 1, uninduced, total protein; lane 2, induced, total protein; lane 3, partially purified E1. Gel used for Western blot ( B) and gel used for Commassie blue staining (A ) are from dierent runs. C, Activity assay of recombinant E1 protein. In a 25 ml reaction, 0.5 mg of partially purified recombinant E1, 1 mg of 125I-labelled ubiquitin, 50 mM Tris–HCl, pH 7.5, 0.2 mM DTT, 5 mM ATP, 5 mM MgCl , two units of inorganic pyrophosphatase were incubated 2 at 37°C for 10 min with ( lanes 5 and 6) or without ( lanes 1–4) the addition of 0.5 mg of recombinant rabbit E . Purified native rabbit E1 was 2–32 used as a positive control. The reactions were stopped with Laemmli sample buer with ( lanes 1, 2 and 5 ) or without ( lanes 3, 4 and 6) 5% bmercaptoethanol and separated by 15% SDS–PAGE. Lanes 1 and 3, native rabbit E1 only; lanes 2 and 4, recombinant E1 only; lanes 5 and 6, recombinant E1 + recombinant E . 2–32
B. Sun et al. / Gene 196 (1997) 19–23
interesting to see that two proteins with similar molecular masses (approx. 116 kDa) were induced as shown in Fig. 3A ( lanes 2 and 3 ). Both of the two bands can be detected by anti-human E1 antibodies (Fig. 3B, lane 2), indicating that both of them might be E1 proteins. The smaller protein probably arose from an internal translation start site. Fig. 3C shows that the bacterially expressed E1 could either form a thiolester bond with ubiquitin in the absence of rabbit E ( lane 4 ) or transfer ubiquitin to 2–32 rabbit E ( lane 6 ). Boiling the samples with loading 2–32 buer containing 5% b-mercaptoethanol eliminated the thiolester bond between E1 and ubiquitin ( lane 2), but only markedly diminished the intensity of the band representing E -ubiquitin ( lane 5). A reasonable 2–32 explanation for this observation is that E might be 2–32 able to ubiquitinate itself, and the isopeptide bond could not be destroyed by boiling in the presence of reducing agent. It has been reported that CDC34, the yeast homologue of rabbit E , can ubiquitinate itself both 2–32 in vitro and in vivo. Yeast CDC34 autoubiquitination forms isopeptide bonds that could not be cleaved by treatment with b-mercaptoethanol ( Banerjee et al., 1993; Goebl et al., 1994 ). 2.4. Conclusion
(1 ) The cDNA encoding full-length rabbit ubiquitinactivating E1 has been cloned and sequenced. (2 ) The deduced amino acid sequence of the cloned E1 cDNA is highly homologous to human and mouse E1. (3 ) The rabbit E1 mRNA exists as a single 3.5 kb transcript in all six tissues examined. (4 ) The recombinant E1 protein has been expressed in a soluble form in E. coli and has been shown to cross-react with anti-human E1 antibodies. (5 ) The bacterially expressed rabbit E1 protein is functional.
Acknowledgement This work was supported by a National University of Singapore research grant RP930311. We thank Dr P. Boon Chock ( Laboratory of Biochemistry, NHLBI, NIH ) for providing us with anity-purified antibodies raised against human E1. We also thank Mrs M.K. Lee for her expert technical assistance.
References Ayusawa, D., Kaneda, S., Itoh, Y., Yasuda, H., Murakami, Y., Sugasawa, K., Hanaoka, F., Seno, T., 1992. Complementation by a
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cloned human ubiquitin-activating enzyme E1 of the S-phasearrested mouse FM3A cell mutant with thermolabile E1. Cell Struct. Funct. 17, 113–122. Banerjee, A., Gregori, L., Xu, Y., Chau, V., 1993. The bacterially expressed yeast CDC34 gene product can undergo autoubiquitination to form a multiubiquitin chain-linked protein. J. Biol. Chem. 268, 5668–5675. Chirgwin, J.W., Przybyla, A.E., MacDonald, R.J., Rutter, W.J., 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294–5299. Ciechanover, A., Schwartz, A.L., 1989. How are substrates recognized by the ubiquitin-mediated proteolytic system? Trends Biochem. Sci. 14, 483–488. Ciechanover, A., 1994. The ubiquitin-proteasome proteolytic pathway. Cell 79, 13–21. Finely, D., Ciechanover, A., Varshavsky, A., 1984. Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85 cell. Cell 37, 43–55. Goebl, M.G., Goetsch, L., Byers, B., 1994. The Ubc3 (CDC34) ubiquitin-conjugating enzyme is ubiquitinated and phosphorylated in vivo. Mol. Cell. Biol. 14, 3022–3029. Haas, A.L., Rose, I.A., 1982. The mechanism of ubiquitin-activating enzyme. J. Biol. Chem. 257, 10329–10337. Handley, P.M., Muecker, M., Siegel, N.R., Ciechanover, A., Schwartz, A.L., 1991. Molecular cloning, sequence and tissue distribution of the human ubiquitin-activating enzyme E1. Proc. Natl. Acad. Sci. USA 88, 258–262. Handley, P.M., Stephen, A.G., Trausch-Azar, J.S., Ciechanover, A., Schwartz, A.L., 1994. Ubiquitin-activating enzyme, E1. Indication of potential nuclear cytoplasmic subpopulations using epitopetagged cDNA constructs. J. Biol. Chem. 269, 33171–33178. Hatfield, P.M., Vierstra, R.D., 1992. Multiple forms of ubiquitin-activating enzyme E1 from wheat. Identification of an essential cysteine by in vitro mutagenesis. J. Biol. Chem. 267, 14799–14803. Imai, N., Kaneda, S., Nagai, Y., Seno, T., Ayusawa, D., Hanaoka, F., Yamao, F., 1992. Cloning and sequence of a functionally active cDNA encoding the mouse ubiquitin-activating enzyme E1. Gene 118, 279–282. Kay, G.F., Ashworth, A., Penny, G.D., Dunlop, M., Swift, S., Brockdorf, N., Rastan, S., 1991. A candidate spermatogenesis gene on the mouse Y chromosome is homologous to ubiquitin-activating enzyme E1. Nature 354, 486–489. Kula, R.G., Rabby, B., Schuster, R., Parag, H.A., Diamond, G., Ciechanover, A., Marcus, M., 1988. A Chinese hamster cell cycle mutant arrested at G2 phase has a temperature-sensitive ubiquitinactivating enzyme, E1. J. Biol. Chem. 263, 15726–15731. McGrath, J.P., Jentch, S., Varshavsky, A., 1991. UBA1, an essential yeast gene encoding ubiquitin-activating enzyme. EMBO J. 10, 227–236. Mitchell, M.J., Woods, D., Tucker, P.K., Opp, J.S., Bishop, C.E., 1991. Homolog of a candidate spermatogenic gene from the mouse Y chromosome to the ubiquitin-activating enzyme E1. Nature 354, 483–486. Mori, M., Eki, T., Takahashi-Kudo, M., Hanaoka, F., Ui, M., Enomoto, T., Characterization of DNA synthesis at a restrictive temperature in the temperature-sensitive mutants, tsFT5 cells, that belongs to the complementation group of ts85 cells containing a thermolabile ubiquitin-activating enzyme E1. Involvement of the ubiquitin-conjugating system in DNA replication. 1993. J. Biol. Chem. 268, 16803–16809. Narasimhan, J., Potter, J.L., Haas, A.L., 1996. Conjugation of the 15-kDa interferon-induced ubiquitin homolog is distinct from that of ubiquitin. J. Biol. Chem. 271, 324–330. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Rabbit ubiquitin-activating enzyme E1: cDNA cloning, sequence and expression Binggang Sun, Kandiah Jeyaseelan, Maxey C.M. Chung, Tian-Seng Teo * Department of Biochemistry, Faculty of Medicine, National University of Singapore, Singapore 119260, Singapore Received 8 November 1996; accepted 8 February 1997; Received by T. Sekiya
Abstract A cDNA clone encoding ubiquitin-activating enzyme E1 has been isolated from a rabbit heart cDNA library and sequenced. The 3.485 kb cDNA contains an open reading frame of 1058 amino acid residues which predicts a protein of approx. 118 kDa. The deduced protein sequence exhibits a very high homology to other ubiquitin-activating enzymes identified in a variety of organisms. Northern blot analysis reveals a single transcript of apporx. 3.5 kb in all the rabbit tissues examined. The entire coding region of the rabbit E1 cDNA has been expressed as a his-tagged protein. The recombinant protein has been verified by its ability to cross-react with anti-human E1 antibodies. Ubiquitin thiolester assay shows that the recombinant rabbit E1 protein is functional. © 1997 Elsevier Science B.V. Keywords: Ubiquitination; Protein degradation; Tissue distribution
1. Introduction Protein ubiquitination plays an important role in a number of cellular processes (Ciechanover, 1994). The initial reaction of protein ubiquitination involves the activation of ubiquitin by E1 in the presence of ATP. This results in the formation of a ubiquitin adenylate intermediate from which the ubiquitin moiety is then transferred to a thiol group of an internal cysteine residue of E1 ( Haas and Rose, 1982). The activated ubiquitin is then transferred to a cysteine residue of E2 which then ligates ubiquitin to the target protein either directly or with the participation of E3 (Ciechanover and Schwartz, 1989). E1 plays a key role in the ubiquitination process as it * Corresponding author. Tel: +65 7723246; Fax: +65 7791453; e-mail: [email protected] Abbreviations: aa, amino acid(s); bp, base pairs (s); CDC, cell division cycle; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin protein ligase; GAPDH, glyceraldehyde3-phosphate dehydrogenase; IPTG, isopropyl b--thiogalactopyranoside; kb, kilobase (s) or 1000 bp; kDa, kilodalton(s); PVDF, polyvinylidine difluoride; RT–PCR, reverse transcription–polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SSC, 0.15 M NaCl/0.015 M Na Ωcitrate (pH 7.6 ); UTR, 3 untranslated region(s). 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0 3 78 - 11 19 ( 9 7 ) 00 15 4 -6
catalyses the first step of the ubiquitin pathway. The gene encoding yeast ubiquitin-activating enzyme UBA1, was cloned and shown to be essential for cell viability (McGrath et al., 1991). Characterization of several mammalian temperature-sensitive mutants of E1 showed that cells defective in E1 may exhibit a variety of abnormalities including cell cycle arrest, inhibition in the degradation of short-lived and abnormal proteins and defects in a number of other cellular functions (Finely et al., 1984; Kula et al., 1988; Ayusawa et al., 1992; Mori et al., 1993 ). Genes encoding E1 from mammals and plants have also been cloned and characterized (Handley et al., 1991; Imai et al., 1992; Hatfield and Vierstra, 1992). All of these genes encode 110–126 kDa proteins which are highly similar in sequence. UBA1 is the only gene encoding E1 in yeast whereas multiform of genes and proteins of E1 were identified in wheat (McGrath et al., 1991; Hatfield and Vierstra, 1992 ). A mouse E1 gene essential for spermatogenesis was identified and shown to be testis-specific ( Kay et al., 1991; Mitchell et al., 1991 ). More recently, it was suggested that unique E1s may be required to activate and transfer ubiquitin-like proteins (Narasimhan et al., 1996 ). The significance of E1 heterogeneity remains unclear. In this paper we report the isolation and expression of a cDNA clone for rabbit E1.
20
B. Sun et al. / Gene 196 (1997) 19–23
Fig. 1. Nucleotide sequence and the deduced amino acid sequence of rabbit E1 cDNA. Sequence of the 3.485 kb cDNA clone was determined by subcloning and sequencing of the overlapping cDNA fragments generated by restriction enzyme digestion of the E1 cDNA. The potential nuclear localization signal is double underlined. The stop codon is given by an asterisk (*). The positions of degenerate primers used to amplify the 560 bp E1 cDNA probe are underlined. The putative polyadenylation site is dotted underlined. This sequence has been deposited in EMBL/GeneBank Data Libraries under accession No. U58653.
B. Sun et al. / Gene 196 (1997) 19–23
2. Experimental and discussion 2.1. Cloning and sequence analysis of the full-length E1 clone
21
based on the existence of the conserved internal start site (Met-41) and a polybasic amino acid sequence KKRR as a potential nuclear localization sequence. 2.2. Tissue distribution of E1
A rabbit heart cDNA library (Stratagene) was screened using a 560 bp rabbit E1 cDNA probe which was obtained by RT–PCR using degenerate primers designed based on the peptide sequence of purified rabbit reticulocyte E1 (positions of the primers are indicated in Fig. 1 ). Approximately 1×106 plaques were screened and 12 positive clones were obtained. The cDNA clone with the largest insert of approx. 3.5 kb was sequenced completely. As shown in Fig. 1, the 3.485 kb cDNA clone contains an uninterrupted open reading frame of 3174 bp, a complete 3∞ UTR with a 50 bp poly(A) and a 68 bp 5∞ UTR. A polyadenylation site (AATAAA) is present at position 3417–3422, 13 bp upstream of the poly(A) tail. Comparison of the deduced amino acid sequence of rabbit E1 with those from other organisms using ClusterW multiple sequence alignment reveals a high homology of 96.7 and 94% to human E1 and mouse E1, respectively. Although the identity between rabbit E1 and yeast E1 is only about 50%, the similarity increases to about 70% with the conservative amino acid substitutions included. To determine the translation initiation site, we compared the 5∞ end of this E1 cDNA clone to that of human and mouse E1 cDNAs. The first ATG of the rabbit E1 cDNA cannot be the translation start site because of the existence of multiple stop codons in this reading frame. The second ATG of the rabbit cDNA clone starts a long open reading frame of 3174 bp which has an overall high homology to human and mouse E1 cDNAs both at the nucleotide and amino acid sequence levels. Sequences outside this reading frame exhibit no homology at all. The corresponding ATG codons (Met-1 ) in human and mouse E1 cDNAs were designated as the translation start sites with an open reading frame encoding a protein of approx. 118 kDa in size ( Handley et al., 1991; Imai et al., 1992 ). It has been shown, however, that human E1 protein exists as two isoforms (116/111 kDa), and translation initiation at the downstream ATG codon (Met-41) of human E1 cDNA could be responsible for the smaller E1 isoform. The larger form contains a nuclear localization sequence KKRR (amino acids 8–11 ) required for the localization of the enzyme to nuclei, and has been shown to have a dierent cellular localization pattern compared to the smaller form. Therefore, both ATG codons (Met-1 and Met-41) in human E1 cDNA represent potential translation start sites, and the two isoforms probably result from alternative translation start sites ( Handley et al., 1994 ). The same assignment of translation start sites may also be applied to rabbit and mouse E1 cDNAs
Northern analysis of total RNA from six rabbit tissues was carried out using the same 560 bp probe as the one used for library screening. A unique transcript with an approximate size of 3.5 kb was identified in all tissues examined ( Fig. 2). Ubiquitous expression of a single 3.5 kb transcript for human E1 was reported previously in a number of human tissues and cell lines ( Handley et al., 1991). These results are consistent with the observation that E1 plays important roles in cell cycle progression as well as in a number of other important cellular processes. Our results also suggest that the 3485 bp cDNA that we have isolated appears to be the intact E1 cDNA. 2.3. Expression, Western blot analysis and activity assay of recombinant E1 The entire E1 cDNA coding region was cloned into the pQE30 vector and transformed into Escherichia coli strain M15 (pREP4) (Qiagen). The resulting construct should permit the expression of a protein with a calculated molecular mass of about 118 kDa excluding the N-terminal 6 histidine tag and six other amino acid
Fig. 2. Northern blot analysis of rabbit E1 mRNA. Total RNA was extracted by the guanidium isothiocyanate/CsCl method (Chirgwin et al., 1979). Total RNA (15 mg) from each tissue was separated on a 1.2% agarose gel containing 2.2 M formaldehyde, transferred to a nitrocellulose membrane and hybridized with 32P-labelled probe as described (Sambrook et al., 1989). The probe used was a 560 bp E1 cDNA fragment labelled with 32P to a specific activity of 1×109 cpm/mg DNA. The positions of 28S and 18S rRNA are indicated. The blot was also stripped o the E1 cDNA probe and reprobed with a human GAPDH probe to normalize the loading of total RNA.
22
B. Sun et al. / Gene 196 (1997) 19–23
residues. The first attempt to purify the recombinant protein by induction with IPTG (0.02–0.5 mM ) at 37°C of E. coli harbouring the construct failed, as most of the protein expressed in this way formed inclusion bodies. Conditions were optimized so as to increase the solubility of the expressed his-tagged E1 protein. As shown in Fig. 3A, a significant amount of protein was expressed in a soluble form at 30°C when cells were induced at a very low IPTG concentration ( 7.5 mM ). The protein was partially purified by Talon metal anity
resin (Clontech) under native conditions with an estimated yield of 50–100 mg per litre of culture. Fig. 3B shows that the recombinant E1 protein could cross-react with antibodies raised against native human E1. Bands which are slightly larger than the 116 kDa molecular mass marker were specifically detected in the induced and partially purified protein samples but not in the uninduced sample. The lower molecular mass bands other than the 116 kDa E1 could be caused by nonspecific binding of antibodies to bacterial proteins. It is
Fig. 3. Expression, immunodetection and activity assay of the recombinant protein. A, recombinant E1 protein expression and SDS–PAGE analysis. An aliquot of 15 ml of overnight culture of a single transformant was diluted into 100 ml of LB containing 100 mg/ml ampicillin and 25 mg/ml kanamycin and incubated at 37°C for 1.5 h. Cells were induced with 7.5 mM IPTG for 2 h at 30°C before harvesting. Cells were collected, lysed by sonication and clarified by centrifugation. Recombinant E1 protein was purified by Talon metal anity resin using a protocol provided by the manufacturer. Protein samples were analysed by 8% SDS–PAGE and stained with Coomassie blue. Lane 1, uninduced; lane 2, induced, total protein; lane 3, induced, soluble protein; lane 4, protein purified. B, Western blot analysis of expressed E1 protein. The protein samples were separated by 8% SDS–PAGE and transferred to PDVF membrane. Blots were blocked with 5% non-fat milk, 0.1% sodium azide in TBST (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20) and probed with rabbit anti-human E1 antibodies. Secondary antibodies used were goat antirabbit IgG labelled with alkaline phosphatase. Blots were visualized using the alkaline phosphatase substrate BCIP/NBT. Lane 1, uninduced, total protein; lane 2, induced, total protein; lane 3, partially purified E1. Gel used for Western blot ( B) and gel used for Commassie blue staining (A ) are from dierent runs. C, Activity assay of recombinant E1 protein. In a 25 ml reaction, 0.5 mg of partially purified recombinant E1, 1 mg of 125I-labelled ubiquitin, 50 mM Tris–HCl, pH 7.5, 0.2 mM DTT, 5 mM ATP, 5 mM MgCl , two units of inorganic pyrophosphatase were incubated 2 at 37°C for 10 min with ( lanes 5 and 6) or without ( lanes 1–4) the addition of 0.5 mg of recombinant rabbit E . Purified native rabbit E1 was 2–32 used as a positive control. The reactions were stopped with Laemmli sample buer with ( lanes 1, 2 and 5 ) or without ( lanes 3, 4 and 6) 5% bmercaptoethanol and separated by 15% SDS–PAGE. Lanes 1 and 3, native rabbit E1 only; lanes 2 and 4, recombinant E1 only; lanes 5 and 6, recombinant E1 + recombinant E . 2–32
B. Sun et al. / Gene 196 (1997) 19–23
interesting to see that two proteins with similar molecular masses (approx. 116 kDa) were induced as shown in Fig. 3A ( lanes 2 and 3 ). Both of the two bands can be detected by anti-human E1 antibodies (Fig. 3B, lane 2), indicating that both of them might be E1 proteins. The smaller protein probably arose from an internal translation start site. Fig. 3C shows that the bacterially expressed E1 could either form a thiolester bond with ubiquitin in the absence of rabbit E ( lane 4 ) or transfer ubiquitin to 2–32 rabbit E ( lane 6 ). Boiling the samples with loading 2–32 buer containing 5% b-mercaptoethanol eliminated the thiolester bond between E1 and ubiquitin ( lane 2), but only markedly diminished the intensity of the band representing E -ubiquitin ( lane 5). A reasonable 2–32 explanation for this observation is that E might be 2–32 able to ubiquitinate itself, and the isopeptide bond could not be destroyed by boiling in the presence of reducing agent. It has been reported that CDC34, the yeast homologue of rabbit E , can ubiquitinate itself both 2–32 in vitro and in vivo. Yeast CDC34 autoubiquitination forms isopeptide bonds that could not be cleaved by treatment with b-mercaptoethanol ( Banerjee et al., 1993; Goebl et al., 1994 ). 2.4. Conclusion
(1 ) The cDNA encoding full-length rabbit ubiquitinactivating E1 has been cloned and sequenced. (2 ) The deduced amino acid sequence of the cloned E1 cDNA is highly homologous to human and mouse E1. (3 ) The rabbit E1 mRNA exists as a single 3.5 kb transcript in all six tissues examined. (4 ) The recombinant E1 protein has been expressed in a soluble form in E. coli and has been shown to cross-react with anti-human E1 antibodies. (5 ) The bacterially expressed rabbit E1 protein is functional.
Acknowledgement This work was supported by a National University of Singapore research grant RP930311. We thank Dr P. Boon Chock ( Laboratory of Biochemistry, NHLBI, NIH ) for providing us with anity-purified antibodies raised against human E1. We also thank Mrs M.K. Lee for her expert technical assistance.
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