Molecular Brain Research 76 2000. 5663 www.elsevier.

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Research report

Isolation, characterization and expression of a human brain mitochondrial glutaminase cDNA

Thomas Holcomb, Lynn Taylor, Jeffrey Trohkimoinen, Norman P. Curthoys
Accepted 16 November 1999 ) Department of Biochemistry and Molecular Biology, Colorado State Uniersity, 316 MRB Bldg., Ft. Collins, CO, 80523-1870, USA

Abstract Various cDNAs that encode overlapping portions of the full-length human brain glutaminase GA. cDNA were cloned and sequenced. The overall nucleotide sequence of hGA has a very high degree of identity with that of the rat kidney-type GA cDNA 77.4%. and the known portion of the cDNA that encodes the 5.0-kb porcine GA mRNA 81.1%.. The identity is even more remarkable at the amino acid level, particularly in the C-terminal half where the three proteins share a 99.7% sequence identity. The hGA cDNA encodes a 73,427-Da protein that contains an N-terminal mitochondrial targeting signal and retains the primary proteolytic cleavage site characterized for the cytosolic precursor of the rat renal mitochondrial glutaminase. The entire coding region was assembled through the use of unique restriction sites and cloned into a baculovirus. Sf 9 cells infected with the recombinant virus express high levels of properly processed and active glutaminase. Thus, expression of the isolated hGA cDNA should provide a means to purify large amounts of the mitochondrial glutaminase, a protein that catalyzes a key reaction in the metabolism of glutamine and the synthesis of important excitatory and inhibitory neurotransmitters. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Glutaminase; cDNA; Baculovirus expression; Mitochondria; Stroke

1. Introduction Clinical studies indicate that glutamate-mediated excitotoxicity contributes to the progressive neuronal cell death that occurs following a stroke w14x. Previous cell culture studies w5,16,21x have suggested that release of the mitochondrial glutaminase contributes to the delayed increase in extracellular glutamate that occurs in vitro. More recent studies w15x indicate that this mechanism may also contribute to the increased in vivo production of glutamate. Thus, selective pharmacologic targeting of the glutaminase that is made accessible by the initial hypoxic-induced neuronal cell damage may selectively inhibit the progressive neuronal atrophy caused by a stroke. To develop such therapeutics will require a readily available source of human brain glutaminase. Mammals typically express two isoforms of the mitochondrial glutaminase w3x. The liver-type glutaminase is expressed only in adult liver tissue, whereas the kidney-type glutaminase is expressed at high levels in kidney, brain,

intestine, the cells of the immune system, and in many transformed cells. A full-length glutaminase GA. cDNA that encodes the rat kidney-type glutaminase has been cloned and characterized w23x. It contains 58 bp of 5X-nontranslated sequence, 2022 bp of an open reading frame and 2446 bp of 3X-nontranslated sequence. The cDNA encodes a 74-kDa precursor protein that is effectively targeted to mitochondria and processed via a 72-kDa intermediate to yield the 68- and 66-kDa peptides which are contained in the mature mitochondrial glutaminase w17,18x. The current study reports the initial cloning and characterization of the human brain GA cDNA. Furthermore, the hGA cDNA was subcloned into a baculovirus. Infection of Sf 9 cells with the recombinant virus resulted in high expression and correct processing of the human mitochondrial glutaminase.

2. Materials and methods 2.1. Materials

Corresponding author. ncurth@lamar.colostate.edu

Fax:

q1-970-491-0494;

e-m ail:

Glutamate dehydrogenase, restriction enzymes and ligase were acquired from Boehringer Mannheim and New

0169-328Xr00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X 9 9 . 0 0 3 3 1 - 9

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England Biolabs. The transfection kit was from Pharmingen. Immobilon-NC transfer membrane was purchased from Millipore. Chemiluminescent detection reagents are products from Pierce Chemical. Tissue culture plates were purchased from Dow Corning. Graces insect cell culture media, lactalbumin hydrolysate and yeastolate were from Life Technologies, Spinner flasks were acquired from Bellco. Tris base was purchased from Research Organics. Chemicals for sodium dodecylsulfate polyacrylamide gel electrophoresis were from Bio-Rad. Secondary antibodies were from Amersham Pharmacia Biotech. All other biochemicals were purchased from Sigma. 2.2. Screening of a l gtll library Primary screens of an oligodT. and random primed human whole cerebral brain l gtll cDNA library Clontech. were carried out using 20 = 20 cm agar dishes as described previously w22x. The 950-bp Bgl IIrEco RI cDNA fragment of rat GA cDNA w23x was w32 Px-labeled with a random primed Oligolabelling Kit Amersham Pharmacia Biotech.. Hybridization of the probe to plaque filter lifts was carried out as described previously w19x. Screening of approximately 250,000 plaques yielded a single clone hGA1. that tested positive in secondary and tertiary screens. The secondary and tertiary screens were performed using the ECL Direct Nucleic Acid Labelling and Detection System Amersham Pharmacia Biotech.. The hGA1 cDNA was amplified by PCR using the Advantage cDNA Polymerase Mix and the l gtll LD Insert Screening Amplimer Set Clontech.. The amplimer set contains primers complimentary to the l gtll vector sequence on either side of the Eco RI cloning site. Thermocycler conditions were as specified in the kit. Amplification of DNA from 6 different positive plaques each produced a single cDNA of approximately 4-kb in length that hybridized with the 950-bp rat GA cDNA probe by Southern analysis. 2.3. AAD20 The AAD20 cDNA, which has a high degree of sequence identity to the 5X-end of the rat GA cDNA w9x, was graciously provided by Dr. Mandel. It had been cloned into the Eco RI site of pSCREEN-1bq. Novagen.. 2.4. Synthesis of hGA3 The hGA3 cDNA was PCR amplified directly from the l gtll library using a 5X primer 5X-TTGGCAACAGCGAGGGCAAAGAGC-3X . designed from the first exon of AAD20 and a 3X primer 5X-CAAACTGCCCTGAGAAGTCATACATGCCAC-3X . from the 5X-portion of hGA1 that is homologous to the rat GA cDNA. The l gtll DNA was initially denatured at 948C for 1 min, followed by 45 cycles of denaturing at 948C for 30 s, primer annealing as described below, and elongation at 658C for 4 min. An-

nealing conditions were started at 678C for 1 min and decreased 0.58C every cycle for 20 cycles. The annealing temperature then remained at 578C for the final 25 cycles. The purpose of starting the annealing temperature high and decreasing it over time was to increase the specificity of the reaction and the yield of the hGA3 cDNA relative to contaminating PCR products. Southern analysis confirmed that the hGA3 cDNA also hybridized to the 950-bp rat GA cDNA probe. Primers were designed using the PCRPLAN program from PCrGene IntelliGenetics.. The hGA3 cDNA was purified on a 1% agarose gel and extracted using a QIAEX II gel extraction kit Qiagen.. 2.5. Assembly of the complete coding region The coding region of hGA1 was isolated by digestion with Ssp I. The hGA3 cDNA and the Ssp I fragment of hGA1 were ligated separately into the Srf I site of pPCRScript using the pPCR-Script Cloning Kit Strategene.. Restriction analysis was used to identify clones in which the 5X-ends of the inserted cDNAs were positioned adjacent to the Eco RI site within the pPCR-Script plasmid. The 5X end of the AAD20 cDNA was isolated by digesting the AAD20 plasmid with Eco RI and Bsu 36I. The resulting 605-bp fragment was ligated into the hGA3 pPCR-Script plasmid that had been digested with Eco RI and Bsu 36I. The 3X-end of the coding region was isolated by restricting the hGA1 pPCR-Script plasmid with Nsi I and Not I and ligating the resulting fragment into the AAD20rhGA3 pPCR-Script plasmid that also been restricted with Nsi I and Not I. The resulting plasmid hGA4. contains the complete coding region of hGA Fig. 1.. 2.6. Sequencing Sequencing of the three hGA plasmids and the AAD20 cDNA was carried out using an ABI Prism 377 DNA Sequencer and primers designed with the PCRPLAN program. In some cases, convenient restriction fragments were subcloned into pBluescript II SKy. Stratagene. and sequenced using the T7 and T3 primers. Plasmids were transformed into XL1-Blue bacteria and purified with Perfect Prep or Bigger Prep kits 5 Prime 3 Prime..

2.7. Synthesis of baculoirus constructs The hGA-BV plasmid was created by ligating a Xho IrNot I fragment of the hGA4 cDNA into the Xho IrNot I sites of the baculovirus transfer vector pAcSG2 Pharmingen.. Thus, the hGA-BV plasmid contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus AcMNPV. and the 5X-nontranslated region and full coding sequence of hGA. The validity of the construct was verified by restriction mapping and by fluorescent dye terminator sequencing.

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Fig. 1. Comparison of rat and human glutaminase cDNAs. The various cDNAs are drawn to scale and are aligned on the basis of homology. The solid bars represent coding sequences and the 950-bp segment of the rat GA cDNA that was used as a probe for the initial screening and for Southern analysis. The indicated restriction sites were used to isolate the 950-bp probe or to combine the indicated segments of the three human brain GA cDNAs to produce hGA4 that contains the entire coding sequence of the human brain glutaminase. The primers used for PCR amplification of hGA3 hydridized to the sites indicated by the arrows. Abbreviations are: ATG, start codon; TAA, stop codon; and pA, polyadenylation signal.

2.8. Cell culture Spodoptera frugiperda Sf 9. cells were obtained from Invitrogen. Cells were cultured on 60 or 100 mm dishes at 278C as recommended by Invitrogen. Media was prepared from powdered Graces insect cell culture media supplemented with 0.35 g NaHCO 3 per liter and adjusted to pH 6.0 with NaOH prior to addition of 3.3 g lactalbumin hydrolysate and 20 ml yeastolate per liter. After filtersterilization, the media was supplemented with 10% fetal bovine serum and 10 m grml gentamycin sulfate. 2.9. Insect cell transfection and protein expression

media at 48 h post infection. The cells were harvested at 3 days and 4 days post infection. 2.10. Enzyme and protein assays Cell pellets were resuspended in 100 m l of buffer containing 10 mM TrisAcetate, pH 8.6, 10 mM tetrapotassium pyrophosphate, 1 mM dithiothreitol and 0.1% Triton X-100. Phosphate-dependent glutaminase activity was assayed as described previously w4x. Protein content was determined by the method of Lowry w13x using bovine serum albumin as the standard. 2.11. Western blotting

Transfection of Sf 9 cells was performed with the Pharmingen Baculogold Transfection Kit, using 2 or 5 m g of the hGA-BV cDNA. Recombinant viral supernatants and cells were harvested 5 d post-transfection by dislodging the cells with the viral supernatant and centrifuging the mixture at 50 = g for 2 min to pellet the cells. The viral supernatant ; 2 ml. was removed to a clean tube and stored at 48C. The cell pellet was frozen at y808C until analyzed for glutaminase activity and protein content. For protein expression, cells grown to confluency on 100 mm dishes were replated at a density of 2 = 10 6 cells per 60 mm dish with 3 ml of complete Graces media and allowed to attach for 15 min. The plating media was removed and replaced with complete media containing 1, 10, or 100 m l of viral supernatant. Dishes were incubated at 278C and supplemented with 1.5 ml complete Graces

Immunoblotting of proteins was performed essentially as described previously w24x. Cellular proteins were resolved on an 8% sodium dodecylsulfate polyacrylamide gel and electroblotted onto an Immobilon-NC membrane. The membrane was blocked overnight at 48C with 5% nonfat dry milk powder dissolved in 20 mM Tris, pH 7.5 and 500 mM NaCl TBS.. The blot was then equilibrated at room temperature for 1 h with gentle rocking and probed with a rabbit polyclonal antibody to rat glutaminase w2x for 1 h. The blot was briefly rinsed twice, then washed once for 15 min and twice for 5 min with TBS. Donkey anti-rabbit antibodies coupled to horseradish peroxidase were allowed to bind to the primary antibodies for 3045 min. The blot was then briefly rinsed three times with TBS, washed once for 15 min and four times for 5 min,

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and allowed to dry until just damp. The various forms of glutaminase were detected using the SuperSignal CL-HRP substrate system Pierce., digitized on a chilled CCD camera and quantified using the 1D analysis program from Phoretix Newcastle, UK..

3. Results 3.1. Characterization of the indiidual hGA cDNAs An initial screen of a human brain l gtll cDNA library yielded a single clone hGA1. that hybridized strongly to a

rat kidney-type GA cDNA Fig. 1.. When amplified by PCR, the insert of the cloned phage produced a 4-kb cDNA that was sequenced in both directions. The initial 283 bp of hGA1 had no significant identity to the rat GA cDNA. This sequence may correspond to intronic sequence since it ends with a consensus 3X splice site data not shown.. In contrast, the remainder of the hGA1 cDNA had a very high degree of identity with the portion of the rat GA cDNA that encodes the C-terminal half of the rat renal glutaminase and the complete 3X-nontranslated region of its mRNA w23x. The coding region, which extended from 2841255 bp, has a 90.6% sequence identity to the corresponding segment 11102081 bp. of the rat GA cDNA.

Fig. 2. Nucleotide sequence of the human brain GA cDNA. The various human brain GA cDNAs were sequenced in both directions and the resulting data were combined to produce the full-length sequence top line.. The sequence is aligned to maximize identity with the rat GA DNA sequence middle line. w23x and the available sequence of the GA cDNA that encodes the 5.0-kb porcine GA mRNA bottom line. Ref. w19x, unpublished data of D. Porter and N.P. Curthoys.. The coding sequences are illustrated with capital letters whereas the non-coding sequences are presented with small letters. Identical nucleotides are illustrated as <.

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Translation of this region yields a 324-amino acid sequence that has a 99.7% identity to the amino acid sequence of the rat renal glutaminase. The single amino acid difference is an asparagine in the human sequence vs. an aspartate at position 659 in the rat glutaminase. AAD20 is a 2-kb cDNA that was isolated by screening a brain expression library with antibodies specific for a polyglutamine sequence w9x. The initial 500 bp of this clone were previously sequenced and found to be homologous to the 5X-end of the rat renal GA cDNA. Thus, the AAD20 cDNA was obtained and both strands were completely sequenced Fig. 1.. The CAG repeats that encode the polyglutamine sequence are located within the initial 230 bp that correspond to the 5X-nontranslated region of the human brain GA cDNA. The AAD20 cDNA contains two stretches with homology to the coding region of the rat GA cDNA. The first stretch, from 231713 bp, has an 85.5% nucleotide identity compared with the rat GA cDNA, whereas the second stretch, from 10701193 bp, was 97.6% identical. However, these segments are homologous to a contiguous segment 59665 bp. of the rat glutaminase cDNA. Furthermore, a consensus 5X splice site and near consensus 3X splice site and branch points are present in the intervening segment of the AAD20 cDNA data not shown.. Thus, the intervening sequence in the AAD20 cDNA may correspond to the initial intron of the human brain glutaminase gene. Removal of the suspected intron sequence and translation of the resulting spliced sequence produced a 202-amino acid sequence that has an 85.1% identity to the N-terminal sequence of the cytosolic precursor of the rat renal mitochondrial glutaminase. The remainder of the AAD20 cDNA has no significant sequence identity to either the rat GA cDNA or the hGA1 cDNA. However, it begins with a near consensus 5X splice site data not shown.. Therefore, the remainder of the AAD20 cDNA may also be intronic sequence. The remainder of the human brain GA cDNA was PCR-amplified from the brain l gtll cDNA library using primers that correspond to coding sequences from the initial exon of the AAD20 cDNA and the 5X-end of the hGA1 cDNA Fig. 1.. Both strands of the resulting cDNA hGA3. were completely sequenced and found to contain a contiguous open reading frame. The overlapping sequences of hGA3 were 100% identical to the 3X- and 5X-ends of the coding sequences contained in the AAD20 and hGA1 cDNAs, respectively. The fact that the PCR product lacked the sequence that corresponds to 7141069 bp of the AAD20 cDNA further supports the conclusion that this segment constitutes the initial intron of the human brain GA gene. The hGA3 also encodes an additional 143-amino acid sequence that is 98.6% identical to the rat renal glutaminase. 3.2. Comparison of arious GA cDNAs The complete nucleotide sequence of the human brain GA cDNA was assembled from the sequences of the

individual hGA cDNAs Fig. 2.. The overall sequence is very homologous to that of the rat renal GA cDNA w23x and the available sequence of the cDNA that encodes the 5.0-kb porcine GA cDNA Ref. w19x; D. Porter and N. P. Curthoys, unpublished data.. For example, the coding region of the hGA cDNA has an 89.2% identity to the coding sequence of the rat GA cDNA. The 3X-nontranslated region of the human GA cDNA has a 66 and 84% sequence identity to the corresponding segments of the rat and porcine GA cDNAs, respectively. The identity between the three genes is even more striking at the amino acid level. The deduced amino acid sequences of the rat and human cDNAs and of the available porcine sequence are compared in Fig. 3. The overall identity between the amino acid sequences of the human and rat glutaminases is

Fig. 3. Amino acid sequence of the human brain glutaminase. The amino acid sequence of the human brain glutaminase was deduced from the nucleotide sequence and is presented using the standard one-letter abbreviations for the amino acids top line.. The sequence is aligned to maximize identity with the rat glutaminase sequence middle line. w23x and the known portion of the porcine glutaminase sequence that is encoded by the 5.0-kb GA mRNA bottom line. Ref. w19x, unpublished data of D. Porter and N.P. Curthoys.. Identical amino acids are illustrated as <.

T. Holcomb et al. r Molecular Brain Research 76 (2000) 5663 Table 1 Expression of human brain glutaminase activity in Sf 9 cells on various days post infection with recombinant baculovirus Days post infection 3 Volume of added virus m l. 1 10 100 1 10 100 non-infected Glutaminase activity unitsrml. 0.50 4.5 34.1 10.6 97.3 85.7 - 0.01 Protein concentration mgrml. 9.1 9.7 9.4 13.4 13.7 9.6 13.3 Specific activity unitsrmg. 0.06 0.46 3.6 0.79 7.1 8.9 - 0.001

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94.9%. The greatest homology occurs in the C-terminal half of the three proteins that share a 99.7% identity. The greatest difference is the finding that the coding sequence of the human glutaminase lacks eight of the nine residues that constitute the polyglutamine stretch which is found near the N-terminus of the fully processed 66-kDa subunit of the rat mitochondrial glutaminase w23,24x. Instead, the human glutaminase contains an additional repeat of a PAA sequence. As a result, the hGA cDNA encodes 669 amino acids that form a 73,427-Da precursor of the mitochondrial glutaminase. 3.3. Baculoirus expression of human brain glutaminase A cDNA hGA4. that contains the complete coding sequence of the human brain glutaminase was assembled by use of the unique restriction sites indicated in Fig. 1. This sequence was then cloned into a baculovirus and used to infect Sf 9 cells Table 1.. The non-infected Sf 9 cells express no detectable glutaminase activity. The level of glutaminase activity expressed in infected cells increases with increasing time and level of viral infectivity. The maximal specific activity 8.9 unitsrmg. reached at 4 days post infection was approximately 30-fold greater than the specific activity of a crude rat brain mitochondrial extract. Western blot analysis Fig. 4. indicates that the rat brain extracts contain predominately the fully processed 66-kDa subunit and small amounts of the 68-kDa subunit. In contrast, the Sf 9 cell extracts contain nearly equivalent amounts of the 73-kDa precursor and the fully processed

66-kDa subunit. Thus, expression of the glutaminase precursor may occur at a rate that exceeds the ability of the Sf 9 mitochondria to translocate andror proteolytically process the precursor.

4. Discussion The full-length hGA cDNA was assembled from three individual cDNAs isolated from cDNA libraries. The 5Xand 3X-segments were obtained by screening and the intermediate sequence was obtained by PCR amplification. Surprisingly, both of the cDNAs screened from libraries contained sequences that lack homology to the rat GA cDNA. In the case of the hGA1 cDNA, the non-homologous sequence constituted the 5X-end of the cDNA. This sequence could represent either an intron or an artifact of cloning. By contrast, the AAD20 cDNA contained an internal segment and a 3X-end that lack homology to the rat GA cDNA. Again, the 3X-end of the AAD20 cDNA could represent either an intron or an artifact of cloning. However, the first non-homologous segment 7141069 bp. contained all of the consensus sequences characteristic of an intron. In addition, the PCR product, hGA3, lacked this sequence and contained a contiguous open reading frame. Together, these observations strongly suggest that base pairs 7141069 of the AAD20 cDNA constitute the initial intron of the hGA gene. The frequent occurrence of potential introns within hGA sequences cloned from cDNA libraries suggests that expression of the hGA gene may be regulated in part at the level of mRNA processing. Both the rat and the hGA cDNAs contain a significant stretch of CAG repeats. However, the CAG repeats are located in different regions of the respective mRNAs. In the hGA mRNA, the CAG repeats are located in the 5X-nontranslated region, whereas in the rat, they are located within the coding region. Within the past decade, trinucleotide repeats have come under intense study. Expansion of these repeats has been shown to be the cause of various neurodegenerative diseases. Among these, Huntingtons disease w20x, spinocerebellar ataxias 1, 2, 3, 6, and 7 w10x, and others involve an increase in the number of CAG repeats within the coding regions of their respective genes. The CAG repeats encode polyglutamine stretches in

Fig. 4. Western blot analysis of the human brain glutaminase expressed in Sf 9 cells infected with the recombinant hGA baculovirus. Samples were separated on an 8% sodium dodecylsulfate polyacrylamide gel and analyzed using polyclonal antibodies raised vs. the rat renal glutaminase. Lane 1 contains 25 m g of a rat brain supernatant and 10 munits of glutaminase activity. Lane 2 contains 2.3 m g of a Sf 9 cell supernatant and 20 munits of glutaminase activity.

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the protein that may be involved in the accumulation of intranuclear inclusions in neural tissue. Other examples of proteins with polyglutamine stretches are the transcription factors, TATA-Binding Protein TBP., Gal 11, and nuclear protein SNF5 w6x. Fragile X syndrome w12x and Myotonic Dystrophy w26x involve expanded trinucleotide repeats in the 5X- and 3X-nontranslated regions, respectively. Thus, rat GA belongs to the class of genes that contain trinucleotide repeats within the coding region and human GA belongs to the class with 5X- or 3X-trinucleotide repeats. At present it is unknown whether the presence of the CAG repeats in either of the two GA genes affects its expression or contributes to a neurological disorder. However, given their different locations, a conserved effect of the CAG repeats in human and rat GA genes must be expressed at the nucleic acid level and not that of the protein. Both the rat and human glutaminase precursors contain a 16-amino acid N-terminal mitochondrial targeting signal that is rich in leucine and arginine residues Fig. 3.. The two sequences differ only by two conservative substitutions. The other region that is highly conserved 100% identity. is the sequence immediately before and after the cleavage site at residue 72. Thus, processing to yield a mature 66-kDa subunit should also be conserved. However, the overall identity of the initial 72-amino acid residues is only 74%. Significant substitutions occur in the regions of the N-terminal extension that may constitute the determinants for the proteolytic reactions that yield the 72-kDa intermediate and the 68-kDa subunit of the rat glutaminase w24,25x. These conclusions are supported by the Western analysis of the human glutaminase expressed in Sf 9 cells Fig. 4.. The glutaminase specific antibodies detected only two peptides that correspond to the 73-kDa precursor and the 66-kDa subunit. Previous studies w7,11,28x have demonstrated that baculovirus expression systems can be used to synthesize and correctly process the precursors of various mitochondrial proteins. The very high level of expression of glutaminase activity Table 1. and the observed formation of a 66-kDa peptide Fig. 4. confirm the validity of the isolated hGA cDNA and the deduced size of its fully processed subunit. The data also indicate that infection of Sf 9 cells with the recombinant hGA baculovirus results in the accurate translocation, processing and folding of the human brain glutaminase. The total activity of recombinant glutaminase expressed in a 1-l culture of Sf 9 cells is greater than that contained in 50 rat brains data not shown.. Based upon the specific activity of the purified rat brain glutaminase w8x, the expressed human brain glutaminase constitutes approximately 3% of the total protein in the Sf 9 cells. The rat w8x and porcine w27x brain glutaminases have been readily purified by making use of the fact that the protein from both species undergoes a reversible polymerization in the presence of borate. If the human glutaminase exhibits the same property, then repeated gel filtration of solubilized extracts of infected Sf 9 cells should yield m g amounts of purified

human brain glutaminase. If not, the coding segment of the hGA cDNA can be cloned with a C-terminal His 6-tag into a recombinant baculovirus. This approach has been used successfully to express and to rapidly purify the rat mitochondrial dihydroorotate dehydrogenase w1x. Alternatively, the pIZrV5-His vector Invitrogen. could be used to isolate stable transfectants of Sf 9 cells that constitutively express the recombinant glutaminase. Thus, one of these approaches should yield homogeneous human brain glutaminase in an amount sufficient to initiate structural studies and the screening of substrate and phosphate analogs. Such studies may eventually lead to the design of a specific inhibitor that can be tested for its efficacy in preventing the propagation of the neuronal degeneration that is associated with stroke.

Acknowledgements This work was supported in part by grants from the Colorado Institute for Research in Biotechnology and the National Institute of Diabetes and Digestive and Kidney Diseases DK-37124. awarded to N.P. Curthoys.

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