©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning of the Mature NAD-dependent Succinic Semialdehyde Dehydrogenase from Rat and Human
cDNA ISOLATION, EVOLUTIONARY HOMOLOGY, AND TISSUE EXPRESSION (*)

(Received for publication, July 11, 1994; and in revised form, October 13, 1994)

Ken L. Chambliss (1) Deborah L. Caudle (1) Debra D. Hinson (1) Carolyn R. Moomaw (2) Clive A. Slaughter (2) Cornelis Jakobs (3) K. Michael Gibson (1)(§)

From the  (1)Metabolic Disease Center, Baylor Research Institute and Baylor University Medical Center, Dallas, Texas 75226, (2)Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9050, and the (3)Department of Pediatrics, Free University of Amsterdam, de Boelelaan 1117, 1081 HV Amsterdam, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Three rat brain cDNA clones 3500, 1465, and 1135 base pairs in length encoding succinic semialdehyde dehydrogenase (SSADH; EC 1.2.1.24) were isolated from two cDNA libraries using a polymerase chain reaction derived probe. Restriction mapping and DNA sequencing revealed that the 3.5-kilobase clone contained an 84-base pair (28 amino acid) insert in the coding region. Composite clones encoding mature SSADH predicted proteins with 488 amino acids (M(r) = 52,188) when including the insert and 460 amino acids (M(r) = 48,854) without the insert. The cDNA clones were confirmed by expression of enzyme activity in bacteria and protein sequence data obtained from sequencing purified rat brain SSADH. Two human liver SSADH cDNA clones of 1091 and 899 base pairs were also isolated. Human and rat SSADH share 83 and 91% identity in nucleotide and protein sequence, respectively. Northern blot analysis revealed two differentially expressed SSADH transcripts of approximately 2.0 and 6.0 kilobases in both rat and human tissues. Human genomic Southern blots indicate that the two SSADH transcripts are encoded by a greater than 20-kilobase single copy gene. Mammalian SSADH contains significant homology to bacterial NADP-succinic semialdehyde dehydrogenase (EC 1.2.1.16) and conserved regions of general aldehyde dehydrogenases (EC 1.2.1.3), suggesting it is a member of the aldehyde dehydrogenase superfamily of proteins.


INTRODUCTION

The metabolism of the inhibitory neurotransmitter GABA (^1)is carried out by three enzymes (Fig. 1). Glutamic acid decarboxylase (EC 4.1.1.15) converts glutamic acid to GABA with stoichiometric production of carbon dioxide. GABA degradation is achieved in a two-step reaction, catalyzed by GABA-transaminase (GABA-T; EC 2.6.1.19) and NAD-dependent succinic semialdehyde dehydrogenase (SSADH; EC 1.2.1.24). The carbon skeleton of GABA thus enters the tricarboxylic acid cycle in the form of succinate. Of these three enzymes, mammalian cDNAs have been isolated for both glutamic acid decarboxylase and GABA-T from different sources, including mouse, pig, and human(1, 2, 3) . More recently, Medina-Kauwe et al.(4) isolated a cDNA encoding rat brain GABA-T which yielded enzymatically active GABA-T after expression.


Figure 1: Metabolic interconversions of succinic semialdehyde (SSA). Additional abbreviations employed: GAD, glutamic acid decarboxylase; 4-HBA, 4-hydroxybutyrate; 4-HBDH, 4-hydroxybutyrate dehydrogenase; 2-KGA, 2-ketoglutaric acid.



SSADH, the final enzyme in GABA catabolism, has been purified to apparent homogeneity from rat and human brain(5, 6) . SSADH is also the site of an inborn error of human metabolism(7) . In autosomal recessively inherited SSADH deficiency, now identified in more than 45 patients who manifest varying degrees of psychomotor retardation with speech delay, the normal oxidative pathway is blocked, thereby resulting in the accumulation of succinic semialdehyde (SSA). Metabolite patterns in physiologic fluids derived from patients show large increases in 4-hydroxybutyric acid, the reduction product of SSA. The biochemical hallmark of SSADH deficiency, 4-hydroxybutyric acid, produces central nervous system effects including altered motor activity and behavior disturbances when administered to animals and humans at pharmacologic levels(8) .

Although the nucleotide sequence of bacterial NADP-linked SSADH (EC 1.2.1.16) has recently been presented(9) , a cDNA encoding mammalian NAD-dependent SSADH has not been reported. We present the isolation of cDNAs encoding SSADH from rat and human and the expression of the rat SSADH cDNAs in bacteria. SSADH cDNAs from both species recognize two differentially expressed mRNAs of approximately 2.0 and 6.0 kb that are transcribed from a single gene. These cDNAs will be useful tools with which to begin investigating the molecular genetics of inherited human SSADH deficiency. The availability of cDNAs encoding GABA-T and SSADH from mammalian sources will enable studies to determine if both genes are coordinately regulated, as has been demonstrated in yeast(10) .


EXPERIMENTAL PROCEDURES

Materials

A rat forebrain ZAP cDNA library (randomly primed) and a rat cerebellum ZAP cDNA library (oligo(dT)-primed) were obtained from the Molecular Neurobiology Laboratory of The Salk Institute for Biological Studies, La Jolla, CA. A ZAP human liver cDNA library (oligo(dT)-primed) was purchased from Stratagene. Northern blots containing 2 µg of polyadenylated mRNA from various rat and human tissues were purchased from Clontech Laboratories, Inc. Other materials were purchased from the manufacturer's as follows: custom-synthesized gel-purified oligonucleotides, National Biosciences; Sequenase DNA sequencing kit, U. S. Biochemical Corp.; Taq DNA polymerase, ProtoBlot Western blot AP System, Promega Biotec.; INValphaF` competent Escherichia coli, Invitrogen; restriction endonucleases, Boehringer Mannheim; Bluescript II plasmid vectors, XL-1 blue E. coli, Stratagene; megaprime random-primed labeling kit, Hybond-N, Rapid-hyb hybridization buffer, [alpha-P]dCTP (3000 Ci/mmol), [alpha-P]dATP (3000 Ci/mmol), and alpha-S-dATP (58 mCi/mmol), Amersham Corp.; Moloney murine leukemia virus reverse transcriptase, OptiMem tissue culture media, Life Technologies, Inc.; deoxynucleotides, Pharmacia Biotech Inc.; Kodak XAR-5 film, VWR; GeneClean glass milk DNA isolation resin, BIO 101. All chemicals were of the highest purity obtainable.

Purification and Amino Acid Analysis of Rat and Human Brain SSADH

Rat and human brain SSADH were purified as reported previously(5) . N-terminal protein sequencing was performed on gel-purified protein with an Applied Biosystems model 470A amino acid sequencer with on-line model 120A phenylthiohydantoin-derivative analyzer. To obtain additional amino acid sequence, the rat brain SSADH protein was digested with trypsin and cyanogen bromide and resulting peptides were isolated on an Applied Biosystems model 130A narrow-bore high performance liquid chromatography system and sequenced.

Cloning Strategy

The mixed oligonucleotides primed amplification of cDNA procedure was used to generate a DNA probe for library screening. First strand cDNA was synthesized with Moloney murine leukemia virus reverse transcriptase using a downstream antisense primer (GCNCCNGCNGCNGG(A/G)TC) according to the protocol of Kawasaki(11) . Thirty cycles of PCR were performed on the cDNA using the upstream primer GGGAATTCGTNGGNGGNCCNGCNGA and the above downstream primer with annealing for 45 s at 42 °C, extension for 1 min at 72 °C, and denaturing for 45 s at 94 °C. A second round of PCR was carried out using 1 µl of the first PCR as template. The same upstream primer was used, but a nested downstream primer with the sequence GGAAGCTTGG(A/G)TC(A/G)TANACNGG(A/G)AA replaced the previous downstream primer. The same temperature profile and cycle number were employed. The PCR product of 117 bp was gel-purified with glass milk and subcloned into the Bluescript II SK vector using the EcoRI and HindIII restriction sites engineered onto the ends of the primers. Six isolates of the clone were sequenced with Sequenase version 2.0 DNA sequencing kit according to the manufacturer's protocols for the sequencing of double-stranded DNA. The SSADH PCR clone was excised from the Bluescript vector with EcoRI and HindIII, gel-purified with glass milk, and labeled with [alpha-P]CTP and [alpha-P]ATP with a random-primed labeling kit from Amersham Corp. according to suggested procedures to a specific activity > 5 times 10^8 cpm/µg. The labeled fragment was purified on Sephadex G-50, denatured at 100 °C, and used as a probe to screen > 10^6 plaques from a ZAP rat forebrain library according to standard protocols provided by Amersham for use with Hybond-N nylon filters. One-hour prehybridization and overnight hybridization were done in 5 times SSPE (1 times SSPE = 0.15 M NaCl, 0.25 M NaH(2)PO(4) (pH 7.4), 1 mM EDTA), 5 times Denhardt's solution (1 times Denhardt's = 0.2% (w/v) Ficoll, 0.2% (w/v) polyvinylpyrrolidone, 0.2% (w/v) bovine serum albumin fraction V), 0.1% (w/v) SDS, and 20 µg/ml yeast tRNA. Filters were washed two times for 10 min at room temperature in 2 times SSPE with 0.1% (w/v) SDS and twice for 15 min at 65 °C in 1 times SSPE with 0.1% (w/v) SDS. Positive plaques were purified through two additional cycles of screening. Positive inserts were subcloned into the Bluescript SK plasmid vector by the ``in vivo excision'' and recircularization process as described by Stratagene and sequenced as described above using XbaI and BglII restriction sites for subcloning.

More than 1.5 times 10^6 plaques of a human liver ZAP cDNA library were screened using the 1465-bp rat forebrain SSADH clone as the probe. Hybridizations, clone isolation, and sequencing were performed as described for rat brain SSADH clones.

DNA and Protein Sequence Analysis

DNA sequence was determined for both strands by the dideoxynucleotide chain termination method(12) . Double-stranded DNA sequencing was performed with the Sequenase modified T7 DNA polymerase following the manufacturer's protocol. DNA sequences which could not be reached from vector priming sites were subcloned using the XbaI and BglII restriction endonuclease sites present within the cDNA clones. Additional primers were synthesized to sequence all regions. DNA and protein sequences were aligned and compared using the Genepro and Align computer programs. Sequences were compared with the GenBank sequences using the Atlas computer program.

Southern and Northern Blot Analysis

Genomic DNA was isolated from human lymphocytes by the method of Steffen et al.(13) . 10 µg of the genomic DNA was digested separately with BamHI, EcoRI, HindIII, and XbaI, separated on a 0.7% agarose gel, and transferred to Hybond-N nylon membrane. DNA was cross-linked to the membrane by alkaline fixation as described by Amersham. The membrane was prehybridized and hybridized in Rapid-hyb blotting buffer from Amersham at 65 °C for the recommended times. The blot was probed with the 1091-bp human liver SSADH cDNA fragment which was random-primed labeled with [alpha-P]dCTP and [alpha-P]dATP to a specific activity >5 times 10^8 cpm/µg and hybridizing bands were visualized by autoradiography with Kodak XAR film. The probe was stripped from the blot by boiling in 0.5% (w/v) SDS, and the membrane was reprobed with a 279-bp BglII-EcoRI fragment from the 3`-end of the human liver cDNA using the same conditions.

Poly(A) RNAs were isolated from various rat and human tissues and 2 µg of each were electrophoresed on 1.2% agarose gels containing 1.1% formaldehyde and transferred to nylon membranes by Clontech. The membranes were prehybridized and hybridized in Amersham's Rapid-hyb buffer as described for Southern blots. The 1091-bp human liver SSADH cDNA EcoRI fragment was used to probe human multiple tissue and brain Northern blots. The 1465-bp rat brain SSADH cDNA containing most of the coding region and part of the 3`-untranslated region was used to probe the rat multiple tissue Northern blot. The rat multiple tissue Northern blot was stripped of this probe by boiling in 0.5% (w/v) SDS and reprobed with a 2-kb XbaI fragment containing mostly downstream 3`-untranslated DNA sequence from the 3.5-kb rat brain SSADH cDNA. Poly(A) RNA was isolated from approximately 5 times 10^8 lymphoblast cells which had been grown in OptiMem liquid media supplemented with 4% fetal bovine serum. 20 µg of the poly(A) RNA was electrophoresed on a 1.2% agarose gel containing 2.2 M formaldehyde. The RNA was electroblotted to Hybond-N membrane, prehybridized and hybridized according to manufacturer's protocols using the human liver 1091-bp SSADH cDNA as probe. All Northern blots (except lymphoblast) were stripped of their respective probes and reprobed with a human beta-actin clone provided by Clontech.

Enzyme Assay

The enzyme assay was modified from the procedures of Manthey et al.(14) and Chambliss and Gibson (5) . The standard enzyme assay contained 0.1 M sodium pyrophosphate buffer (pH 9.0), 0.1 mM EDTA, 500 µM NAD, and 20 µM succinic semialdehyde. Measurements of initial reaction velocity were carried out spectrophotometrically at 340 nm and 25 °C in 3 ml total volume. The components in blank assays were identical except that SSA was omitted. The reaction was started by addition of SSA, and the increase in the optical density at 340 nm was measured for 2-10 min. The extinction coefficient of NADH used was 6.22 mM cm at 340 nm. The data of initial reaction velocities showed that reaction rates were proportional to protein concentration. Protein concentrations were determined by the method of Lowry et al.(15) using bovine serum albumin as a standard.

Expression Analysis

Two composite rat brain SSADH cDNA clones were constructed by ligating the PCR amplified 117-bp sequence (encoding the mature N terminus) onto the 5`-end of the 3.5-kb and the 1465-bp rat brain SSADH partial cDNAs using the NotI restriction site at base pair 55 (Fig. 3). The EcoRI fragment of the composite clones was then transferred into the EcoRI site of the glutathione S-transferase fusion vector pGEX-5X-1 (Pharmacia). DH5alpha E. coli were transformed and grown overnight at 37 °C in TB media (12 g of Bacto-tryptone, 24 g of Bacto-yeast extract, 2 g of casamino acids/liter). The next day, 95 ml of TB media supplemented with 0.1 M potassium phosphate (pH 7.5), 1 ml of 50% glycerol, and 100 µg/ml ampicillin was inoculated with 5 ml of the overnight culture and grown at 37 °C with shaking at 200 rpm for two h. Gene expression was induced by addition of isopropylthio-beta-D-galactoside to a final concentration of 1 mM, and the cultures were grown at 28 °C overnight with gentle shaking. The cells were pelleted at 1500 times g and washed twice in phosphate-buffered saline. The cell pellet was then resuspended in 1 ml of 0.1 M sodium pyrophosphate buffer, pH 9.0, 1% (v/v) 2-mercaptoethanol, and 1% (v/v) Triton X-100 and sonicated for 2 min while chilled in an ice bath. Cell debris was removed by centrifugation at 12,000 times g for 5 min, and the supernatants were assayed for SSADH activity as described above. A rat brain extract was prepared in the same buffer and assayed as an additional parallel control.


Figure 3: Nucleotide and deduced amino acid sequences of rat brain (R) and human liver (H) SSADH. Nucleotides are numbered to the right, and amino acids (in three letter code) are numbered above with position 1 being assigned to the first nucleotide and residue of the rat brain cDNA. Underlined amino acid residues were determined by amino acid sequencing of rat brain SSADH prior to cloning. The codons and amino acid residues in bold (residues 72-99) comprise the 84-bp (28 amino acid) insert and are absent from two of the rat brain cDNAs. The cDNA from the rat brain SSADH 2.0-kb transcript ends at the bold underlined cytosine at position 1573 and an additional 158 bp of the approximately 2 kb 3`-untranslated tail from the 6.0-kb transcript is shown. The human liver sequence begins at amino acid 166 and is indicated only where it differs from the rat brain sequence. The consensus polyadenylation signal (AATAAA) of each sequence is underlined.




RESULTS

Reverse Transcription-PCR of Rat Brain SSADH

The rat brain SSADH N-terminal peptide sequence (VGGPADLHADLLRGDSFVGGRWLPTPA) and an overlapping tryptic fragment (WLPTPATFPVYDPASGAK) provided information for designing oligonucleotide primers for PCR amplification. Reverse transcription of rat brain total RNA followed by PCR amplification was performed as described under ``Experimental Procedures'' and produced a single product of 117 base pairs. The amplified DNA was cloned and several isolates were sequenced and shown to encode the exact amino acids predicted from the peptide sequence of this region.

Isolation and Characterization of Rat Brain SSADH cDNAs

Two rat brain SSADH cDNAs were isolated after screening approximately 10^6 clones from a rat forebrain ZAP cDNA library with the 117-bp rat brain SSADH PCR product. The sizes of the cDNA inserts in these clones were 1465 and 1135 base pairs. Complete DNA sequencing of the clones revealed that the 1135- and 1465-bp clones were 100% homologous, with the larger clone having 24 more bases 5` and 306 more bases 3` than the 1135 bp clone (Fig. 2). The DNA sequence contained an open reading frame which included most of the known amino acid sequence obtained from sequencing the mature SSADH protein (Fig. 3). The 1465-bp cDNA began at the 8th amino acid after the mature N terminus of the SSADH protein and contained 106 bp of the 3`-untranslated region, including an AATAAA polyadenylation signal 92 bp upstream from the end of the cDNA. Only 6 amino acid residues from a tryptic peptide sequence were not found in the cDNA. The deduced molecular weight of a polypeptide comprised of amino acids from the mature N terminus through the end of the open reading frame was 48,854 daltons.


Figure 2: Schematic representation of cloned SSADH cDNAs. A, 117-bp PCR-amplified rat brain SSADH encoding the mature N terminus; B-D, rat brain SSADH cDNAs of 1135, 1465, and 3500 bp, respectively. The dotted area represents the 84-bp region not present in clones B and C but present in clone D. E and F, composite rat brain SSADH cDNAs made from splicing clone A with clones D or C, respectively. The hatched bar represents the open reading frame. G and H, human liver SSADH cDNAs of 1091 and 899 bp, respectively.



The 1465-bp insert was used to screen a second rat brain ZAP cDNA library in an attempt to isolate a more complete cDNA. One additional clone was obtained from a screen of approximately 10^6 clones. This clone had a cDNA insert of approximately 3.5 kb. Restriction enzyme mapping and DNA sequencing of the clone revealed a 5`-end which began 12 bp downstream from the 1465-bp clone and a 3`-tail with approximately 2 kb of additional 3`-untranslated DNA as compared with the 1465-bp clone (Fig. 2). The 3.5-kb clone had 100% homology with aligned regions of the 1465-bp clone with the exception of an additional 84-bp insert 214 bp from the 5`-end of the 3.5-kb clone that was not present in the other two cDNAs (Fig. 3). This insert encoded 28 additional amino acids, starting at amino acid 72, and included the 6 amino acids from the sequenced tryptic fragment that were not present in the 1465-bp cDNA. The presence of a consensus splice site at the insertion position suggests that the extra sequence in the 3.5-kb clone is due to an alternative RNA splicing mechanism. The deduced molecular mass of the protein encoded by this clone was 52,188 daltons, which was in good agreement with the estimated molecular weight of 54,000 for purified rat brain SSADH(5) .

Isolation and Characterization of Human Liver SSADH cDNA

A human liver ZAP cDNA library was screened with the 1465-bp rat brain SSADH cDNA. Approximately 10^6 clones were screened and two positive clones were isolated. The cDNA inserts of these clones were 1091 and 899 bp. Complete sequencing and comparison of the clones revealed identical 3`-ends and 100% homology, with the longer clone extending 192 bp further upstream than the shorter (Fig. 2). The 1091-bp human clone contained a 972-bp open reading frame and 119-bp 3`-untranslated tail with an AATAAA consensus polyadenylation signal 45 bp from the end of the cDNA (Fig. 3). Comparison of the human liver SSADH cDNA with the rat brain SSADH cDNAs revealed an 83% identity at the DNA level over the coding region and 91% homology at the amino acid level. Most of the observed base changes occurred at the third position of codons and do not result in a change of the amino acid residue. Many of the amino acid differences are due to the change of a single base within the codon.

Tissue Distribution of SSADH mRNA

Northern blot analysis of mRNA from rat and human tissues revealed two differentially expressed transcripts of approximately 2.0 and 6.0 kb (Fig. 4, A and B). Some tissues such as brain and pancreas express predominantly the 6.0-kb transcript with relatively low amounts of the 2.0-kb transcript. Other tissues such as heart, liver, skeletal muscle, and human kidney have appreciable amounts of both transcripts. Human placenta and rat spleen, lung, kidney, and testis have only trace amounts of either transcript relative to other tissues. No tissues examined expressed the 2.0-kb transcript alone. Analysis of mRNA from different regions of human brain showed a consistent level of expression of the two SSADH transcripts throughout that organ (Fig. 4C). Northern blot analysis of cultured lymphoblasts showed expression of both the 2.0- and 6.0-kb transcripts (Fig. 4D).


Figure 4: Northern blot analysis of poly(A) RNA from rat and human tissues. A, rat tissues; B, human tissues; C, human brain regions; D, cultured human lymphoblasts. Approximately 2 µg of poly(A) RNA was loaded in each lane, except for lymphoblast which contained 20 µg. The probe for A was the 1465-bp rat brain SSADH cDNA and for B-D was the 1091-bp human liver cDNA. The position of molecular weight size standards are shown to the left. A-C were stripped and rehybridized with a human beta-actin probe to check levels of RNA between lanes (small panel below the blots). Abbreviation: skel mus, skeletal muscle.



Size considerations suggested that the 3.5-kb rat brain SSADH cDNA was produced from the 6.0-kb SSADH transcript. To verify this, the multiple tissue Northern blots were stripped and reprobed with a radiolabeled DNA fragment containing only the 3`-terminal 2-kb tail of the 3.5-kb cDNA. Only the 6.0-kb SSADH transcript hybridized with this 3`-fragment (data not shown).

Expression of Rat Brain SSADH

To verify that the isolated cDNAs encoded a protein with SSADH activity, two composite clones were constructed which included the PCR amplified mature N terminus coding region ligated to the 3.5-kb and 1465-bp rat brain SSADH cDNAs. The larger clone constructed with the 3.5-kb cDNA contained the 84 bp (28 amino acid) insert near the 5`-end, whereas the shorter clone did not contain the insert. The composite clones were placed in frame into a pGEX-5X bacterial expression vector. Table 1shows results of the expression of these glutathione S-transferase-SSADH fusion composite clones as well as a control clone that was not in frame and parent vector alone. Bacterial homogenates made from cells with in frame composite rat brain SSADH clones displayed an approximate 300-fold greater SSADH activity when the 84-bp insert was present and an approximate 150-fold greater SSADH activity without the insert in comparison to homogenates made from cells containing vector only or the inverted larger composite clone.



Southern Hybridization Analysis of Human Genomic DNA

To estimate the size of the genomic SSADH gene and to establish whether the two SSADH transcripts were from one gene or two different genes, genomic Southern hybridization was performed. Genomic DNA was isolated from human lymphocytes and digested with four separate restriction endonucleases. Southern blot hybridization studies using the 1091-bp human SSADH cDNA as probe revealed several restriction fragments with each endonuclease used and indicated that the SSADH gene covers at least 20 kb in the human genome (Fig. 5). Upon reprobing with a 296-bp radiolabeled fragment from the 3`-end of the human SSADH cDNA (containing 172 bp of C-terminal coding region), a single restriction fragment was detected in each digest. This data suggests that human SSADH is a single copy gene.


Figure 5: Southern blot analysis of human lymphocyte genomic DNA. Lymphocyte genomic DNA was digested with the indicated restriction endonucleases. A Southern blot was prepared and hybridized to the radiolabeled 1091-bp human liver SSADH cDNA (left panel). The nylon filter was stripped of the probe and rehybridized with a radiolabeled 279-bp BglII-EcoRI restriction fragment from the 3`-end of the 1091-bp human liver cDNA (right panel). Molecular weight markers are shown to the left.



Comparison of SSADH with Other Aldehyde Dehydrogenases

Human and rat SSADH share substantial homology to the recently presented E. coli gabD gene, an NADP-dependent succinic semialdehyde dehydrogenase (EC 1.2.1.16) (9) (Fig. 6). Human and rat SSADH share 56 and 54% homology, respectively, to this protein. A search of the GenBank reveals that human and rat NAD-dependent SSADH described in this paper and the bacterial NADP-dependent SSADH also share 36-38% homology at the protein level to mammalian general aldehyde dehydrogenases (EC 1.2.1.3) and 36% homology with bacterial 2-hydroxymuconic semialdehyde dehydrogenase. SSADH shares only slight amino acid homology to other bacterial semialdehyde dehydrogenases, including glutaric semialdehyde dehydrogenase, N-acetylglutamate--semialdehyde dehydrogenase and aspartate-beta-semialdehyde dehydrogenase at 18, 15, and 10%, respectively.


Figure 6: Comparison of other aldehyde dehydrogenases with SSADH. RSDH refers to rat brain succinic semialdehyde dehydrogenase, BSDH to bacterial NADP-dependent succinic semialdehyde dehydrogenase, RCYT to rat cytoplasmic aldehyde dehydrogenase, HADH to human mitochondrial aldehyde dehydrogenase, and HSDH to bacterial 2-hydroxymuconic semialdehyde dehydrogenase. Identical residues are capitalized and indicated by a closed box below the sequences(). Four of five amino acid matches are indicated by a shaded box below the sequences (&cjs2108;). Amino acid residues are numbered in parenthesis.




DISCUSSION

We report the sequence of cDNAs encoding the mature form (minus the mitochondrial entry sequence) of rat brain SSADH and a significant amount (323 amino acids) of the coding region of human liver SSADH. The clones were confirmed by matching deduced amino acid sequence of the rat brain cDNA with 120 amino acid residues sequenced from the N terminus and seven different peptides of the purified rat brain protein and by expression of composite clones encoding the mature rat brain protein. Although rat and human NAD-dependent SSADH share greater than 50% homology with NADP-dependent SSADH of E. coli, these enzymes represent two different classes of semialdehyde dehydrogenases. The sequence of the bacterial NAD-dependent SSADH, the product of the sad gene, has not been reported.

Alignment of SSADH with other aldehyde and semialdehyde dehydrogenases reveals greater homology with cytosolic and mitochondrial general aldehyde dehydrogenases (EC 1.2.1.3) than with most other semialdehyde dehydrogenases, except the bacterial NADP-SSADH described above and 2-hydroxymuconic semialdehyde dehydrogenase. SSADH possesses many of the conserved residues found among aldehyde dehydrogenases. Glycines 245 and 250 of cytosolic aldehyde dehydrogenase are believed to play a role in NAD binding and correspond to glycines 237 and 242 of rat SSADH. The consensus active site motif (SAG)XFXXXGQXCX(AGN) containing the important Cys-302 (16) is present in SSADH at residues 282 through 297. General aldehyde dehydrogenases have been shown to contain a conserved VTLELGGK motif at amino acids 265-274 which was identified by inactivation with bromoacetophenone(17) . The SSADH enzymes have a 5 of 8 match to this sequence with maintenance of the putative active site Glu-268 (Glu-259 in rat SSADH). Most other sequenced aldehyde dehydrogenases contain an EEIFGP sequence near their C-terminal end, whereas the deduced amino acid sequences of the three SSADH proteins have a new version of this conserved sequence EETFGP. These consensus sequences, and others, clearly identify SSADH as a member of the aldehyde dehydrogenase superfamily.

Northern and Southern blot data reveal two SSADH messages which are transcribed from a single gene. The larger transcript of rat was shown to have a much longer 3`-untranslated tail (at least 2 kb) presumably due to the selection of a different 3`-processing and polyadenylation site. Because no cDNAs were found that contained the 5`-end of an SSADH transcript, it is not known whether the two messages share the same transcriptional start point; however, it seems unlikely, based upon size considerations of RNA transcripts and isolated cDNAs. Also, the two transcripts are expressed at varying ratios in the different tissues examined; however, no tissue expresses only the 2.0-kb transcript.

Another difference in SSADH transcripts appears to be the presence or absence of an 84-base pair segment capable of encoding 28 amino acids. We know that the purified rat brain SSADH protein was translated from a transcript that contained the 84-bp segment, because 6 deduced amino acid residues encoded by this segment correspond to 6 amino acid residues of a sequenced tryptic peptide. It remains possible that the absence of the segment is an artifact of the reverse transcription and subsequent cloning steps in library construction; however, there are several observations which suggest that the lack of the segment is not an artifact. First, two separate and unique cDNA clones lack the 84-bp segment, and both of these clones have identical sequences at the site where the segment is missing. Also, the site of insertion of the segment is flanked by a consensus splice site motif (AG/G), and the reading frame of the protein is preserved both with and without the segment. Last, bacterial expression analysis shows high levels of SSADH activity in composite cDNA clones with and without the 84-bp segment. It is possible that the 84-bp segment is unique to the 6.0-kb SSADH transcript, since the rat brain cDNA clone that contains the segment was reverse transcribed from the larger transcript (known because of the long 3`-tail); however, the absolute presence (or absence) of the 84-bp segment in the different SSADH transcripts is still under investigation.

An important question that remains is why the two SSADH mRNAs are at different ratios in the various tissues. Although they may encode two different subunits of SSADH, most of our data in rat argue in favor of a single subunit comprising the SSADH protein. We observe a single protein band on SDS-polyacrylamide gels of purified rat brain SSADH, and we detect only one cross-reactive band on Western blots of tissue homogenates(5, 18) . Also, our expression data show that an enzymatically active SSADH protein can be formed from a single subunit encoded by a cDNA transcribed from the 2.0- or 6.0-kb transcript. Also in support of the single subunit theory is the observation that rat brain SSADH can be resolved to a single band by isoelectric focusing, suggesting one isozyme(5) . Complete cloning of the human SSADH cDNA may shed light on the subunit structure of the protein.

Although the composite cDNAs reported here encode the mature and functionally active rat brain SSADH protein, we were unable to obtain a full-length cDNA containing the 5`-untranslated region and encoding the mitochondrial signal sequence. Both screening of several rat and human cDNA libraries and attempts at rapid amplification of cDNA ends have thus far been unsuccessful. Similar results have been reported by other groups attempting to obtain the 5`-ends of aldehyde dehydrogenase cDNAs (20, 21, 22) . The problem may be due to a high degree of secondary structure in the aldehyde dehydrogenase RNAs which lead to premature termination of cDNA synthesis by reverse transcriptase. All of the rat brain SSADH cDNA clones reported in this paper have a 5` terminus within a 12-bp region which is a few codons downstream of the first codon of the mature protein. Primer extension analysis performed in this laboratory reveals a strong stop which maps to this region and further indicates secondary structure of the RNA at that site.

This paper represents the first demonstration by Northern blot data of the presence of SSADH in non-neural tissues as well as neural tissue. Other groups have demonstrated SSA oxidizing activity in non-neural tissues; however, the presence in mammalian tissues of other aldehyde dehydrogenases which can oxidize SSA leaves doubt to the actual presence of SSADH, especially in tissues with low levels of activity. However, most tissues examined have some amount of SSADH message. In conjunction with the demonstration that GABA and GABA-T are located in a number of non-neural tissues(23) , our data would suggest that catabolism of GABA through GABA-T and SSADH is an active metabolic pathway in mammalian non-neural tissues.

Isolation of rat and human cDNAs encoding SSADH are important steps to begin an analysis of the molecular genetics of SSADH deficiency. SSADH deficiency is believed to be caused by a mutation in the gene encoding SSADH. The information in the present report should be of value in isolating a full-length human cDNA encoding SSADH, which will be important for mutation studies in cells from affected individuals. Mutation analysis in SSADH deficiency may help to explain the pronounced clinical heterogeneity of the disease.


FOOTNOTES

*
Portions of this work have been presented in abstract form (19) . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L34820 [GenBank]and L34821[GenBank].

§
To whom correspondence should be directed: Baylor Research Institute, 3812 Elm St., Dallas, TX 75226. Tel.: 214-820-2687; Fax: 214-820-4952.

(^1)
The abbreviations are: GABA, -aminobutyric acid; GABA-T, -aminobutyric acid transaminase; SSA, succinic semialdehyde; SSADH, succinic semialdehyde dehydrogenase; bp, base pair(s); kb, kilobase(s); PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We thank Dr. J. Boulter for the brain cDNA libraries and Terry Munn for assistance in GenBank sequence comparisons.


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