J. Biochem. 112, 28-32 (1992)

cDNA Cloning and Tissue Distribution of a Rat Ubiquitin Carboxyl-Terminal Hydrolase PGP9.51 Yasuo Kajimoto,* Takeshi Hashimoto,* Yutaka Shirai,* Naoki Nishino,*' Takayoshi Kuno,* and Chikako Tanaka*2 Departments of 'Pharmacology and "Psychiatry and Neurology, Kobe University School of Medicine, Chuo-ku, Kobe, Hyogo 650 - Received for publication, January 27, 1992 '['.

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We have isolated a cDNA clone encoding ubiquitin carboxyl-terminal hydrolase PGP9.5 from a rat brain cDNA library and examined the tissue distribution. The primary structure of the cDNA consists of 856 nucleotides including the entire coding region for 223 amino acids, and the calculated molecular mass is 24,782 Da. The rat PGP9.5 is strikingly homologous to the human PGP9.5, 75.2% of nucleic acids and 95.1% of amino acids being identical. The mRNA of PGP9.5 is most abundant in the rat brain and to a lesser degree in the testis. In other peripheral tissues we tested, the mRNA was undetectable. Western blotting using an anti-rat PGP9.5 antibody revealed the parallel distribution of mRNA and protein in various brain regions and testis. The availability of the rat PGP9.5 clone provides a new approach to examine the function of PGP9.5 and the role that it plays in the pathology of neurodegenerative diseases.

Ubiquitin is implicated in the regulation of a wide variety of cellular functions, one of the major functions being its involvement in protein degradation (1). Ubiquitinylation of proteins has been shown to target a subset of cellular proteins for rapid degradation by an ATP-dependent pathway. Ubiquitin carboxyl-terminal hydrolytic activity is probably a necessary step in the regeneration of the ubiquitin monomer from ubiquitin-protein conjugates. A new enzyme family of mechanistically related hydrolases has been proposed: the ubiquitin carboxyl-terminal hydrolases (2). Protein gene product 9.5 (PGP9.5) is among the most abundant proteins in the brain, representing 1-2% of total soluble brain proteins (3). A recent report concluded that PGP9.5 is ubiquitin carboxyl-terminal hydrolase isozyme LI (4). Immunohistochemical studies demonstrated that PGP9.5 is highly expressed in neuronal and neuroendocrine tissues (3, 5, 6). Ubiquitin is a component of the intermediate filament inclusion bodies characteristic of neurodegenerative diseases such as the neurofibrillary tangles of Alzheimer's disease and the Lewy body of Parkinson's disease (7-9). Immunocytochemistry, using a polyclonal antibody against PGP9.5, revealed that PGP9.5 was selectively present in ubiquitinated inclusion bodies characteristic of human neurodegenerative diseases (10). These observations suggest that PGP9.5 as well as ubiquitin may contribute to the pathology of these diseases. A human cDNA clone encoding 1 This study was supported by research grants from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare of Japan. The nucleotide sequence data reported in this paper have been submitted to the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases under accession number D01109. 1 To whom correspondence should be addressed. Abbreviations: lxSSC=0.16M NaCl, 15 mM sodium citrate (pH 8.0); lxSSPE = 0.18MNaCl, 10 mM sodium phosphate (pH 7.4), 1 mMEDTA.

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PGP9.5 has been isolated (11). As a tool to examine the role that it plays in the pathology of neurodegenerative diseases, a rat cDNA clone for PGP9.5 may be useful. We have now cloned and sequenced rat PGP9.5 cDNA, and examined its tissue distribution. MATERIALS AND METHODS cDNA Cloning—To isolate a clone encoding ubiquitin carboxyl-terminal hydrolase PGP9.5, a rat brain cDNA library within a A.gtll vector was immunoscreened using rabbit antiserum to PGP9.5 protein purified from bovine brain by the method of Doran et al. (3). The phages were plated on the Y1090 strain of Eacherichia coli at a density of 106 per plate and the protocol of Young and Davis (12) was followed for plaque lifting and antibody screening. Positive clones were selected from the master plates and purified through several additional rounds of screening. Fifteen different clones were thus identified. Sequence analysis indicated that one of the clones, called pKH9, encoded ubiquitin carboxyl-terminal hydrolase PGP9.5. Because the clone contained only short DNA sequences, it was used as a plaque hybridization probe to isolate a longer cDNA clone. A phage (AgtlO) cDNA library was constructed using rat brain poly(A)+ RNA, and was screened on nitrocellulose filters with the randomly primed cDNA probe. The filters were prehybridized at 40"C in solution containing 20% (v/v) formamide, 5 X SSPE and 0.5% (w/v) SDS. Hybridization was carried out with 32P-labeled probes in the same solution at 42'C overnight. After hybridization the filters were washed with O.lxSSC and 0.1% SDS at 60"C. The filters were then dried and autoradiographed. In the case of positive signals, several rounds of screening were carried out until 100% of plaques gave positive signals. Nucleotide Sequencing—The nucleotide sequence was J. Biochem.

cDNA Cloning of Rat PGP9.5

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determined using the dideoxynucleotide chain-termination was induced after 3.5 h of plaque growth by placing a method (13) after subcloning into M13mpl8 and M13mpl9 nitrocellulose filter containing 10 mM IPTG (isopropyl ySvectors. D-thiogalactopyranoside) onto the plate and continuing Northern Blot Analysis—Total cellular RNA was isogrowth at 37'C. Two hundred microhters of crude antiselated from rat tissues by the guanidine thiocyanate method rum against PGP9.5 was diluted with a solution of 3% {14), electrophoresed in a formaldehyde-1% (w/v) agarose normal goat serum (NGS) in PBST (0.1% phosphategel, and blotted onto nitrocellulose paper. After baking at buffered saline+ 0.03% Triton X) and incubated with the 80'C for 2 h and prehybridization, the blot was hybridized filters for 1.5 h. The filters were washed seven times in in 4xSSC, 40% formamide, 10% (w/v) dextran sulfate, PBST for 3min each. Specific antibody was eluted sepa0.02 M Tris-HCl (pH7.4), lxDenhardt's solution, 250 rately from each filter with two 1 min washes in a solution Mg/ml yeast tRNA, and 20 ^g/ml salmon sperm DNA with of 5 mM glycine-HCl (pH 2.3), 150 mM NaCl, 0.5% Triton the randomly primed cDNA probes at 42'C overnight. The X, and 100 //g/ml BSA. Products of the combined washes blots were washed twice in 0.1 X SSC, 0.1% SDS for 30 min were immediately neutralized with 1 M Tris-HCl (pH 7.4) each at 60°C and the resulting blots were visualized by to a final concentration of 50 mM. These antibodies were autoradiography. then used to probe Western blots. Antibody Purification—Antiserum against bovine PGP Western Blot Analysis—Tissue samples were homoge9.5 protein was further purified for specific detection of the nized in 20 mM Tris-HCl (pH 7.4), 2 mM EDTA, and 25 protein in Western blotting. The clone (pK119) isolated mM mercaptoethanol and loaded onto an SDS/15% (w/v) from the A g t l l library was used to infect E. coli (Y1090). polyacrylamide gel. After electrophoresis, protein was Expression of the /S-galactosidase-cDNA fusion proteins transferred from the gel to a nitrocellulose membrane. The 10 20 30 40 50 60 GGCGCTTGTTCTTCCCTGTGCCACTCCGCAAAGAT£CAGCTGAAACCGATGGAGATTAAC M Q L K P M E I N 70 80 90 100 110 120 CCCGAGATGCTGAACAAAGTGTTGGCCAAGCTGGGGGTCGCCGGGCAGTGGCGCTTTGCC P E M L N K V L A K L G V A G Q W R F A 130 140 150 160 170 180 GACGTGCTAGGGCTGGAGGAGGAGACTCTGGGCTCAGTGCCATCTCCTGCCTGCGCCCTG D V L G L E E E T L G S V P S P A C A L 190 200 210 220 230 240 CTGCTGCTGTTTCCCCTCACGGCCCAGCATGAAAACTTCAGGAAAAAACAAATTGAGGAA L L L F P L T A Q H E N F R K K Q I E E 250 260 270 280 290 300 CTGAAGGGACAAGAAGTTAGCCCTAAAGTTTACTTCATGAAGCAGACCATCGGGAACTCC L K G Q E V S P K V Y F H K Q T I G N S 310 320 330 340 350 360 TGTGGTACCATTGGGCTGATGCACGCAGTGGCCAATAACCAAGACAACCTGGGATTTGAG C G T I G L M H A V A N N Q D N L G F E 370 380 390 400 410 420 GATGGATCAGTCCTGAAACAGTTTCTGTCTGAAACGGAGAAGTTGTCCCCTGAAGACAGA D G S V L K Q F L S E T E K L S P E D R

430

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450

460

470

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GCCAAGTGTTTCGAGAAGAACGAGGCCATTCAGGCAGCCCATGACTCCGTGGCCCAGGAG A K C F E K N E A I Q A A H D S V A Q E

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GGCCAGTGCCGGGTAGACGACAAAGTGAATTTCCATTTTATCCTGTTCAATAATGTGGAC G Q C R V D D K V N F H F I L F N N V D 550 560 570 580 590 600 GGCCACCTCTACGAGCTCGATGGGCGAATGCCTTTCCCGGTGAACCATGGCGCCAGTTCA G H L Y E L D G R H P F P V N H G A S S 610

620

630

640

650

660

GAGGACTCTCTGCTGCAGGATGCCGCCAAGGTCTGCAGAGAATTCACTGAGCGCGAGCAG E D S L L Q D A A K V C R E F T E R E Q

670

680

690

700

710

720

GGAGAGGTCCGCTTCTCCGCAGTGGCTCTCTGCAAAGCAGCC1AAGTAGGGAGAGAGAAC G E V R F S A V A L C K A A * Fig. 1. Nucleotide and deduced amlno acid sequence ofthe rat PGPS.ScDNA clone. The numbers 730 740 750 760 770 780 indicate nucleotide positions. The initiation and tor- CAGCCGCTCCCCCATCCCTGGGCAGGTGCGCGCTGCCCTGCCCTTGGTTTGCAGCTTTAG mination codons are underlined. The deduced amino CACTTAACAACCACAGCrGTCTTCTTGCGTTCTACTGCCCCGTCCCCTCCACCCCACCCA acid sequence is shown in the one-letter code below 950 the nucleotide sequence. GGCCACCAGGGAGCTC Vol. 112, No. 1, 1992 Downloaded from https://academic.oup.com/jb/article-abstract/112/1/28/837907 by Insead user on 19 July 2018

Y. Kajimoto et al. membrane was blocked by washing in PBST containing 3% gelatin and incubated with anti-PGP9.5 antibody. Immunoreactive bands were visualized by the peroxidaseantiperoxidase method (15). RESULTS

Cloning and Sequencing—Making use of an antiserum to ubiquitin carboxyl-terminal hydrolase PGP9.5, we screened a rat brain cDNA library within a Agtll vector and isolated 15 positive clones. Sequence analysis revealed that one of the clones, called pK119, encoded ubiquitin carboxyl-terminal hydrolase PGP9.5. Since the insert contained only short DNA sequences, we screened a XgtlO library prepared from rat brain cDNA with the "P-labeled insert of A.gtll clone and obtained a single clone (pK151) containing the full-length coding sequence. The nucleotide sequence for rat PGP9.5 cDNA and the predicted amino acid sequence are shown in Fig. 1. The cDNA insert consists of 856 base pairs (bp) and has 33 bp of 5'-non-coding region, an open reading frame of 669 bp and 154 bp of 3'-non-coding region. The open reading frame

begins with the initiating ATG codon at nucleotide 34 and is terminated by a TAA stop codon at nucleotide 703. The ATG codon at 34 is thought to be an initiator, for the following reasons: (i) the region on the 5'side of the ATG codon is G+C-rich; (ii) the nucleotide sequence surrounding this ATG agrees with the consensus sequence related to the initiation of translation often found in eukaryotes (16). The open reading frame predicts a protein containing 223 amino acids with a molecular mass of 24,782 Da. Tissue Distribution—We examined tissue expression of PGP9.5 mRNA by Northern blot analysis (Fig. 2). The 0.21 -kb EcoBl fragment (amino acids 646-856) consisting of the terminal portion of the coding sequence and the 3'-untranslated sequence was used as a probe for hybridization to avoid cross-hybridization with other isoforms of PGP9.5. Northern blot analysis revealed the presence of 1.1-kb RNA species at a high level in the brain and to a lesser degree in the testis (Fig. 2A). No detectable RNA species were observed in the heart, lung, pancreas, small intestine, liver, kidney, spleen, or adrenal tissues. Northern blot analysis revealed the presence of 1.1-kb RNA species in various regions of the rat brain, with the

B

A 28S-

28S18S-1.1

18S-

Fig. 2. Northern blot analysis of PGP9.5 mHNA. Total RNA (several rat tissues [A], 25 fxg; rat brain areas [B], 15 fig) was blotted onto a nitrocellulose filter and the blotted filter was hybridized with "P-labeled cDNA probe corresponding to the 0.21-kb EcoBI fragment. The positions of 18 S and 28 S rRNAs are indicated.

B kOa

kDa

44282818-

18-

Fig. 3. Western blot analysis of PGP9.5. Tissue samples (several rat tissues [A]; rat brain areas [B]) were homogenized and subjected to SDSpolyacrylamide gel electrophoresis, then the separated proteins were transferred to nitrocellulose. The Western blots were incubated sequentially with an anti-rat PGP9.5 antibody. Protein sizes in kilodaltons are indicated on the left. J. Biochem.

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cDNA Cloning of Rat PGP9.5 Fig. 4. Amlno acid sequence rat comparison between the rat PGP9.5 and the human PGP9.5. human The deduced amino acid sequences are shown in one-letter code. The amino acid sequence of rat the human PGP9.5 is from the report by Day et al {11, i7).The human matched residues are connected by asterisks. Conservative substitutions are indicated by dots. rat

MQLKPMEINPEMLNKVLAKLGVAGQWRFAnVLGLEEEILGSVPSPACALLLLFPLTAQHE

human

TEKMSPEDRAKCFEKNEAIQAAnDAVAQEGQCRVDDKVNFnFILFNNVDGHLYELDGRMP

rat

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MQLKPMEINPEMLNKVLSRLGVAGQWRFVDVLGLEEESLGSVPAPACALLLLFPLTAQHE NFRKKQIEELKGQEVSPKVYFMKQTIGNSCGTIGLMnAVANNQDNLGFEDGSVLKQFLSE

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NFRKKQIEELKGQEVSPKVYFMKQTIGNSCGTIGLIHAVANNQDKLGFEDGSVLKQFLSE TEKLSPEDRAKCFEKNEAIQAAHDSVAQEGQCRVDDKVNFHFILFNNVDGHLYELDGRMP

FPVNHGASSEDSLLQDAAKVCREFTEREQGEVRFSAVALCKAA •

human

60

11111,1111111111111111

180

223

IIIIIMMI.II.IIIIIIIIItlllllllUHIIIIlll

FPVNnGASSEDTLLKDAAKVCREFTEREQGEVHFSAVALCKAA

midbrain area showing the highest concentration (Fig. 2B). PGP9.5 mRNA was found in high amounts in the rat cortex, hippocampus, hypothalamus, thalamus, olfactory bulb, and striatum and to a lesser degree in the cerebellum and pons-medulla. The fusion proteins expressed by E. coli Y1090 infected with the PGP9.5 clone pK119 were absorbed on nitrocellulose filters and incubated with crude antiserum to PGP 9.5. Affinity-purified antibodies immobilized on each filter were eluted and used to detect the PGP9.5 protein by Western blot analysis. A single band with a molecular mass of ~ 2 5 kDa was detected in Western blots (Fig. 3). The observed molecular mass of the PGP9.5 protein agrees well with calculated value of 24,782 Da. The abundance of the PGP9.5 protein was estimated by Western blotting of the proteins from several rat tissues by using an anti-rat PGP9.5 antibody (Fig. 3A). The PGP9.5 protein was found in many rat brain regions with the highest level in the mid-brain area and a lower level in the cerebellum and pons-medulla (Fig. 3B). It was also detected in the testis, but the concentration was below the level of detection in the brain. These results indicate the parallel distribution of the PGP9.5 protein and mRNA. DISCUSSION We isolated a cDNA clone encoding ubiquitin carboxylterminal hydrolase PGP9.5 from a rat brain cDNA library. The nucleic acid sequence of rat PGP9.5 is 75.2% identical to the sequence of the cDNA encoding human PGP9.5 {11, 17). Within the coding region there is 88.3% nucleotide identity, with the majority of differences being third-baseposition changes. At the amino acid level, the two sequences are 95.1% identical (Fig. 4). Immunohistochemical studies demonstrated that PGP 9.5 is highly expressed in neuronal and neuroendocrine tissues. Wilson et al. (6) examined immunolocalization of PGP9.5 in human, rat, and guinea-pig tissues, using polyclonal and monoclonal antibodies. They noted immunoreactive PGP9.5 in neurons and nerve fibers at all levels of the central and peripheral nervous system, in numerous neuroendocrine cells, in part of the renal tubule, in spermatogonia and Leydig cells of the testis, and in ova and in some cells of the corpus luteum. We report here the first evidence of the tissue distribution of the PGP9.5 mRNA. It Vol. 112, No. 1, 1992 Downloaded from https://academic.oup.com/jb/article-abstract/112/1/28/837907 by Insead user on 19 July 2018

is interesting that the distribution of the PGP9.5 mRNA was parallel to that of a 25 kDa protein band detected by Western analysis using specific antibody. Western blot analysis indicated that the antibody did not cross-react with any additional PGP9.5 isoforms. The mRNA for PGP9.5 and the PGP9.5 protein was most abundantly expressed in the brain and to a lesser extent in the testis. In the heart, lung, pancreas, small intestine, liver, kidney, spleen, and adrenal, mRNA and protein were undetectable. Thus, the distribution of PGP9.5 mRNA and protein does not agree with all of the results of immunohistochemical studies. TmmiinnBtjiining for PGP9.5 was evident in the renal tubules, pancreatic islets and adrenal medulla in addition to the brain and testis. It is possible that the amount of message present may have been below the level of detection of our assays. Alternatively, there may be additional PGP9.5 isoforms in the kidney, pancreas, and adrenal that were not detectable with our antibody. The expression of PGP9.5 proved to be tissue-specific and distinct in various brain regions. These observations suggest that the role of PGP9.5 may vary according to tissue or neuronal type. As PGP9.5 may be involved in the pathology of several human neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, the cloning of the rat PGP9.5 provides a new tool for use in studies on the function of PGP9.5. We thank Mariko Ohara for critical comments. REFERENCES 1. Herahko, A. (1988) J. BioL Chan. 263, 15237-15240 2. Mayer, A.N. & Wilkinson, K.D. (1989) Biochemistry 28, 166172 3. Doran, J.F., Jackson, P., Kynoch, P.A.M., & Thompson, R.J. (1983) J. Neurochem. 40, 1542-1547 4. Wilkinson, K.D., Lee, K., Deshpandei S., Duerksen-Hughes, P., Boss, J.M., & Paul, J. (1989) Science 246, 670-673 5. Thompson, R.J., Doran, J.F., Jackson, P., Dhillon, A.P., & Rode, J. (1983) Brain Res. 278, 224-228 6. Wilson, P.O.G., Barber, P.C., Hamid, Q.A., Power, B.F., Dhillon, A.P., Rode, J., Day, I.N.M., Thompson, R.J., & Polak, J.M. (1988) Br. J. Exp. PathoL 69, 91-104 7. Mori, H., Kondo, J., & Ihara, Y. (1987) Science 235, 1641-1644 8. Kuzuhara, S., Mori, H., Izumiyama, N., YoshimuraM., & Ihara, Y. (1988) Ada NeuropathoL 75, 345-353 9. Lowe, J., Blanchard, A., Morrell, K., Lennox, G., Reynolds, L.,

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Y. Kajimoto et al. Billett, M., Landon, M.( & Mayer, R J . (1988) J. PathoL 155, 915 Lowe, J., McDermott, H., Landon, M., Mayer, RJ., & Wilkinson, K.D. (1990) J. Nathol. 161, 153-160 Day, I.N.M. ft Thompson, R J . (1987) FEBS Lett. 210, 157-160 Young, R.A. & Davis, R.W. (1983) Proc. Nad. Acad. Sd. USA 80, 1194-1198 Sanger, F., Nicklen, S., &Coulson, A.R. (1977) Proc. Nad. Acad.

Sci. USA 74, 5463-5467 14. Chirgwin, J.M., Pryzbala, A.E., MacDonald, R.Y., & Rutter, W.J. (1979) Biochemistry 18, 5294-5299 15. Stemberger, L.A., Hardy, P.H., Cuculis, J.J., & Meter, H.G. (1970) J. Histochem. Cytochem. 18, 314-333 16. Kozak, M. (1987) Nucleic Adds Res. 15, 8125-8148 17. Day, I.N.M., Hinks, L.J., & Thompson, R J . (1990) Biochem. J. 268, 521-524

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