(Received for publication, July 11, 1994; and in revised form, October 13, 1994)
From the
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
= 52,188) when including the insert and 460 amino acids (M
= 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.
The metabolism of the inhibitory neurotransmitter GABA ()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) .
More than 1.5 10
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.
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
10
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
-actin clone provided by Clontech.
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.
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
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) .
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
-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).
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.
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.
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.
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].