Biochimica et Bioph.vsica Acta, 1089(1991) 250-253 ~ 1991 ElsevierSciencePublishersB.V. 0167-4781/91/$03.50 ADONIS 016747819100155W

250

BBAEXP 9{)229

Short Sequence-Paper

Molecular cloning, structure and expression analysis of a full-length mouse brain glutamate dehydrogenase c D N A George Tzimagiorgis and Nicholas K. Moschonas Department of Biology, Unicersity of Crete and Institute of Molecular Biology and Biotechnology, Foundation ]'or Research and Technology, Heraklion, Crete (Greece)

(Received 14 February1991)

Key words: Glutamatedehydrogenase;Olivoponto-cerebellaratrophy:cDNA cloning:Aminoacid sequence: Signalpeptide; Gene expression:(Mouse) We isolated and analysed a full-length mouse brain glutamate dehydrogenase (GLUD) cDNA as a preliminary step to use the mouse model for the investigation of GLUD function in neurotransmission and neurodegeneration. GLUD coding sequences were found highly conserved among mouse, human and rat. Northern blots revealed two transcripts with different ratios in different mouse organs implying some mechanism of tissue-specific exp.'ession. In contrast to human, mouse GLUD gene family appears not to contain an intronless member.

Glutamate dehydrogenase (GLUD; EC 1.4.1.3.), a mitochondrial matrix enzyme, catalyzes the reversible oxidative deamination of L-glutamate to a-ketoglutarate using NAD a n d / o r NADP as cofactors [1]. GLUD is predominantly expres,,ed in mammalian organs such as brain, liver, kidney and leucocytes [1,2]. The study of the mammalian brain G L U D is of particular interest. Recent stt:,iies suggested that GLUD is important in the biology of the nervous system and the pathogenesis of certain neurodegenerative disorders characterized by multisystem atrophy [2,3]. Glutamate, the main substrate of GLUD, is a well-characterized neurotransmitter [4]. Because of its neuroexcitotoxic potentials, it may mediate neuronal damage in response to various metabolic insults [5]. The direct role of GLUD in neurodegcneration was suggested by studies showing partial enzymatic deficiency and abnormal glutamate metabolism in patients with olivopontocerebellar atrophies (OPCAs) [3,6]. Similarly, it was found that rat brain GLUD mRNA levels were seri-

Abbreviations: GLUD, glutamate dehydrogenase; NAD, nicotinamide adenine dinucleotide;NADP, nicotinamideadenine dinucleotide phosphate;OPCAs, olivoponto-cerebellaratrophies. The sequence data in this paper have been submitted to the EMBL/Genbank under the accessionnumber X57024. Correspondence: N.K. Moschonas, Institute of Molecular Biology and Biotechnolngy,Foundation of Research and Technology, P.O. Box 1527, Heraklion711 10, Crete, Greece.

ously decreased in an animal model of encephalopathy, a neurological disorder caused by liver dysfunction associated with brain hyperammonemia [7]. We have isolated human G L U D cDNAs and predicted that the enzyme is encoded by a multigene family [8]. Because of the large number of neurological mutations described in the mouse, we have initiated molecular studies of the G L U D system in this species to facilitate the understanding of mammalian G L U D gene structure and expression in health and disease. Here, we report the characterization of a full-length mouse brain G L U D eDNA clone. Results on mouse GLUD gene organization and expression are also presented. A series of clones were isolated from a ~tgtl0 mouse ( B A L B / c ) brain eDNA library (a generous gift of Dr. G. Grosveld) using as a probe a full-length human liver G L U D eDNA [8] radiolabeled by random priming [9]. DNA inserts were subcloned in pUCI9 and both strands were sequenced by the double-strand dideoxy chain termination method [10] using universal sequencing primers and home-designed oligonucleotide primers synthesized on an Applied Biosystems DNA synthesizer. Mouse genomie D N A and total cellular RNA preparations, and blotting analyses were performed as described elsewhere [8]. A set of five mouse brain cDNAs, selected on their size and restriction map, were fully sequenced. All clones were found to be identical at their overlapping regions. The larger (2.94 kb) clone, MBG-1, repre-

251

sented a full-length GLUD cDNA (Fig. 1) according to its apparent high degree of homology to the human [8] and rat [11] liver GLUD cDNA sequences. It included a 76 bp 5' untranslated region, a 1674 bp open reading frame encoding for a single 558 amino acid polypeptide (including a 53 amino acid mitochondrial signal pep-

tide), a 1212 bp 3' untranslated region and a 9 bp poly(A) tail. Five putative polyadenylation signals were identified. Nucleotide sequence alignment of MBG-1 with the human and the rat GLUD cDNAs revealed a striking homology at the coding region (alignment not shown), i.e., 92.3% (mouse vs. human) and 95% (mouse

CCTCCCCGCGGTCCAGGCCTGCGAGCTCCGGTCTTTACAGCTCCCGCCGCACTCGCCTCAGCCCGCCC-eCC~CcCC

CGC GCC GGG CCC GCT GCC CTG GGC TCT GCG GCT GCA CAC TCA GCC GCG CTG CTG GGC

Arg Ala GIF Pro Ala Ala Leu Gly Ser Ala Ala H:Ser GGG CTC ACG CCG GTC GCC CGA CGC CAC TAC AGC GIy Leu Thr Pro Val Ala Arg Arg His Tyr 5er H:AZa Leu A l a GAC CGC GGC GCC AGC ATC GTA GAG GAC /tAG CTG Asp Arq Gly A1a Set Ile Val Glu Asp Lys Leu

Ala Asp Ser Ala Ala Leu Leu GIy

GAA GCG GCC GO= GAC Glu A l a Ala A l a ASp 8:Val R:Thr GTG C~A GAC CTG AAG Val Glu ASp Leu Lys M:Arq CTG AGG ATC ATC AAG CCC TGC AAC CAT GTG TTG AGC CTC TCC T1"C CO= Leu Arq Ile l l e Lys Pro Cys Asn His Val Leu Set Leu Set Phe Pro

CGC GAA GAC

Arq GIu ASp

ATG TAC CC,C CGT CTG C,GC GAA GCG CTG CTG CTG TCC Met Tyr Art] A r g Leu G l y GIu A l a Leu Leu Leu Ser tl: T y r R: Val TGG GCC CGC GGA CAG CCC TCC GCC GCC CCG CAA Trp A l a A r q GIg Gln P r o S e r A l a A l a Pro Gin H:AIa R:Val GAC CCC AAC TIC. TTC AAG ATG GTG GAG GGC TTC Asp Pro ASh Phe Phe Lys Her; V a l Glu Gly Phe

ACC CGG GAG AGC .'hE A r g GIU Ser R:ASn ATC CGG CGC GAC lle Arq A/q Asp

112

12

CCC Pro

205 43

TI~

298 74

Phe

GAG GAG CAG AAG CGG AAC CGG GTG CGC GGC ATC

391

GIU GIu Gin Lys A~g Asn Arg V a l Arg Gly I l e

105

GAC GGC TCC TGG ~

484

GTC ATC GAA GGC TAC CGG

Asp Gly Set T r p Glu Val Ile Glu Gly Tyr A~g

136

GCC CAG CAC AGC CAG CAC CGC ACG CCC TGC AAG GGA GGT ATC CGT TAC AGC ACT CAC GTG AGT GTG GAT GAA GTA AAA GC.A CTG GCT TCC Ti"A Ala Gln His Ser Gln His Arg Thr Pro Cys Lys Gly Gly Ile Arg Tyr Set Thr Asp Val Set Val Asp Glu Val Ly$ Ala Leu Ala Set Leu

577 167

ATG ACA TAC AAG TGC GCT GTG GTC GAT GTA CCG TTT GGA GGT GCT AAA GCA GGC GTT AAG ATC AAC CCC AAG AAC TAT ACA GAT AAT GAA TTA Met Th~ Tyr Lys Cys Ala Val Val ASp Val Pro Phe Gly Gly A l a Lys A l a Gly V a l Lys Ile Ash Pro Lys Ash Tyr Thr Asp Ash Glu Leu

670 198

GAA AAA ATT ACA CGG AGG ~ C ACT ATG GAG CTG GCC AAG AAG GGT TTT ATT GGT CCT GGC ATT GAT GTG CCT GCC CCA GAC ATG AGC ACG GGT Glu Lys Ile Thr Arg Arg Phe Thr Met Glu Leu Ala Lys Lys Gly Phe I l e G l y Pro Gly I l e Asp V a l Pro A l a Pro Asp Met Set Thc Gly

763 229

GAG CGG GAG ATG TCC TGG ATC GCT GAC ACC TAT GCC AGC ACC ATA GGG CAC TAT GAT ATC ~AT GCG CAT GCC TGT GTT ACT GGG AAA CCC ATC GIu Arg GIu Met Set Trp Ile Ala Asp Thr Tyr Ala Set Thr Ile GIy 8is Tyr Asp lle ASn Ala His Ala Cys VOI Thr Gly Lys Pro Ile

856 260

AGT CAA GGA GGC ATC CAC GGG CGC ATC TCC GCT ACT GGC CGG GGT GTC TTC CAT GGA ATT GAA AAC TTC ATC AAT GAG GCT TCT TAC ATG AGC Ser Gln GIy Gly Ile His Gly Arg Ile Set A/a Thr Gly Arg Gly Val Phe His Gly Ile G1u ASh Fhe lle Ash GII: Ala Ser Tyr Met Set

949 291

ATT TTA GGA ATG H~A CCA GGC TTT Ile Leo Gly Met Thr Pro Gly Phe R:Leu TTT GGT GCT AAA TGT GTT GGT GTT Phe G l y A l a Lys Cys V a l Gly V a l H:Ile A l a CAA CAT GGA TCA ATT CTG GGC TTC Gln Hls GIy Set Ile Leu GIy Phe

GGC GAT AAG ACA TTT GTT GTT CAG GGA TTT GGT AAT GTG GGC CTG CAC TCT ATG AGA TAT TTA CAT CGT 1042 322 Gly Asp Lys Thr Phe Val Val Gln Gly Phe GIy Ash Val GIy Leu His 5er Met Arg Tyr Leu His Arg GGA GAG TCT GAT GGG AGT ATA TGG AAT CCG CAT GGG ATT GAC CCA AAA GAA CTG GAA GAC ~ C AAG TTG Gly Glu Set Asp Gly Set Ile Trp ASh Pro ASp Gly I l e ASp Pro Lys Glu Leu Glu Asp Phe Lys Leu

1135 353

CCC A A A GCC AAG GTC TAT GRA GGA AGC ATC TTG GAG GCT GAC TGT GAC ATT CTG ATT CCT GCT GCC AC-C 1228 384 Pro Lys Ala Lys Val Tyr Glu GIy Set lle Leu GIu Ala Asp Cys ASp Ile Leu lle Pro Ala Ala Set

GGT GCC AAT GGG CCA ACC ACT CCA GAG GCT GAT AAG GAG AAG CAG TTG ACC AAA TCC AAT GCA CCC ~ A GTC AAA GCC AAG AT(: ATT GCT ~ Glu Lys Gin Leu Thr Lys Set ASn A1a Pro Arg Val Lys Ala Lys lle lle Ala GIu Gly Ala ASn Gly PrO ThE Thr Pro GIu Ala Asp Lys

1321 415

ATT TTC CTG GAA AGA AAC ATC ATG GTT ATT CCC GAT CTC TAC ~'rA AAT GCT GGA GGA GTA ACA GTG TCT TAC TTT GAG TGG CTA AAG AAT CTA Ile Phe Leu GIu Arg Asn Ile Met Val Ile Pro Asp Leu Tyr Leu Ash Ala Gly GIy Val Thr Val Ser Tyr Phe Glu Trp Leu Lys As~ Leu

1414 446

AAT CAT GTC AGC TAC GGC CGA TTG ACC TTC AAA TAT GAA AGG GAC TCT AAC TAC CAC TTG CTC ATG TCT GTT CAA GAG AGT TTA GAG AGA AAG Asn His Val Set Tyr Gly Arq Leu Thr Phe Lys Tyr Glu Arg ASp Set ASh Tyr His Leu Leu Met Set Val Gln Glu 5er Leu Glu Arg Lys

1507 477

GTC CCC ACA GCA GAG TTC CAG GAC AGG ATA TCG GGT GCA TCT GAG AAA GAC ATT GTG CAC TCT Val Pro Thr Ala Glu Phe Gln Asp Arg Ile Set Giy Ala SeE GIU Lys Asp Ile Val His 5e~

1600 508

AGG CAA ATT ATG CGC ACA GCC ATG AAG TAT AAC CTG GGA TTG GAC CTG AGA ACA GCT GCC TAT Arg Gin Ile Met Arg Tht Ala Met Lys Tyr Asn Leu Gly Leu Asp Leu Arg Thr Ala Ala Tyr

1693 539

TTT GGA AAG CAT GGT GGA ACT ATT CCT GTG Phe Gly Lys His Gly G1y Thr Ile Pro Val H:lle GGC TTG GCC TAC ACA ATG GAG AGA TCT GCC Gly Leu Ala Tyr Thr Met Glu Arg Set Ala

GTC AAT GCT ATC GAG AAA GTC TTC AAG GTG TAC AAT GAA GCT GGT GTG ACC TTC ACA TAG ACAGCTCACAGCCGACTr CYITACCACCTCTIV~ACCTATAATT Val Ash Ala Ile Glu Lys Val Phe Lys Val Tyr ASh GIu Ala GIy Val Thr Phe Thr "'"

1 ~96 558

1920 TCTGCAGACCTGTCACAAGTTr~CATATAACCATAGAAA~TGACTCATTAGTTAACGGACA~GTCAGTTGGAATCAGC~C~-£~TTAAGTTA 2044 GAGGATCATGTACAAGCTGATGGTATAAAAGTAGGAATCACGTGTAT~GTTA~TVi~x~AGGTAAAGTCTTCL`TCCTGTGCTG~CC1~1X~C~GAG TCAGTGCTG ~ T , ~ % C , RAGTAGGTGTGT GGC~I-t-I~..AP~GGTGGCATGt~ ~t~-~..tCAAATC~AG 2168 2292 TTATTGTGGTTTTCCAT~ T A R C T C A ~ - r vI'L'TAA TAAAi%GCTATGTCTCATGTATTTTATTCrC[EAGAATAAN:EAGTACAATGCTGCTGTaATAbJLTTGC 2416 C TT C A A C C A C T T A A G C C T A A C C T T T A C I T A A ~ C A C C T A R A A T ~ ~ ~ ' t -t-rt-a'~A T H ~ C T A T A A ~ A C A G T A T C A C T T G T A G C I T A ~ T C A T T G T 2540 CCAGCAGnAATA~A GTAGCACTATGGGGCGC TCTC CTCAGACC TGCAAGGGACAAGGG~L- ILT~GGGANY£GGTAATGCAGTCCAGAGAGTAGAG TAACCACTTTACCATGAARACTACTI~'TT CTTCCCATTTGAAGGTCTATTCTGTTGAGTAATTGAACCATAATTCAAATCGAGTGT ~ G T T T GGAGTCFCq;CAGGCI~ ~GTGGCCAG 2664 GCTCAACCTGTGTGTCCCCTTCCAAGGCTTTGCATTCTGCrCACCGeTTCA~CAGCAGCAGGCTAGAGACTGCCTGCCTGGGCTTGALT~.r ~ ~ A C 2788 2912 AGGAATGTCCCACAGAAGTACCAGACATTATTTATATAAGGATGAA~GCTI~1Ti-1-ri~AACAGT~CAGAAATTTCTAACTACTTTGTA~CTC~ATGATT~TAAAAGCAGT 2942 TATTAAAAGTCTAC~~

Fig. I. Nuclcotide and derived protein sequence o f mouse brain G L U D c D N A . Signal p e p t i d e is marked in italics. A m i n o acid substitutions are shown for h u m a n (H) and rat (R) G L U D polypeptides. U n d e r l i n e d are five putatiVe polyadenylation signals; d o u b l e - u n d e r l i n e d is the ribosome

attachment site [18].

252 vs. rat) implying a high degree of evolutionary conservation of GLUD mammalian molecules. The homology is extended at the 3' untranslated region approaching 83% and 95%, respectively. Comparison of the eDNA-deduced polypeptide sequences among the three species (Fig. 1) revealed 11 amino acid substitutions between mouse and human (98% homology) and 5 substitutions between mouse and rat (99% homology). About half of them were accumulated at the mitochondrial signal peptide. Apparently, these substitutions do not affect the conformation of the signal peptide. To investigate the GLUD mRNA content of various mouse organs, total cellular brain, liver and kidney RNA was extracted and 20 /tg of the preparations were blot-hybridized to MBG-1. As in human, monkey and rabbit [9], two major GLUD-specific transcripts of about 3.3 and 2.8 kb, were detected in mouse (Fig. 2). In contrast to human [8], these bands differed significantly in their ratio among different organs. The 3.3 kb transcript represented more than 95% of brain GLUD mRNA, whereas the abundance ratio between the 3.3 and the 2.8 kb transcripts was about 2:1 in liver and about 5:1 in kidney, a moderate GLUD mRNA producing organ. GLUD mRNA bands could either represent products of alternative processing of a single primary RNA molecule or derived from different functional GLUD genes. In any case, a tissue-specific mechanism regarding the differential expression of GLUD gene(s) should be envisaged. The identification of multiple GLUD transcrips is consistent with the determination of two physico-chemically distinct GLUD isoforms in human [12], rat [13] and frog [14] and the identification of at least three distinct GLUD polypeptides in human [3]. Human GLUD is encoded by a multigene family [8]. This family includes at least three members, i.e., two split and an intronless gene [8,15,16], located on, at least, two distinct chromosomal regions (Ref. 15 and Anagnou et al., unpublished data). We have also mapped mouse GLUD genes and identified, again, two different G l u d loci [17]. However, extensive mouse

KD BN • ,~

= ~.

LV KD

o

~"1---

16.4 kb el5. 8 ,D -3.12 kb * -0.96

q

Fig. 3. MousegenomicSouthernanalysesusing EcoRV and Hincll. A 745 bp EcoRV and a 620 bp Hincll fragmentsof MBG-I were used as probes, respectively.A third experimentusing Arall, produced resultsconsistentto the above(not shown). genomic DNA blot hybridization analysis was consistent with a GLUD-specific sequence pattern less complex than in human, and suggested the presence of only two mouse GLUD genes (Ref. 17, and our unpublished data). To test for the presence (or absence) of a mouse intronless GLUD gene, we proceeded in preliminary DNA blot hybridization using restriction enzymes known to produce fragments of defined size within MGB-I cDNA (Fig. 3). Hybridization of the filters to the respective MBG-1 restriction fragments produced, exclusively, larger hybridization bands than the expected from the probe sizes. This finding suggested the .lack of an intronless mouse GLUD gene. This may suggest that the integration of an intronless GLUD gene in the human genome is a recent genetic event that occurred after the karyotypic divergence of man and mouse. This work was supported by University of Crete grant 142, by NIH grant NS-16871 and by IMBB. We thank G. Grosveld for the cDNA library, A. Babaratsas for excellent technical assistance and Y. Tselepidis for photography.

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References

3.3 kb-*. 2.8 kb.,.

Ac* g ~

rr 0 (.) W

I~

Fig. 2. RNA blot hybridizationanalysis of adult mouse brain (BN), liver (LV) and kidney (KD). Filters were hybridized to MBG-I and a human /3-actin probe (Ac). Sizing is according to the migration of 28S and 18S rRNAs (not shown).

1 Smith. E.L., Austen, B.M., Blumenthal, K.M. and Nyc, J.F. (1975) in The Enzymes,(Boyer, P.D., ed.), Vol. 11, pp. 293-367, Academic Press, New York. 2 Plaitakis,A., Nicklas,WJ. and Desnick,R.J. (1980)Ann.Neurol. 7, 297-303. 3 Hussain, M.M., Zannis, V.I. and Plaitakis, A. (1989) J. Biol. Chem. 264, 20730-20735. 4 Fonnum,F. (1984)J. Neurochem.42, 1-11. 5 Wieloch,T. (1985) Science230, 681-683. 6 Finocchiaro,G., Taroni, F. and Didonato, S. (1986) Neurology 36, 550-553.

253 7 Thomas, J.W., Whitman, J., Mullen, K. and Banner, C. (1987) in Advances in gene technology: the molecular biology of development, (Voellmy, R.W., Ahmad, F., Black, S., Burgess, D.R., Rotundo, R., Scott, W.A. and Whelan, W.J., eds.). Vol. 7, p. 102, ICSU Press, New York. 8 Mavrothalassitis, G., Tzimagiorgis, G., Mitsialis, A., Zannis, V., Papamatheakis, J. and Moschonas, N.K. (1988) Proc. Natl. Acad. Sci. USA 85. 3494-3498. 9 Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13. 10 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Aead. Sci. USA 74, 5463 5467. I1 Das, T.A., Moerer, P., Charles, R., Moorman A.F.M. and Lamers, W.H. (1989) Nucleic Acids Res. 17, 2355.

'2 Plaitakis, A., [left, S. and Yahr, M.D. (1984) Ann. Neurol. 15, 144-153. 13 Colon, A.. Plaitakis, A., Perakis, A., Berl and Clarke, D.D. (1986) J. Neurochem. 46, 1811-1819. 14 Wiggert, B.O. and Cohen, P.P. (1966) J. Biol. Chem. 241,210-216. 15 Jung, K.Y., Warter, S. and Rumpler, Y. (1989) Ann. Genet. 32, 109-110. 16 Amuro, N., Goto, Y. and Okazaki, T. (1990) Biochim. Biophys. Acta 1049 216-218. 17 Tzimagiorgis, G.. Adamson, M-C., Kozak. C. and Moschonas N.K. (1991) Genomics, in press. 18 Kozak, M. (1987) Nucleic Acids Res. 15, 8125-8132.