Molecular Enzymology of Mammalian
1-Pyrroline-5-carboxylate Synthase
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Chien-an A.
Hu
§,
Wei-Wen
Lin¶
,
Cassandra
Obie
, and
David
Valle
**
From the
Howard Hughes Medical Institute, Department
of Pediatrics and Institute of Genetic Medicine and the
¶ Predoctoral Training Program in Human Genetics, Johns Hopkins
University School of Medicine, Baltimore, Maryland 21205
 |
ABSTRACT |
1-Pyrroline-5-carboxylate
synthase (P5CS; EC not assigned), a mitochondrial inner membrane, ATP-
and NADPH-dependent, bifunctional enzyme, catalyzes the
reduction of glutamate to
1-pyrroline-5-carboxylate, a
critical step in the de novo biosynthesis of proline and
ornithine. We utilized published plant P5CS sequence to search the
expressed sequence tag data base and cloned two full-length human P5CS
cDNAs differing in length by 6 base pairs (bp) in the open reading
frame. The short cDNA has a 2379-bp open reading frame encoding a
protein of 793 residues; the long cDNA, generated by "exon
sliding," a form of alternative splicing, contains an additional 6-bp
insert following bp +711 of the short form resulting in inclusion of
two additional amino acids in the region predicted to be the
-glutamyl kinase active site of P5CS. The long form predominates in
all tissues examined except gut. We also isolated the corresponding
long and short murine P5CS transcripts. To confirm the identity of the
putative P5CS cDNAs, we expressed both human forms in
-glutamyl
kinase- and
-glutamyl phosphate reductase-deficient strains of
Saccharomyces cerevisiae and showed that they conferred the
proline prototrophy. Additionally, we found expression of the murine
putative P5CS cDNAs conferred proline prototrophy to P5CS-deficient
Chinese hamster ovary cells (CHO-K1). We utilized stable CHO-K1 cell
transformants to compare the biochemical characteristics of the long
and short murine P5CS isoforms. We found that both confer P5CS activity
and that the short isoform is inhibited by L-ornithine with
a Ki of ~0.25 mM. Surprisingly, the long isoform is insensitive to ornithine inhibition. Thus, the two
amino acid insert in the long isoform abolishes feedback inhibition of
P5CS activity by L-ornithine.
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INTRODUCTION |
Proline can be synthesized either from glutamate or ornithine and
is nonessential for full-term human infants and adults (1, 2) but
conditionally indispensable for premature neonates (3, 4).
1-Pyrroline-5-carboxylate
(P5C),1 which is in
nonenzymatic equilibrium with glutamic
-semialdehyde, is a common
intermediate in these pathways (5). Formation of P5C from glutamate in
humans has been proposed to be catalyzed either by P5C synthase, a
bifunctional enzyme with both
-glutamyl kinase (
-GK) and
-glutamyl phosphate reductase (
-GPR) activities or a complex of
separate
-GK and
-GPR enzymes (6). Hu et al. (7)
showed that plant P5CS is bifunctional, catalyzing the conversion of
glutamate to P5C. By contrast, P5CS activity in prokaryotes and lower
eukaryotes like Saccharomyces cerevisiae requires the
combined functions of separate
-GK and
-GPR enzymes. In rats,
P5CS activity is highest in small intestine with measurable activity in
colon, pancreas, and thymus (8). Since proline may be a
neurotransmitter and because the concentration of proline in the
extracellular fluid of the central nervous system is low (<5
µM) (9-14), P5CS may be of particular importance in
certain regions of the central nervous system. Wakabayashi et
al. (8) showed that detectable P5CS activity is present in rat
cerebrum and cerebellum; however, the regional distribution of this
enzyme in the central nervous system has not been well characterized. P5C can also be synthesized from ornithine in a reaction catalyzed by
ornithine
-aminotransferase (OAT) (15).
The P5CS genes of plants (7, 16-18) and the
-GK and
-GPR genes
of prokaryotes (e.g. in Escherichia coli; see
Ref. 19) and S. cerevisiae (20, 21) have been cloned and
sequenced. Mutant strains of S. cerevisiae auxotrophic for
proline because of mutations in
-GK or
-GPR genes are known and
provide a potential system for expression and analysis of human P5CS.
Additionally, CHO-K1 cells are proline auxotrophs and lack both P5CS
and OAT activities (22-24).
Early studies of Chinese hamster-human somatic cell hybrids (25) and
chromosome localization of glutamate oxaloacetate transaminase (26), a
marker linked to P5CS, suggested that the human P5CS gene is on
chromosome 10. During the course of our work, Aral et al.
(27) reported the sequence of a human cDNA that, based on sequence
similarity, appeared to encode P5CS. Direct evidence showing that this
cDNA encodes a functional enzyme was not provided. This group also
localized the structural gene encoding this cDNA to chromosome 10 in the interval between q24.3 to q24.6 (28).
To better understand the molecular enzymology and regulation of
mammalian P5CS, we utilized homology to Vigna P5CS to clone two full-length human P5CS cDNAs and the corresponding murine P5CS
cDNAs. Expressing these cDNAs in mutant yeast strains and in
CHO-K1 cells, we showed both forms of mammalian P5CS encode bifunctional enzymes and complement growth phenotype of mutant cells.
More interestingly, we found that the short but not the long P5CS
isoform is sensitive to feedback regulation of ornithine. Our results
suggest a mechanism for differential regulation of P5CS activity.
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EXPERIMENTAL PROCEDURES |
cDNA Libraries and Plasmids--
We obtained a human small
intestine 5'-STRETCH cDNA library from CLONTECH
and a human kidney cDNA from Dr. G. Bell. pBluescript II KS(+) was
used for all cloning manipulations.
Cloning by Homology Probing and cDNA Library
Screening--
We utilized the deduced amino acid sequence of
Vigna aconitifolia P5CS (7) as a probe to screen the
GenBankTM (EST) data base (National Center for
Biotechnology Information, Bethesda, MD) with the BLAST algorithm (29).
We obtained a candidate human EST clone (GenBankTM
accession no. R13516) from the National Genome Center, Lawrence Livermore National Laboratory (Livermore, CA) and sequenced the cDNA insert. Using this putative P5CS EST clone as a probe, we screened >5 × 105 plaques from both the human kidney
and small intestine cDNA library as described (30, 31). We purified
two positive plaques, one (HsP5CS.K5) from kidney cDNA
library and the other (HsP5CS.G1) from the small intestine
cDNA library, through two additional cycles of screening, subcloned
the inserts (HsP5CS.K5, 2.7 kb; HsP5CS.G1, 2.8 kb) into pBluescript II KS(+), and sequenced them. Using the
full-length human P5CS cDNA sequence as a probe, we also identified
a full-length murine P5CS long form cDNA (GenBankTM
accession no. AA1042889) and confirmed its identity by sequencing the
insert (MmP5CS.long.1; 3.3 kb).
Genomic and Reverse Transcription PCR--
We performed PCR
amplification on either human or murine genomic DNA (100 ng) with
primers DV2375 (5'-GGATTTCCATGATGAGCAGAAGC) and DV2374
(5'-CAGTCGGGCAGCCAGGCTATCATTATC), corresponding to sequences flanking
the 6-bp insert in HsP5CS.long with an initial denaturation
step of 6 min at 96 °C, followed by 30 cycles of 1 min at 94 °C,
2 min at 65 °C, and 2 min at 72 °C. The amplified products were
electrophoresed in 1% agarose, excised, gel-purified, and sequenced
directly with the 5' nested primer DV1585 (5'-GCCGAACCTCAATGGAACAC). Reverse transcription PCR (RT-PCR) was done with 1 µg of poly(A) RNA
from human small intestine, placenta, or cerebellum
(CLONTECH) with a antisense primer DV1537
(5'-TCCACAATGTCTGTGATGACGTGC) and a cDNA cycle kit (Invitrogen) for
the reverse transcription reaction followed by PCR with primers DV2375
and DV1537 with the same conditions as utilized for PCR of genomic DNA.
We cloned the amplified DNA fragments in pCR II (Invitrogen) and
transfected the recombinant molecules into DH5
-competent cells. To
quantify the ratio of the long and short forms of the inserts, we
performed a nested PCR amplification with primers complementary to
internal sequence DV1475 (5'-CCCAGCTGAGCCCAACAGTGACC) and DV2374 and
bacterial transformants harboring recombinant plasmids as template DNA.
The PCR conditions were as utilized for PCR of genomic DNA. The
amplified DNA fragments were electrophoresed in 4% Nusieve agarose to
resolve the long and short products differing in length by 6 bp.
DNA and Deduced Amino Acid Sequence Analysis--
We sequenced
all cDNA clones in both directions using the Sequenase version 2.0 DNA sequencing kit (U.S. Biochemical Corp.) according to the
manufacturer's protocols. DNA sequences were aligned and compared
using the MacVector (Eastman Kodak Co.) and the BLAST programs. We
compared the deduced amino acid sequences of the putative full-length
human P5CS cDNA clones to that of S. cerevisiae
-GP,
-GPR, and additional kinases with the MegAlign program (DNASTAR).
Ribonuclease Protection Assay on Human RNA--
We cloned a PCR
fragment of the HsP5CS cDNA corresponding to bp +474 to
+852 of the long form into pCR II (Invitrogen) and sequenced the
insert. To provide a template for synthesis of the riboprobe, we
linearized the insert by digestion with EcoRI and utilized
[
-32P]UTP, T7 RNA polymerase (Ambion) to make cRNA and
the RPA II kit (Ambion) to conduct ribonuclease protection assay (RPA).
The cRNA consists of a sequence 100% complementary to a 379-nucleotide fragment of the HsP5CS.long plus 12 nucleotides of the
vector. We hybridized human poly(A) RNA overnight at 45 °C with the
cRNA (2 × 105 cpm) and then digested with an RNase
mixture (1 unit of RNase A and 200 units of RNase T1, RPA II kit,
Ambion) at 37 °C for 30 min. We determined the size of the protected
fragments by electrophoresis on a 6% polyacrylamide sequencing gel in
parallel with a sequencing ladder and determined the intensity with a
PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
Northern Blot Analysis--
We hybridized two commercial human
multiple tissue Northern blot filters (CLONTECH)
under high stringency conditions (final wash in 0.1% SDS, 0.1× SSC,
50 °C for 40 min) as suggested by the manufacturer. Human
-actin
was used as a probe to verify RNA quality and quantity. Hybridization
and autoradiography were performed according to Hu et al.
(30).
Construction of Human P5CS Yeast Expression Vectors--
We used
the 2µ-based p424GAL1 (32) E. coli/yeast shuttle vector
plasmid containing TRP1- and
AmpR-selectable markers and a multiple cloning
site downstream of an S. cerevisiae galactokinase
(GAL1) promoter. pTB26, a plasmid containing the S. cerevisiae PRO1 gene, was a gift from M. Brandriss. We subcloned
the yeast PRO1 gene from pTB26 to p424GAL1 to form pScPRO1.
To replace the S. cerevisiae PRO1 ORF with those of the long
and short forms of human P5CSs, we used a homologous
recombination-based cloning strategy (33) to construct
pHsP5CS.short.2. Briefly, we designed two oligonucleotide
primers to amplify the entire human P5CS ORF flanking with S. cerevisiae PRO1 5'- and 3'-untranslated sequences. The 5' sense
primer contained 67 nt of sequence identical to the sense strand of
S. cerevisiae PRO1 beginning 67 nt 5' to the initiation ATG
(
67 to
1) followed by 27 nt identical to the sense strand of the
5'-end of the human P5CS ORF (+1 to +27). The 3' antisense primer
contained 60 nt of sequence corresponding to the antisense strand
beginning 60 bp downstream of the stop codon of S. cerevisiae
PRO1 ORF (+1686 to +1627) followed by 34 nt of sequence identical
to the antisense strand of the 3'-end of the human P5CS.short ORF
(+2382 to +2349). We used a combination of Pfu and
Taq polymerases (Stratagene) to amplify
HsP5CS.short. Homologous recombination in yeast was done by
cotransfecting the BglII- and HpaI-linearized
pScPRO1 and the yeast/human P5CS fragment into yeast strain
DT1103 (see below). We synthesized pHsP5CS.long.2 by
replacing the relevant region of P5CS.short with the counterpart of the
long form containing the 6-bp insert. The sequence at the recombination
junctions of these recombinant plasmids was vetted by sequencing.
Functional Complementation of Yeast Auxotrophs--
DT1103
(MATa, ura3-52, trp1, pro1::URA3) and DT1102
(MATa, ura3-52, trp1, pro2::URA3) were gifts from
M. Brandriss (34). We transformed yeast by the lithium acetate method
as described (30). Strains HV10, HV11, HV13, and HV14 were constructed
by introduction of recombinant vectors pScPRO1, p424GAL1,
pHsP5CS.short.2 and pHsP5CS.long.2 into DT1103,
respectively. Strains HV12, HV14, HV15, and HV16 were derived from
DT1102 transfected with p424GAL1, pHsP5CS.short.2,
pScPRO1, and pHsP5CS.long.2, respectively. The transformation reactions were plated on minimal glucose medium containing 0.1% ammonium sulfate without tryptophan
(MGA-trp
) or minimal glucose medium containing 0.1%
ammonium sulfate and 1% proline without tryptophan
(MGAP-trp
) (30). After growth for 10 days at 30 °C,
plasmid DNA was isolated from yeast transformants and used to transform
E. coli 294-competent cells (30).
Construction of Murine P5CS Expression Vectors--
We used the
pcDNA3 (Invitrogen) mammalian expression plasmid containing
NeoR- and AmpR-selectable markers and a
multiple cloning site downstream of human cytomegalovirus promoter. We
digested the murine EST clone, designated as pMmP5CS.long.1
containing the full-length murine P5CS long form cDNA with
EcoRI and ApaI and subcloned the insert into
pcDNA3 to form pMmP5CS.long.2.
To introduce MmP5CS.short into pcDNA3, we first made an
intermediate construct, designated as pMmP5CS.short.1, by
replacing the region containing the 6-bp insert of the long form with
the corresponding sequence of the short form. We took advantage of the
unique restriction enzyme sites BsgI (+482) and
StuI (+807) to engineer pMmP5CS.long.1.
Initially, we amplified the short form cDNA by RT-PCR of murine
small intestine poly(A) RNA (1 µg) with a antisense primer DV2231
(5'-AGGAGGTGAAGAATGCGGTTGCTG) and a cDNA cycle kit followed by PCR
with primers DV2967 (5'-CCTGCACTCAGGACAGAACCATCTGAA) and DV2899
(5'-GGGCTGGCAATCTTCCCTCTG) with the same conditions as utilized for PCR
of genomic DNA (see above). We digested the amplified DNA fragments
with BsgI and StuI and ligated them into BsgI/StuI-linearized pMmP5CS.long.1 to
produce pMmP5CS.short.1. Subsequently, we subcloned the
insert of pMmP5CS.short.1 into pcDNA3 to form
pMmP5CS.short.2. We vetted the replaced segment of the
recombinant plasmid pMmP5CS.short.2 by sequencing.
Transfection and Functional Complementation of CHO-K1
Cells--
We transfected pMmP5CS.short.2 or
pMmP5CS.long.2 into a subclone (CHO-K1-NC5) of the proline
auxotrophic cell line CHO-K1 (ATCC no. CCL61) by electroporation as
described by Melkonyan et al. (35). Briefly, we
electroporated 7 × 106 cells resuspended in the
growth medium containing 1.25% Me2SO (300 µl) and 30 µg of plasmid DNA with a Gene Pulser (Bio-Rad) using the setting 960 microfarads, 100 ohms, and 350 V. We selected stable transformants in
the minimal essential medium with 10% (v/v) dialyzed fetal calf serum
lacking L-proline (22).
P5CS Enzyme Assay and End Product Regulation--
We assayed
P5CS activity radioisotopically as described by Wakabayashi et
al. (8). In preliminary experiments (not shown) we verified that
P5CS activity is dependent on the presence of ATP, MgCl2,
and NADPH and increases linearly with added protein up to a
concentration of 1 mg/ml and with time up to 60 min.
 |
RESULTS |
Isolation and Characterization of Human and Murine P5CS
cDNAs--
We utilized the C-terminal 200-amino acid sequence of
Vigna (mothbean) P5CS to perform a BLAST searches of the
GenBankTM EST data base and identified a single human
candidate clone (GenBankTM accession no. R13516). The
deduced amino acid sequence of this clone had 59% identity to the
corresponding region of mothbean P5CS (data not shown). A second BLASTP
search utilizing the EST as probe retrieved several cDNAs including
Arabidopsis P5CS, mothbean P5CS, a Caenorhabditis
elegans EST sequence (GenBankTM accession no. D35184)
and
-GPR genes for S. cerevisiae and several prokaryotes
(data not shown). We obtained this human putative P5CS EST clone,
sequenced the entire insert for confirmation, and utilized it to probe
human kidney and small intestine cDNA libraries. One cDNA clone
(P5CS.K5) had an insert size of 2726 bp extending from +472 (where +1
is the A of the initiation methionine codon) to a poly(A) tail. The
second clone (P5CS.G1) lacked the 3'-most 503 bp of P5CS.K5, had a
2225-bp overlap with the 5'-end of P5CS.K5, and extended an additional
529 bp 5' to
57. Using a similar strategy, we also obtained a murine
putative P5CS EST clone (GenBankTM accession no. AA1042889)
isolated from 13-day-old mouse heart cDNA library had an insert
size of 3343 bp extending from
53 (where +1 is the A of the
initiation methionine codon) to a poly(A) tail. It has a 53-bp 5'-UTR,
2385-bp ORF, and 805-bp 3'-UTR. The mouse ORF encodes a polypeptide
with 95% sequence identity to its human counterpart.
The overall amino acid sequence of the human putative P5CS has 43 and
40% identity to those of Arabidopsis thaliana and V. aconitifolia P5CSs, respectively. Examination of the human P5CS sequence indicates that the enzyme is bifunctional. The N-terminal half
has 36% identity to S. cerevisiae
-GK, while the
C-terminal half has 48% identity to S. cerevisiae
-GPR
(Fig. 1). The N-terminal half of human
P5CS also has strong similarity to lower eukaryotic and prokaryotic
-GKs, and the C-terminal half is similar to lower eukaryotic and
prokaryotic
-GPRs (data not shown). As expected for a mitochondrial
inner membrane protein, the N terminus of both the human and the murine
P5CS sequences has features characteristic of a mitochondrial targeting
sequence with frequent basic and no acidic residues (36). Based on the
consensus for cleavage of mitochondrial targeting sequences (36-38),
there are potential cleavage sites following amino acids 56 and 64 (Fig. 1). Using BLASTP and Blocks data base (39) searches, we
identified a 32-amino acid motif (between Asp245 and
Pro276) with similarity to a sequence thought to be the
active site motif in several other kinases in the aspartokinase family
(see Figs. 1 and 2). In this motif, two
aspartate residues (Asp245 and Asp258) are
conserved in a comparison of eight aspartokinases including one from
Vigna, two from yeast, and five from prokaryotes. Five other
residues are identical in at least seven of the examples. Additionally,
the human and murine P5CS sequences have three conserved residues
(Asn191, Asp193, and Phe194)
predicted by homology to Vigna P5CS and E. coli
-GK to be involved in proline feedback regulation of P5CS activity
(7, 38, 40) (Fig. 1). Also, there are four putative
N-glycosylation sites and a putative NADP(H) binding domain
(41) (Fig. 1).

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Fig. 1.
Comparison of the deduced amino acid sequence
of human P5CS with S. cerevisiae -GK
and -GPR. Residues identical in the human
and yeast proteins are shown in black. The
vertical arrows indicate potential sites of
cleavage of the mitochondrial targeting sequence (see text). The
asterisks indicate three codons shown to be involved in the
proline feedback inhibition based on studies of Vigna P5CS
(40) and corresponding to Asn191, Asp193, and
Phe194 of HsP5CS.short. The solid
rectangles indicate the location of potential
N-glycosylation sites. The thick
overline indicates the conserved putative aspartokinase
active site motif, and the thin overline
indicates the NADP(H) binding site motif (41). The location of the
2-amino acid (VN) insert in the long form of HsP5CS is also
indicated.
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Fig. 2.
Comparison of the putative aspartokinase
active site motif of human P5CS with those of V. aconitifolia, S. cerevisiae, and E. coli and other kinases. The conserved 32-amino acid
motif (overline) and flanking residues are shown. The
location of the 2-amino acid insert resulting from alternative splicing
of HsP5CS is shown in boldface type at
the N-terminal end of the sequence. Residues are numbered to the
left. Residues identical in five or more of the sequences
are shown in black. HsP5CS (GenBankTM
accession no. U76542), human P5CS; VaP5CS
(GenBankTM accession no. P32296), mothbean P5CS;
Sc -GK (GenBankTM accession no. P32264),
S. cerevisiae -GK; Ec -GK
(GenBankTM accession no. P07005), E. coli
-GK; ScAK (GenBankTM accession no. P10869),
S. cerevisiae aspartokinase; EcAK
(GenBankTM accession no. P00561), E. coli
aspartokinase; EcUK, E. coli uridylate kinase
(GenBankTM accession no. D13334); PaCK
(GenBankTM accession no. P13982), P. aeruginosa
carbamate kinase. The asterisks indicate completely
conserved residues.
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Comparing the sequences of the various human putative P5CS cDNAs,
we noted that some had a 6-bp insert following bp +711 while others did
not. To determine if this insert was the result of some form of
alternative splicing, we amplified genomic DNA with primers
complementary to cDNA sequences flanking the region of the insert
(5' DV1475; 3' DV2374). Amplification of cDNA with these primers
produces a 74-bp fragment. However, with genomic DNA as template, the
product was ~500 bp. Sequence analysis showed that this fragment
contains a portion of a 5' exon followed by a 400-bp intron and a
portion of a 3' exon. We sequenced the amplified P5CS structural gene
fragment and found that the 6-bp insert derives from the 5'-end of an
intron with two potential donor splice sites in tandem (Fig.
3). Alternative utilization of these two
donor splice sites generates two different mature transcripts: a short form, utilizing the most 5' donor site and lacking the insert (Fig. 3),
and a long form, utilizing the most 3' donor site and containing the
6-bp insert following position +711.

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Fig. 3.
Genomic and cDNA sequences of the human
P5CS showing the two alternative donor splice sites in the structural
gene in the region corresponding to the 6-bp insert in the
cDNA. The two additional amino acids resulting from use of the
more 3' donor splice site are indicated at the bottom.
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To examine the tissue expression of the two P5SC isoforms, we conducted
RT-PCR and RPAs with poly(A) RNA isolated from human small intestine,
placenta, and cerebellum. The former has the advantage that individual
clones can be scored to give a quantitative result; the latter has the
advantage that it avoids PCR. In the RT-PCR assay, we cloned the
amplified fragments and performed a second PCR amplification with a
pair of nested primers surrounding the region containing the insert. In
clones containing the insert, the amplified product is 80 bp; in clones
without, the amplified product is 74 bp. We found that the ratio of the
short to long forms in small intestinal RNA was 0.3 (58/19), whereas
the ratio in placental (15/62) and cerebellum (15/59) RNAs was ~0.25
(Fig. 4). The RPA data are generally
consistent with this result. The antisense riboprobe (cRNA) used for
RPA is 391 bp and has 6 bp of vector sequence at each end flanking
379-bp HsP5CS.long sequence complementary to bp +474 to
+852. When the probe hybridizes to the mature HsP5CS.long
transcript, a 379-bp fragment is protected. Alternatively, when the
probe hybridizes to a P5CS.short transcript, a single-stranded loop
structure will form at the site of the insert yielding two products of
238 and 135 bp following RNase digestion. As seen in Fig. 4, this assay
confirms the short form is predominant in the gut, whereas the converse
is true in placenta and cerebellum. PhosphorImager analysis of the
intensity of these bands gave short to long ratios of 3.75 in gut, 0.83 in placenta, and 0.55 in cerebellum.

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Fig. 4.
Ratios of HsP5CS.long and
HsP5CS.short as revealed by RPA and RT-PCR.
A shows an autoradiogram of an RPA performed as described
under "Experimental Procedures." Lane 1, labeled
riboprobe only; lane 2, riboprobe digested with
RNase mixture; lanes 3-6, riboprobe hybridized with tissue
RNA digested with RNase mixture; lane 3, yeast
total RNA (10 µg); lane 4, human small
intestine poly(A) RNA (1 µg); lane 5, human
placenta poly(A) RNA (1 µg); lane 6, human
cerebellum poly(A) RNA (1 µg). B, agarose gel
electrophoresis of PCR-amplified fragments of cloned RT-PCR products
from RNA isolated from the indicated tissue. The long fragment is 80 bp; the short one is 74 bp.
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Tissue Distribution and Size of Human P5CS
Transcripts--
Northern blots of mRNA from multiple human
tissues showed a single predominant P5CS transcript of about 3.6 kb
(Fig. 5). Pancreas, ovary, testis, and
kidney had the highest expression, followed by colon, small intestine,
placenta, heart, and skeletal muscle.

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Fig. 5.
Northern blot analysis of P5CS expression in
various human tissues. The same blot was probed with radiolabeled
P5CS.short (top) or human -actin (bottom). The
position of size markers (kb) is indicated. We trimmed the images of
the Northern blots to save space; no other hybridizing transcripts were
present.
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Functional Complementation of
-GK- and
-GPR-deficient
Yeasts--
To confirm the identity and function of the two P5CS
cDNAs, we constructed composite human P5CS cDNA clones,
designated HsP5CS.short.1 and HsP5CS.long.1,
respectively. The HsP5CS.short.1 cDNA is 3256 bp in
length including 57 bp of 5'-untranslated sequence, a 2379-bp ORF, and
820 bp of 3'-UTR extending to the poly(A) tail. The
HsP5CS.short.1 ORF encodes a 793-amino acid protein with a
predicted Mr of 90. HsP5CS.long.1 is
identical to HsP5CS.short.1 except that it includes the 6-bp
insert following position +711, resulting in a 2385-bp ORF encoding a
795-amino acid protein. To express these in yeast, we utilized PCR and
homologous recombination to construct yeast/human P5CS chimeric
minigenes (pHsP5CS.short.2 and pHsP5CS.long.2), comprising the S. cerevisiae PRO1 promoter and 5'- and
3'-UTR sequences surrounding the intact human P5CS.short and P5CS.long ORFs. Previous work by ourselves and others has shown that
substitution of yeast 5'- and 3'-UTR sequences may improve the
expression of mammalian genes in yeast (30, 42). DT1103, a proline
auxotroph with a partially deleted
-GK gene (pro1), is
unable to grow on minimal medium without proline. We transfected this
strain with either pHsP5CS.short.2 or
pHsP5CS.long.2 and selected transformants on medium lacking
both proline and tryptophan (MGA-trp
). Both
pHsP5CS.short.2 and pHsP5CS.long.2 restored the
PRO+ growth phenotype in DT1103 (see strains HV13 and HV14; Fig.
6) although not as efficiently as a
construct containing the yeast
-GK gene (pScPRO1; see
HV10, Fig. 6). To confirm that both P5CS cDNAs encode bifunctional
enzymes, we performed a second complementation experiment, transfecting
pHsP5CS.short.2 and pHsP5CS.long.2 into DT1102, a mutant yeast strain auxotrophic for proline because it has a partial deletion of the
-GPR gene (pro2). As shown by the DT1102
transformants, HV15 and HV17, pHsP5CS.short.2 and
pHsP5CS.long.2 also complement deficiency of
-GPR (Fig.
6). As predicted by the S. cerevisiae requirement for the
combined activities of
-GK and
-GPR for proline prototrophy,
pScPRO1 does not complement the proline auxotrophy of DT1102
(see strain HV16, Fig. 6). We also measured the growth rates of these
strains in proline-deficient liquid medium. The doubling time was
2.7 h for strain HV10 and about 6 h for strains HV13, HV14,
HV15, and HV17. DT1103 and DT1102 transfected with vector alone do not
grow in this medium (data not shown). Thus, as predicted by the
sequence, these results confirm that both human P5CS cDNAs encode
bifunctional enzymes with
-GK and
-GPR activities.

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Fig. 6.
Culture of S. cerevisiae
strains HV10, HV11, HV12, HV13, HV14, HV15, HV16, and HV17 on
minimal glucose medium with ammonium sulfate without tryptophan
(MGA-trp ) or minimal glucose medium with ammonium sulfate
and proline without tryptophan (MGAP-trp ) for 12 days at
30 °C. Strain information is shown in the accompanying
table.
|
|
Both Murine P5CS cDNA Isoforms Complement the CHO-K1 Cell
Growth Phenotype--
Although the human P5CS cDNAs complement the
growth phenotype of the
-GK- and
-GPR-deficient yeast strains, we
were not able to measure P5CS activity in extracts of these yeast
transformants. We reasoned that this might reflect the heterologous
environment. To do biochemical studies, therefore, we transfected
murine P5CS cDNAs into a subclone (NC5) of CHO-K1 (CHO-K1-NC5)
cells previously shown to be auxotrophic for proline (43) because of
deficiency of both P5CS and OAT activities (22, 23). We selected stable transformants in medium lacking proline and assayed P5CS activity on
individual clones. To rule out reversion at the hamster P5CS locus, we
confirmed that the P5CS expressed by the transformants was murine
rather than hamster by RT-PCR and sequence analysis (not shown). We
found that both murine P5CS cDNAs expressed RNA transcripts and
complemented the growth phenotype of CHO-K1-NC5 cells. Assay of P5CS
activity in extracts of stable transformants expressing either
MmP5CS.short or MmP5CS.long showed that both constructs conferred measurable P5CS activity. Consistent with the role
of P5CS in ornithine and citrulline synthesis, previous studies using
extracts of rat small intestine showed that P5CS activity is
noncompetitively inhibited by ornithine (44, 45). To determine if both
isoforms of murine P5CS had similar sensitivity to ornithine
inhibition, we assayed their activity in the presence of 5 mM ornithine. We found that P5CS.short, the predominant
isoform in gut, is inhibited >75% by 5 mM ornithine with
a Ki in this assay of ~0.25 mM (Fig.
7). In agreement with the earlier work,
we obtained similar results using mouse small intestine as the source
of P5CS activity (Fig. 7). By contrast, P5CS.long, the predominant
isoform in peripheral tissues, is completely insensitive to 5 mM ornithine (Fig. 7).

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Fig. 7.
Effect of ornithine on murine P5CS
activity. Ornithine (5 mM) inhibits >75% of murine
gut P5CS and MmP5CS.short activities. Inhibition of
MmP5CS.short by ornithine is concentration-dependent
with a Ki of ~0.25 mM. In contrast,
the MmP5CS.long is insensitive to 5 mM
ornithine.
|
|
 |
DISCUSSION |
Utilizing homology to Vigna P5CS, we isolated two
splice forms of cDNAs encoding human P5CS: a short form
(P5CS.short) encoding a 793-amino acid protein and a long form
(P5CS.long), with an additional 6-bp insert following bp +711, encoding
a 795-amino acid protein. The human P5CS polypeptides have high
sequence identity to P5CS of plants and to
-GK and
-GPR enzymes
of lower eukaryotes and prokaryotes. Expression of human P5CSs restores
proline prototrophy in two mutant yeast strains, one deficient in
-GK (pro1), the other in
-GPR (pro2),
confirming the bifunctional nature of the mammalian enzyme. To study
the biochemistry and regulation of mammalian P5CS isoforms, we
expressed murine P5CS cDNAs in CHO-K1 cells that lack endogenous
P5CS activity. Both murine P5CS cDNAs restored proline prototrophy,
and stable transformants expressed measurable P5CS activity. Using
extracts of these stable transformants, we showed that the two isoforms
differ dramatically in their sensitivity to inhibition by ornithine.
During the course of our studies, Aral et al. (27) also
reported the sequence for a human P5CS cDNA. As we did, they
identified a partial length EST clone containing the 3' 438 bp of the
ORF. They used the sequence of this EST to initiate a series of
5'-rapid amplification of cDNA end reactions followed by direct
sequencing of the products to extend the cDNA sequence 138 bp into
the 5'-UTR. The nucleotide sequence of our P5CS cDNAs has 14 single
bp differences with that of Aral et al. (27), all in the ORF
and resulting in 13 amino acid changes. Some of these differences may
represent polymorphisms; others may reflect sequence errors. Aral
et al. (27) utilized direct sequencing of amplified
material. Our composite sequence derives from sequencing multiple
cDNA clones in both directions. The overlapping sequences of our
various cDNAs are 100% identical (for example, clones
HsP5CS.G1 and HsP5CS.K5 have a 2225-bp overlap in
perfect agreement).
In addition to the overall similarity of the N-terminal half of
HsP5CS to
-GKs from a variety of organisms
(e.g. 36% identity with S. cerevisiae
-GK),
BLASTP and Blocks data base (39) searches utilizing the
-GK domain
of the human P5CS as a probe revealed a 32-amino acid motif (from
Asp245 to Pro276) in the N-terminal half of
human P5CS with similarity to a conserved sequence characteristic for
members of the aspartokinase family including aspartokinases, uridylate
kinases, and carbamate kinases from a variety of species (46-49).
Aspartokinases catalyze phosphorylation of substrates with a carboxyl
group as the nucleophilic acceptor (50). The conserved sequence
overlaps with and extends more 5' of a region of sequence similarity
between E. coli and Serratia marcescens
aspartokinases and
-glutamyl kinases identified by Omori et
al. (46), who suggested it is the active site of these related
enzymes. Three residues in this motif corresponding to Asp245, Asp260, and Pro276 in
HsP5CS.long are conserved in all
-GKs and nearly all
members of the aspartokinase family (Fig. 2). Another region likely to be important for HsP5CS function is a cluster of three amino
acids (Asn191, Asp193, and Phe194)
about 50 residues N-terminal of the putative aspartokinase motif. These
are conserved with Vigna P5CS, where mutagenesis experiments have shown that they are essential for feedback regulation of P5CS
activity by proline (40) (Fig. 1).
We identified two forms of P5CS cDNAs differing only by a 6-bp
insertion at position +711. Two lines of evidence indicate that this
insert is generated by a variant of alternative splicing called "exon
sliding" (51). First, the insert sequence is colinear with the
genomic sequence of the human P5CS structural gene and has the
consensus sequence of a 5' splice site. The same colinearility is also
present in murine P5CS (data not shown). Thus, the choice between the
two tandem splice donor sequences determines whether the mature
transcript will contain the 6-bp insert. The fact that we see markedly
different ratios of short/long transcripts in various tissues suggests
either that there is tissue-specific stability of the two products or
that the choice of splice donor is somehow regulated. How this choice
might be regulated is unclear. Based on the calculation of the
consensus value of the 5' splice donor site (28, 52-54), both of these
tandem donor sites are relatively "weak" with the consensus values
of 77.7 and 73.2, respectively, where the value for a typical donor
site ranges from 0.7 to 1.0 with the mean of 0.85. This suggests that
there might be tissue- or development-specific factor(s) influencing the relative use of the two competing 5' splice sites. Multiple mechanisms including cis elements and
trans-acting factors influence 5' splice site selection
(53-61). Exonic splicing enhancers are positive acting, cis
RNA sequences capable of inducing the assembly of the splicesome at
weak 5' splice sites and are generally purine-rich. Interestingly, we
note several purine-rich sequences in both HsP5CS and
MmP5CS cDNAs 20-500 bp 3' of the 6-bp insert, for
example, a GGAAATGAAAA 47 bp 3' (bp +765 to +775), a GGAGGGGAAGAAG 314 bp 3' (bp +1032 to +1045), and an AGCAGGGAGAAA 376 bp 3' (bp +1094 to
+1105) of the 6-bp insert of the HsP5CS long cDNA. Other
examples of generating short (<20 codons) inserts in mature
transcripts by "exon sliding" have been described in the
Drosophila Ultrabithorax gene (62), porcine urokinase-like
plasminogen activator (63), human
-adducin gene (64), chicken growth
hormone-releasing hormone gene (65), and human presenilin-1 gene
(66).
Interestingly, the 2-amino acid insert (Val238 and
Asn239 in P5CS.long) is immediately N-terminal of the
putative aspartokinase motif (amino acids 245-276) and 43 amino acids
C-terminal of the cluster of residues (amino acids 191-194) predicted
to be involved in the feedback regulation of plant P5CS by proline
(Fig. 1). This location in or near the active site and the apparent
variation in the ratio of short versus long isoforms in
different tissues suggests that the presence or absence of the insert
may play a role in the regulation of P5CS activity. Previous work
utilizing extracts of rat small intestinal mucosa showed that mammalian P5CS is inhibited by ornithine with a Ki of about
0.4 mM (44, 45). We found similar results for the isoform
encoded by the predominant gut transcript (P5CS.short). In mice and
humans, plasma ornithine is about 75 µM with a
distribution ratio of intracellular/extracellular ornithine of about 5, predicting an intracellular ornithine concentration of ~0.37
mM (15). This suggests that physiologic concentrations of
ornithine are in the range predicted to regulate P5CS.short activity.
By contrast, we found the long P5CS isoform is insensitive to ornithine
inhibition. These results correlate functional differences in the
protein with the choice of splice sites. As far as we know, this is the
first demonstration of functional differences in protein isoforms
generated by exon sliding.
The P5CS reaction serves two physiologic functions in mammals: proline
biosynthesis and arginine biosynthesis. In tissues with a high protein
synthetic requirement for proline (e.g. cartilage and
possibly central nervous system), P5CS catalyzes the first committed
step in the two-step pathway synthesizing proline from glutamate (5).
In prokaryotes and plants, this pathway is regulated by allosteric
inhibition of P5CS by the end product, proline (7, 40, 67, 68). In
contrast to prokaryotes, yeast, and plants, the mammalian P5CS reaction
is also the first committed step in the pathway of de novo
arginine biosynthesis. In this pathway, the product of the P5CS
reaction is converted to ornithine by OAT and then to citrulline and
arginine by the enzymes of the urea cycle (15, 69, 70). Arginine
synthesis is active in the epithelium of small intestine, particularly
in early postnatal life (69-75). De Jonge et al. (71)
showed that genes encoding arginine synthetic enzymes including OAT,
carbamyl phosphate synthetase, ornithine transcarbamylase,
argininosuccinate synthetase, and argininosuccinate lyase are highly
expressed in rat small intestine during perinatal development, and the
pattern of expression of these genes is spatiotemporally and
zonationally regulated as follows. mRNAs for OAT, carbamyl
phosphate synthetase, and ornithine transcarbamylase are concentrated
at the base of the villi and developing crypts, while mRNAs for
argininosuccinate synthetase and argininosuccinate lyase are detected
only in the upper part of the villi, suggesting that the basal
enterocytes synthesize citrulline and that the enterocytes in the upper
half of the villus synthesize arginine. The physiologic significance of
this arginine synthetic pathway is highlighted by the observations that
mice with targeted disruption of OAT die as a result of arginine
deficiency in the neonatal period and that human infants with inherited
deficiency of OAT develop severe hypoargininemia (76). The variable
sensitivity to end product inhibition of the two isoforms of mammalian
P5CS (P5CS.short and P5CS.long) provides a mechanism for this
differential regulation. Additionally, mammalian P5C reductase, which
catalyzes the first committed step in proline biosynthesis, is
sensitive to inhibition by proline (77). Combining our results with
this information suggests a tissue-specific model for regulation of the
pathway converting glutamate to either arginine or proline. In gut,
where the emphasis is on arginine biosynthesis, P5CS.short is the
predominant isoform, and ornithine regulates the pathway by its
inhibitory effect on P5CS. In peripheral tissues where proline
biosynthesis is important, P5CS.long predominates, and the pathway is
insensitive to ornithine but is regulated at the level of mammalian P5C
reductase by proline (Fig. 8).

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Fig. 8.
Hypothetical model for the regulation
of mammalian P5CS in gut (predominantly P5CS.short) and other tissues
(predominantly P5CS.long).
|
|
Aral et al. (27) and Kamoun et al. (78) describe
two siblings with joint hyperlaxity, skin hyperelasticity, cataracts, and mental retardation who are the product of a consanguineous union.
Their biochemical phenotype (low plasma proline, citrulline, and
ornithine) suggests P5CS deficiency, and both were shown to be
homozygous for the P5CS missense mutation, L396S. The functional consequence of this mutation on P5CS activity was not tested. Availability of the human P5CS cDNA sequences and the expression systems provided by P5CS-deficient CHO-K1 cells and yeast strains provides the molecular resources to study the P5CS genes of these and
similar patients.
 |
ACKNOWLEDGEMENTS |
We thank Dr. M. Brandriss for the mutant
strains of S. cerevisiae and Sandy Muscelli for assistance
with manuscript preparation.
 |
FOOTNOTES |
*
This work was supported in part by National Eye Institute
Grant (5RO1EY02948).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be 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 GenBankTM/EMBL Data Bank with accession number(s) U68758 (HsP5CS.short), U76542 (HsP5CS.long), AF056573 (MmP5CS.long), and AF056574 (MmP5CS.short).
§
A Research Associate in Institute of Genetic Medicine, Johns
Hopkins University School of Medicine.
Present address: Tri-Service General Hospital, National
Defense Medical Center, Taipei, Taiwan, ROC.
**
An Investigator in the Howard Hughes Medical Institute. To whom
correspondence should be addressed: PCTB 802, 725 N. Wolfe St., Johns
Hopkins University School of Medicine, Baltimore, MD 21205. Tel.:
410-955-4260; Fax: 410-955-7397; E-mail: dvalle{at}jhmi.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
P5C,
1-pyrroline-5-carboxylate;
-GK,
-glutamyl kinase;
CHO-K1, Chinese hamster ovary cells;
P5CS,
1-pyrroline-5-carboxylate synthase;
ORF, open reading
frame;
-GPR,
-glutamyl phosphate reductase;
OAT, ornithine
-aminotransferase;
EST, expressed sequence tag;
PCR, polymerase
chain reaction;
RT-PCR, reverse transcription PCR;
bp, base pair(s);
kb, kilobase pair(s);
nt, nucleotide(s);
UTR, untranslated region;
RPA, ribonuclease protection assay.
 |
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