From the Janssen Research Foundation, Departments of
Biotechnology, ¶ Biochemistry, and
Neuropsychopharmacology, B2340 Beerse, Belgium
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ABSTRACT |
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Hydrolysis of the neuropeptide
N-acetyl-L-aspartyl-L-glutamate
(NAAG) by N-acetylated The neuropeptide
N-acetyl-L-aspartate-L-glutamate
(NAAG)1 is expressed both in
the central nervous system and in the periphery. NAAG has been
localized to specific sub-populations of neurones and is one of the
most abundant peptides in brain, being present in millimolar
concentrations in certain brain regions (for review see Ref. 1).
Clarification of the physiological role of NAAG has been difficult
because it is co-localized with glutamate, however, it does fulfill
some of the criteria for a neurotransmitter. It is localized in
synaptic vesicles and is released in
Ca2+-dependent manner from nerve terminals
(1).
In the central nervous system NAAG has been shown to act as a weak
partial agonist at N-methyl-D-aspartate
receptors but not at The N-acetylated In this study we describe the cloning, expression, and characterization
of a third novel member of this enzyme family, human NAALADase II, that
is similar to but distinct from NAALADase I and NAALADase L. In
addition we have identified, expressed, and characterized the full
coding sequence of human NAALADase L.
Sequence Similarity Searching for NAALADase L
Molecules--
Using the complete human (GenBankTM
accession number M99487), rat (GenBankTM accession number
RNU75973), and mouse (GenBankTM accession number AF026380)
NAALADase I protein sequences, the complete rat NAALADase L
(GenBankTM accession number AF009921) protein sequence and
a partial human NAALADase L protein sequence (GenBankTM
accession number AF010141) as query sequences, a Basic Local Alignment
Search Tool (17) search was performed on the WashU Merck expressed
sequence tag (EST) data base and on a proprietary LifeSeqTM
human EST data base. Five EST clones 4190746, 1547649, 3448872, 3608639, and 1333965 were ordered from Incyte Pharmaceuticals. The DNA
insert of each clone was sequenced on both strands using Applied
Biosystems prism BigDye Terminator Cycle sequencing kits and an Applied
Biosystems 377XL sequencer (Perkin-Elmer).
NAALADase Cloning
NAALADase I--
Sequence data from human NAALADase I
(GenBankTM accession number M99487) was used to design
primers to amplify the complete coding sequence of NAALADase I by PCR.
Primers used were NAALD1S2 (BamHI) = 5'-CCC
GGATCC GAG ATG TGG ATT CTC CTT CAC GAA AC-3' and NAALD1AS2(XhoI) = 5'-CCC CTCGAG
TTA GGC TAC TTC ACT CAA AGT CTC TGC -3' (restriction sites to be
introduced are underlined). PCR amplification was performed using
Marathon-Ready human prostate cDNA (CLONTECH)
and the Expand High Fidelity PCR system (Boehringer Mannheim) with
primers NAALD1S1(BamHI) and NAALD1AS1(XhoI)
according to the manufacturer's instructions. All PCR reactions began
with an initial denaturation step (94 °C for 5 min) prior to
addition of enzyme, followed by 30 cycles of amplification (45 s at
94 °C, 1 min at 55 °C, and 1 min 48 s at 68 °C) and ended
with a final extension step (7 min at 72 °C). A 2303-bp PCR fragment was cloned into pCR2.1 according to manufacturer's instructions (Invitrogen). A full-length clone containing a single PCR-induced error, at position 1183, was corrected by site-directed mutagenesis (SDM) using the QuickChange SDM Kit (Stratagene). Primers designed for
the SDM reactions were NAALD1-SDM-S1 = 5'-CCC TCA GAG TGG AGC AGC
TGT TGT TCA TGA AAT TGT GAG G-3' and NAALD1-SDM-AS1 = 5'-CCT CAC
AAT TTC ATG AAC AAC AGC TGC TCC ACT CTG AGG G-3'.
NAALADase L--
Sequence data from partial human NAALADase L
(GenBankTM accession number AF10141) was used to design
primers to amplify the 3' end of NAALADase L by PCR. First round PCR
amplification was performed with NAALDLS1 = 5'-GTT CTT CAA CAA GCT
GCA GGA GCG -3' and NAALDLAS1(XhoI) = 5'- CCC
CTCGAG CCG GAG TAA AGG GAG GGC TGA AG-3'.
Second round PCR amplification was performed with nested primers
NAALDLS2 = 5'-GGC GAC CTG AGC ATC TAC GAC AAC-3' and NAALDLAS2 (XhoI) = 5'- CCC CTCGAG TCC CCT CAG
AGG TCA GCC ACA G-3'. Cycling conditions were similar to those
described above (30 cycles of 45 s at 94 °C, 1 min at 57 °C,
and 1 min at 72 °C). PCR products derived from Marathon-Ready human
small intestine cDNA were cloned into pCR2.1 and extended to the
translation termination codon of NAALADase L. To obtain unknown 5'
coding sequence for human NAALADase L, antisense primers were designed
for the 5'-rapid amplification of cDNA ends (5'-RACE); antisense
primers NAALDLAS5 = 5'-CTG CAG CTT GTT GAA CTC TTC TGT G-3' and
NAALDLAS6 = 5'-CAA ACA CGA TTG ATC TGC GAG GAC-3' were synthesized
for amplifications using various human Marathon-ReadyTM
cDNAs. Cycling conditions were as described previously. PCR
products derived the small intestine cDNA amplifications were
cloned into pCR2.1 and found to extend the coding sequence beyond the
putative translation start codon and into part of the 5'-untranslated region.
To construct a full-length NAALADase L clone, primers were designed to
introduce a unique restriction site (MunI) into the DNA
sequence of NAALADase L without changing the amino acid sequence of the
ORF. The first primer set was NAALDLS3 (EcoRV) = 5'-CGGATATCC GCA GGA TGC AGT GGA
CGA AG-3' and NAALDLAS8 (MunI) = 5'-CAA ACA CAATTG ATC TGC GAG GAC GC-3' and the
second primer set was NAALDLS8 (MunI) = 5'-GCG TCC TCG CAG
ATCAATTGT GTT TG-3' and
NAALDLAS1 (XhoI). The base change introducing the
MunI site is marked in bold. PCR amplification was performed
on the 3' end clone with primers NAALDLS3 (EcoRV) and
NAALDLAS8 (MunI) and on the 5' end clone with primers
NAALDLS8 (MunI) and NAALDLAS1 (XhoI). PCR
products were cloned into pCR2.1 and sequence-verified as described previously.
It should also be noted that amplifications using NAALADase L-specific
primers resulted in a number of PCR products of unexpected size using
different cDNAs. These PCR products were cloned and sequenced in
order to identify possible splice variants.
NAALADase II--
Sequencing results from Incyte clone 3608639, derived from a human lung cDNA, suggested that this clone contained
a DNA sequence spanning the complete coding region of a putative
NAALADase-like molecule (NAALADase II) similar to but distinct from
NAALADase I and NAALADase L. Sequential 5'-RACE PCR performed with
antisense primers NAALD2AS1 = 5'-CTT TGA TGA TAG CGC ACA GAA GTG
G-3' and NAALD2AS2 = 5'-GGA AAG ATG CCA GCG CAG GAC-3' failed to
identify any potential upstream initiation codons in amplifications
using brain, fetal brain, prostate, small intestine, and colon human Marathon-Ready cDNAs.
Activity Determinations of NAALADases Transiently Expressed
in COS Cells
Subcloning of NAALADases into Expression Vectors--
NAALADase
I, II, and L clones were subcloned into the cytomegalovirus
promoter-based plasmid pcDNA3. NAALADase I/pCR2.1 was digested with
BamHI/XhoI to excise the complete NAALADase I
sequence. NAALADase II/pINCYTE was digested with EcoRI to
excise the complete NAALADase II sequence. NAALADase L-5'/pCR2.1 was
digested with EcoRV/MunI, and NAALADase
L-3'/pCR2.1 was digested with MunI/XhoI to excise
the two halves of complete NAALADase L sequence. All expression
constructs subcloned into pcDNA3 were verified by full sequence analysis.
Transient Transfection into COS Cells--
COS cells were
maintained in complete medium (defined minimal essential medium
supplemented with 10% fetal calf serum, 1× non-essential amino acids,
and a 1× streptomycin/penicillin/glutamine mix). Cell titer was
determined in a Coulter counter, and cells were seeded in six-well
plates at a density of 15,000 cells/cm2 and allowed to
reach approximately 80% confluence.
For each transfection 6 µl of FuGENE6 (Boehringer Mannheim, Germany)
was added to 96 µl of serum-free medium and incubated for 5 min at
20 °C. This preparation was added to a second tube containing 1 µg
of NAALADase/pcDNA3 DNA, mixed gently, and allowed to stand for 15 min at room temperature. The DNA/FuGENE6/serum-free medium mix was
pipetted into a well containing 2 ml of fresh complete medium. Cells
were incubated for 72 h in a 37 °C incubator before harvesting.
Determination of Biological Activity of NAALADase
Homologues--
Transfected COS cell pellets were scraped with 50 mM Tris-HCl (pH 7.4), 0.1% Triton X-100 and vortexed.
Homogenates were put through at least one freeze/thaw cycle in liquid
N2 before assay. Assay were performed as described
previously (9) for each NAALADase, using equivalent numbers of cells or
equivalent protein content, using
N-acetyl-L-aspartyl-L-3,4-[3H]glutamate
([3H]NAAG). Assays were initiated by the addition of the
membrane homogenates to the pre-warmed assay mixture and incubated at
37 °C for various times. Reactions were terminated by addition of ice-cold 250 mM potassium phosphate and loaded onto 4-cm
anion exchange mini-columns (Bio-Rad AG1-X8).
[3H]Glutamate was eluted off the column with 0.5 M formic acid and counted in a scintillation counter
(Packard). Inhibition curves were performed under similar conditions
with increasing concentrations of QA. Assays were performed on cell
samples from at least three independent transient transfections.
DPP IV activity was determined by fluorescent analysis (excitation at
335 nm and emission 450 nm) of the hydrolysis of Gly-Pro-AMC as
described previously (16). Assays were initiated by the addition of
cellular homogenates (135 µg of total protein per reaction) to the
buffered substrate solution (100 µM in 150 mM
glycine, pH 8.5) in a total reaction volume of 100 µl and followed
for 40 min at 37 °C.
Chromosomal Localization of NAALADases by Fluorescent in Situ
Hybridization (FISH) Analysis--
Chromosomal mapping studies were
carried out by SeeDNA Biotech (Ontario, Canada) using FISH analysis.
Slide Preparation--
Lymphocytes isolated from human blood
were cultured in Slide Preparation--
Lymphocytes isolated from human blood
were cultured in FISH Detection--
NAALADase I (bp 1-2252) and NAALADase II
(bp 1-2503) probes and a partial NAALADase L probe (bp 1220-2276)
were biotinylated with dATP for 1 h at 15 °C using the BioNick
labeling system (Life Technologies, Inc.). The procedure for FISH
detection was performed as described previously (17, 18).
Tissue Distribution of NAALADases
NAALADase II Gene Expression by Northern Blot--
Human
multiple tissue Northern blots (CLONTECH)
containing 2 µg of poly(A)+ RNA derived from non-neuronal
tissues were hybridized in ExpressHyb hybridization solution
(CLONTECH) for 2 h at 68 °C. A 546-bp
NAALADase II fragment, isolated from NAALADase II/pcDNA3 by
digestion with EcoRI and BglII, was radiolabeled
using a Rapid Multiprime Labeling kit (Amersham Pharmacia Biotech) and
[32P]dCTP (NEN Dupont). Unincorporated label was removed
using a Microspin S-200 column (Amersham Pharmacia Biotech), and the
denatured probe (specific activity = 2.6 × 108
dpm/µg) was incubated overnight at 68 °C in ExpressHyb. Washes were performed at high stringency (55 °C in 0.1× sodium chloride sodium citrate, 0.1% SDS) and blots exposed to X-Omat AR Film (Eastman
Kodak) for 2 days at NAALADase I, II, and L Gene Expression Analysis by
RT-PCR--
0ligonucleotide primers were designed for the specific
amplification of a PCR fragment for each NAALADase; NAALADase I primers were NAALD1S3 5'-GGG AAA CAA ACA AAT TCA GCG GC-3' and NAALD1AS3 5'-GTC
AAA GTC CTG GAG TCT CTC ACT GAA C-3' yielding a 341-bp product;
NAALADase II primers were NAALD2S4 5'-CAC TAA GAA TAA GAA AAC AGA TAA
GTA CAG C-3' and NAALD2AS4 5'-GAT CAA CTT GTA TAA GTC GTT TAT GAA AAT
CTG-3' yielding a 353-bp product; and NAALADase L primers were NAALDLS7
5'-GAC CGG AGC AAG ACT TCA GCC AG-3' and NAALDLAS7 5'-GTG TTG ATA TGC
GTT GGC CCA AG-3' yielding a 330-bp product. Each primer set was tested
for its ability to specifically amplify the desired NAALADase and not
to cross-react in amplification reactions with the other two family
members (data not shown). These primers sets were used for PCR
amplifications on human multiple tissue cDNA panels
(CLONTECH) normalized to the mRNA expression
levels of six different housekeeping genes. Control PCR amplifications
using GAPDH-specific primers (CLONTECH) were also
used. Human cDNAs from carefully dissected brain regions, transformed prostate tumor cell lines, and a prostate tumor tissue sample were prepared from mRNA using an Expand Reverse
Transcriptase kit (Boehringer Mannheim) and normalized to the mRNA
expression levels of three different housekeeping genes, GAPDH,
clathrin, and actin. PCR reactions with NAALADase or GAPDH-specific
primers were performed with Advantage Taq polymerase mix
(95 °C for 30 s, 68 °C for 1 min 30 s). Upon completion
of 25 cycles, the PCR machine was paused at 80 °C, and a 15-µl
aliquot was removed from each PCR tube. Tubes were returned to the
machine, and the cycling method was continued. Further aliquots were
removed after 30 and 35 cycles. Samples were analyzed agarose by gel
electrophoresis, and images of the ethidium bromide-stained gels were
captured using an Eagle Eye II system (Stratagene).
Molecular Cloning and Sequence Analysis of NAALADase II and
NAALADase L
NAALADase II--
Incyte Pharmaceutical clones 1547649, 3448872, 3608639, and 1333965 contained sequences originating from a single gene
similar to but not identical to NAALADase I or NAALADase L. Clone
3608639, from a lung carcinoma cDNA library, contained a DNA
sequence with a 2223-bp ORF coding for a 740-amino acid residue
protein, which we termed NAALADase II (Fig.
1). Analysis of this open reading frame
predicted a calculated molecular mass of 83,590 kDa and isoelectric
point of 8.53. The putative ATG translation start codon is in a
favorable context for translation initiation (19), and no ATG codons
were detected upstream. NAALADase II was predicted to be a type II
integral membrane protein containing a hydrophobic membrane spanning
domain extending from amino acid residues 8-31 (20). There are also
seven potential N-glycosylation sites (N × S/T) as indicated in Fig. 1.
NAALADase L--
Similarity searching of the LifeSeqTM
data base (Incyte Pharmaceuticals, Palo Alto, CA) with the human, rat,
and mouse NAALADase I sequences and with rat and partial human
NAALADase L protein sequences yielded 13 EST sequences, some of which
were overlapping, encoding for a novel protein sequence similar to
NAALADase I. DNA obtained from six of the clones were sequenced.
Incyte Pharmaceuticals clone 4190746, isolated from a cerebellar
cDNA library, contained known sequences corresponding to human
NAALADase L (16). However, because this DNA sequence also contained two
segments of intronic sequence, it was not suitable for further cloning
experiments. PCR reactions were performed to amplify a PCR product
containing the 3'-half of the NAALADase L coding region from small
intestine cDNA. To identify the remaining unknown human
5'-NAALADase L sequence, 5'-RACE PCR was performed on a number of
cDNAs. Sequencing of the amplification products obtained from
reactions using small intestine cDNA yielded a further 1344-bp
fragment covering the complete coding sequence of NAALADase L. The full
cDNA sequence contained an open reading frame of 2223 bp encoding a
protein of 740 amino acid residues with a calculated molecular mass of
80,638 Da and isoelectric point of 5.26 (Fig. 2). The putative ATG translation start
codon is in a favorable context for translation initiation (19) with no
ATG codons detected upstream. Analysis of the human NAALADase L ORF
suggests that it is a type II integral membrane protein containing a
single hydrophobic membrane spanning domain extending from amino acid residues 6-27, with lysine residues bordering either side of the potential membrane spanning domain (20). There are seven potential N-linked glycosylation sites (N × S/T) as
indicated in Fig. 2. The predicted protein sequences of human NAALADase
L were compared with that of rat using the alignment program Genedoc.
Human NAALADase L protein sequence was 78% identical and 87% similar
to rat NAALADase L.
Alternative Splicing of NAALADase L--
In the course of our
cloning and RT-PCR gene expression analysis of NAALADase L, a number of
amplified PCR products of unexpected size were observed, isolated, and
sequenced in order to identify possible splice variants. We found both
the splicing out of putative exon sequences as well as the presence of
intronic sequences (as judged by the presence of G(T/A)G donor/acceptor
sites) that were repeatedly amplified from our cDNA preparations
(Fig. 3). When performing 5'-RACE
amplifications, deletions between bases 497-619 and 903-1007 were
identified in small intestine and colon that resulted in two in-frame
amino acid residue deletions. In addition, PCR products have been
obtained containing a 153-bp intron insertion at base 1094 resulting in
an in-frame amino acid insertion of 51 amino acid residues. This
insertion is most likely an intron as it has the consensus G(T/A)G
donor acceptor sites at its 5' and 3' ends, respectively (21). In the
PCR amplifications of the 3' end of NAALADase L, several variants were
also identified from small intestine, colon, brain, and fetal brain.
These consisted of either a deletion of bases 1525-1615 or a larger
deletion between bases 1525 and 1697 (Fig. 3). Both these deletions
resulted in frameshifts and the premature termination of the protein
sequence. Finally, in every cDNA sample examined, two intronic
sequences were found to be inserted at either base 1697 and/or at base
1870. Inclusion of one or both of these intronic sequences into the ORF
of NAALADase L accounted for PCR products of unexpected size seen in
RT-PCR experiments migrating at 420 and 500 bp. Introduction of one or
both these sequences results in a frameshift and therefore in altered
amino acid sequence and premature stop (Fig. 3).
The predicted protein sequences of NAALADase I, II, and L were compared
with each other using the alignment program BESTFIT (Genetics Computer
Group Software, WI), and the percent identity and percent similarity
between each pair of sequences were calculated by the Genedoc program.
NAALADase I sequence was 67% identical (81% similar) to NAALADase II
and 35% identical (54% similar) to NAALADase L, whereas NAALADase II
and L sequences were 37% identical (54% similar). The three protein
sequences for NAALADase I, II, and L were aligned using the ClustalW
alignment program (EMBL, Heidelberg, Germany) and are shown in Fig.
4. A phylogram of NAALADase I, II, and L
was constructed using the GCG Distances program with standard
parameters and the Growtree program with the UPGMA method and is
depicted in Fig. 5. From this phylogram it is clear that NAALADase I and II are the most closely related proteins. Two putative catalytic domains have previously been identified in rat NAALADase I and L sequences by comparison to other
peptidases (12, 16). By using multiple sequence alignments of NAALADase
I, II, and L, we have identified similar putative catalytic domains in
human NAALADase II and L (Fig. 4 and 6). The first catalytic domain is related to bacterial and yeast
Zn2+-dependent peptidase domains (12), and the
second catalytic domain is related to members of the -linked acidic dipeptidase
(NAALADase) to release glutamate may be important in a number of
neurodegenerative disorders in which excitotoxic mechanisms are
implicated. The gene coding for human prostate-specific membrane
antigen, a marker of prostatic carcinomas, and its rat homologue
glutamate carboxypeptidase II have recently been shown to possess such
NAALADase activity. In contrast, a closely related member of this gene
family, rat ileal 100-kDa protein, possesses a dipeptidyl peptidase IV
activity. Here, we describe the cloning of human ileal 100-kDa protein, which we have called a NAALADase- "like" (NAALADase L)
peptidase based on its sequence similarity to other members of this
gene family, and its inability to hydrolyze NAAG in transient
transfection experiments. Furthermore, we describe the cloning of a
third novel member of this gene family, NAALADase II, which codes for a
type II integral membrane protein and which we have localized to
chromosome 11 by fluorescent in situ hybridization
analysis. Transient transfection of NAALADase II cDNA confers both
NAALADase and dipeptidyl peptidase IV activity to COS cells. Expression
studies using reverse transcription-polymerase chain reaction and
Northern blot hybridization show that NAALADase II is highly expressed
in ovary and testis as well as within discrete brain areas.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid or kainate receptors (2-4). NAAG attenuates
N-methyl-D-aspartate- or glutamate-induced neurodegeneration, and its addition to hippocampal slices or mixed cortical cultures results in neuroprotection following addition of
excitotoxins, via a mechanism distinct from the action of NAAG on the
N-methyl-D-aspartate receptor (5). In this case
it is postulated that NAAG, which is poorly transported or actively taken up, diffuses from the synaptic cleft and binds as an agonist to
type II metabotropic glutamate receptors (mGluR), such as mGluR 3, leading to reduced glutaminergic neurotransmission (6, 7). This
hypothesis is supported by studies using the mGluR 3 antagonist, ethyl
glutamate, which eliminated the neuroprotective actions of NAAG
(5).
-linked acidic dipeptidase (NAALADase)
cleavage of NAAG was first reported by Robinson et al. (8,
9) and shown to be sensitive to the synthetic glutamatergic agonist quisqualic acid (QA). Subsequently, a 94-kDa membrane glycoprotein purified from rat brain was shown to possess an NAALADase-type activity
similar to that previously described from brain extracts (10). Antisera
raised to this protein were used by Carter et al. (11) to
screen rat brain expression libraries resulting in the cloning of a
novel rat partial cDNA. The full-length coding sequence was
cloned, and the protein it encoded was termed glutamate carboxypeptidase II (EC 3.4.17.21; also called NAAG peptidase; 12, 13).
This rat gene sequence was 86% identical to the human prostate-specific membrane antigen (PSM), a cDNA of previously unknown function, which is highly expressed in prostate tumors (14).
Transfection studies with PSM into NAALADase-negative cell lines
conferred a NAAG-hydrolyzing activity to these cells that could be
inhibited by the NAALADase inhibitor QA, demonstrating the first
functional expression of a NAALADase cDNA clone (11). PSM has also
been shown to possess a second enzymatic activity, that of a
pteroyl-poly-
-glutamylcarboxypeptidase (15). Recently, a novel
100-kDa glycoprotein has been cloned, from rat ileal brush border
membranes, which is homologous to but distinct from human PSM and rat
glutamate carboxypeptidase II based on amino acid sequence. This
protein possesses a dipeptidyl peptidase IV (DPP IV) type activity,
being able to hydrolyze Gly-Pro 7-amido-4-methylcoumarin, but has
no reported NAALADase activity (16). For ease of understanding we have
simplified the nomenclature used in this report to reflect the
relatedness of these protein sequences. Human PSM and rat glutamate
carboxypeptidase II (or rat NAAG peptidase) are termed human and rat
NAALADase I, respectively. We have also termed ileal 100-kDa protein an
NAALADase-"like" peptidase (NAALADase L) based on its sequence
similarity to NAALADase I but lack of any functional NAALADase activity.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimal essential medium supplemented with 10%
fetal calf serum and phytohemagglutinin at 37 °C for 68-72 h. The
lymphocyte cultures were treated with bromodeoxyuridine (0.18 mg/ml,
Sigma) to synchronize the cell population. The synchronized cells were
washed three times with serum-free medium to release the block and
re-cultured at 37 °C for 6 h in
-minimal essential medium supplemented with 10%
fetal calf serum and phytohemagglutinin at 37 °C for 68-72 h. The
lymphocyte cultures were treated with bromodeoxyuridine with thymidine
(2.5 µg/ml). Cells were harvested and slides were prepared using
standard procedures including hypotonic treatment, fixations, and air drying.
70 °C with two intensifying screens.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide sequence and amino acid sequence
of human NAALADase II. The nucleotide and predicted one-letter
code amino acid sequence are shown. The putative membrane spanning
domain, deduced from hydrophilicity plots, is marked by a
line. Potential N-linked glycosylation sites are
in shaded squares. A putative Zn2+ binding
domain is marked within open boxes, and residues important
in a putative /
hydrolase catalytic site are within shaded
circles. Base pairs are numbered in the right
margin.
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Fig. 2.
Nucleotide sequence and amino acid sequence
of human NAALADase L. The nucleotide and predicted single letter
code amino acid sequence are shown. The putative membrane spanning
domain, deduced from hydrophilicity plots, is marked by a
line. Potential N-linked glycosylation sites are
shaded. A putative Zn2+ binding domain is marked
within open boxes, and residues important in a putative
/
hydrolase catalytic site are within shaded circles.
Base pairs are numbered in the right margin.
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Fig. 3.
Alternative splicing of NAALADase L. Amino acid sequence for NAALADase L is shown. Sites where putative DNA
sequences are spliced out are marked by an arrow with the
resulting (in-frame) amino acid deletions highlighted in bold
italics. Sites of putative intronic DNA insertion are marked by
triangles, with the intronic DNA sequence shown
above. Resulting changes to the amino acid sequence are
highlighted in bold italics. Amino acid residues are
numbered in the right margin.
/
hydrolase
fold family of proteins (16).
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Fig. 4.
Alignment of the predicted protein sequences
for human NAALADases I, II, and L. The amino acid sequences were
aligned using the ClustalW alignment program. Amino acid residues
identical to all three proteins are shaded in
black. Amino acid residues identical to two of the proteins
are shaded in gray. A putative Zn2+
peptidase domain is highlighted between arrows
and was identified by comparison to yeast and bacterial
aminopeptidases. Putative residues involved the catalytic site of the
/
hydrolase fold family of proteins are marked by three
arrows (nucleophile acid base). Amino acid residues are numbered
in the right margin.
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Fig. 5.
Phylogram of NAALADase I, II, and L. The
human and rat sequences were used, and the alignments were performed
with the ClustalW program. The tree was constructed using the GCG
Distances program with standard parameters and the Growtree program
with the UPGMA method.
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Fig. 6.
Alignment of the NAALADase peptidase domains
with related peptidases. Amino acid sequences were aligned using
the standard settings of ClustalW alignment program. Similar amino acid
residues conserved in proteins are shaded in
black. Similar amino acid residues conserved in 80% of the
proteins are shaded in dark gray. Similar amino
acid residues conserved in 60-79% of the proteins are
shaded in light gray. Amino acid residues are
numbered to the right. Putative residues involved in zinc
binding are marked by asterisks. The base residue thought to
be important in catalysis is marked by an arrow. Sequence
names other than NAALADases correspond to sequence accession numbers in
Swiss-Prot and SPTREMBL; Ape 3 yeast,
Saccharomyces cervisiae aminopeptidase Y; P96152,
Vibrio cholerae aminopeptidase; Ampx vibpr,
Aeromonas proteolyitca aminopeptidase; Apx strgr,
Streptomyces griseus aminopeptidase. Putative residues
involved in zinc binding are marked by asterisks. A general
base residue thought to be important in catalysis is marked by an
arrow. Amino acid residues are numbered in the right
margin.
Expression and Functional Activity of NAALADases
To determine if the newly identified NAALADases had peptidase
activity, mammalian expression constructs were transiently transfected into COS cells and cellular homogenates prepared. Expression of NAALADase I in COS cells was performed as a positive control to establish the working conditions of the assay, and homogenates from
mock transfections were used in parallel as negative controls. Hydrolysis of [3H]NAAG by recombinant NAALADases,
measured by elution of [3H]glutamate, occurred in a
time-dependent manner. Activity was observed in homogenates
from NAALADase I and II but not NAALADase L transfections (Fig.
7A). Addition of 30 µM quisqualic acid to the reaction inhibited this
activity by over 50% after 60 min (data not shown). Inhibition curves
with increasing concentrations of QA gave IC50 values of
1.2 × 105 and 1.7 × 10
5
M for NAALADase I and II, respectively (Fig.
7B).
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DPP IV-like activity was assayed using homogenates from COS cells transiently transfected with a particular NAALADase cDNA. Enhanced DPP IV-like activity was observed in all samples tested when compared with mock-transfected cells. NAALADase I and II showed the higher levels of DPP IV activity than NAALADase L in repeated experiments (Fig. 7C).
Chromosomal Localization
The complete coding sequence of NAALADase I was used as a probe for FISH analysis. Under the conditions used, the hybridization efficiency was approximately 71% for this probe (among 100 checked mitotic figures, 71 of them showed signals on one pair of the chromosomes). DAPI banding was used to identify the specific chromosome, and an assignment between the signal from the probe and the short arm of chromosome 11 was made. The detailed position to region p11.21 was further determined based upon summary data from 10 photographs. A weak hybridization signal was also detected in the region of 11q14.3 with low frequency. From the mapping data obtained, it was concluded that this weak signal was a result of cross-hybridization to NAALADase II. Examples of the mapping results are presented in Fig. 8, A and B. For NAALADase II the hybridization efficiency was approximately 74%, and DAPI banding was used to identify the signal to human chromosome 11, region q14.3-q21 (Fig. 8, C and D). For NAALADase L the hybridization efficiency was approximately 71%, and DAPI banding was used to identify the signal to the long arm of chromosome 11 region q12 (Fig. 8, E and F).
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Tissue Distribution of NAALADase II as Determined by Northern Blot
Northern blot analysis was performed on mRNA derived from
different human tissues (Fig. 9). A
NAALADase II-specific probe indicated the presence of transcripts in
testis ovary, spleen > prostate gland, heart, and placenta
with no signal observed in other tissues. In testis, four transcripts
were represented. The most predominant transcript was of approximately
3.4 kb, consistent with the approximate expected size of a NAALADase II
message. Two transcripts of 2.4 and 4.4 kb, respectively, and a weaker transcript of about 7.5 kb were also present. In the other tissues the
3.4-kb transcript was the only signal detected, apart from ovary where
a weak 7.5-kb signal could also be seen. The precise nature of these
transcripts awaits further elucidation but may be due to alternative
splicing of the message.
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Analysis of NAALADase Gene Expression by RT-PCR
To examine the detailed tissue distribution of all NAALADases, PCR
was performed on normalized cDNAs from 16 different tissues. Fig.
10A shows the results from
PCR reactions performed with NAALADase I-specific primers, yielding
amplification products of the expected size (~341 bp). The highest
expression of NAALADase I appeared to be in prostate gland. Rank order
of expression after 25 cycles was prostate liver and kidney > small intestine > brain and spleen, with no product
amplification observed in the other tissues. At 30 cycles amplification
products could be seen in most other tissues with the exception of
muscle, blood, and thymus in which products could only be observed
after 35 cycles of amplification. NAALADase II-specific primers yielded
a 353-bp amplification product of the expected size (Fig.
10B). NAALADase II expression was highest in ovary, testis,
and spleen with PCR products detected after 25 cycles of amplification.
After 30 cycles amplification products could be detected from all
tissue cDNAs apart from lung, muscle, blood, and thymus in which a
product could only be seen after 35 cycles. These results are in good
accord with the expression data obtained with the multiple tissue
Northern blots. NAALADase L-specific primers yielded a 330-bp
amplification product of the expected size, as well as two products
migrating with slightly higher sizes of 420 and 500 bp (Fig.
10C). NAALADase L expression was highest in small intestine,
spleen, and testis with PCR products detected after 25 cycles of
amplification, whereas products in heart, ovary, colon, blood, and
prostate could be seen after 30 cycles. Some amplification products
following 35 cycles were observed in all tissues, with brain and muscle
showing the lowest levels. The 420- and 500-bp bands were due to
amplification of NAALADase L sequences containing one or two intronic
sequences that were commonly found in all our amplification reactions
(see above). Control amplification reactions using GAPDH-specific
primers demonstrated comparable levels of amplification products for
each cDNA (Fig. 10D). Comparison of the relative
abundance between the four messages was also possible from these
experiments, since the same cDNAs were used for each set of
amplifications. Abundance of NAALADase I message was greater than
NAALADase II which was greater than NAALADase L, as judged by the
relative amount of amplification products detected at 25 and 30 cycles.
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PCR reactions using the same NAALADase primers as in the above experiments were performed on 13 different brain cDNAs normalized to the expression levels of three housekeeping genes. NAALADase I-specific amplification products were detected with highest levels in ventral striatum and brain stem after 30 cycles. After 35 cycles NAALADase I-specific amplification products could clearly be detected in all brain areas studied (Fig. 11A). NAALADase II-specific primers yielded a 353-bp amplification product of the expected size (Fig. 11B). Amplification products were observed after 30 cycles in striatum, parietal cortex, and ventral striatum with lower levels of amplification product detected in hippocampus, brain stem, putamen, and superior colliculus. After 35 cycles the presence of NAALADase II-specific products could be detected in all cDNAs apart from inferior colliculus. NAALADase L-specific primers yielded a 330-bp amplification product of the expected size, as well as a product migrating at a higher size of 500 bp (Fig. 11C). Amplification of the 500-bp product was observed after 35 cycles in brain stem, amygdala, thalamus, ventral striatum, and to a lesser extent in striatum and hippocampus, whereas the expected 330-bp product was only seen in brain stem and ventral striatum. Control amplification reactions using GAPDH-specific primers demonstrated comparable levels of amplification products for each cDNA apart from brain stem which yielded relatively more GAPDH-specific product (Fig. 11D). Overall expression of NAALADase L appears to be lower in these brain areas relative to NAALADase I and II.
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Finally, NAALADase expression was investigated in cDNAs prepared from either prostate tumor cell lines or prostate tumor tissue that had been normalized against three different housekeeping genes. NAALADase I expression was highest in LNCaP and prostate tumor (Fig. 12A). Amplification products were also detected in PC-3 cDNA after 30 and 35 cycles but not in DU145 cDNA. NAALADase II expression was higher in LNCaP than prostate tumor. A faint amplification product could also be detected in PC-3 after 35 cycles but not in DU145 cDNA (Fig. 12B). The 330-bp NAALADase L product was detected in highest amounts in cDNA from prostate tumor and less in PC-3 and DU145 samples after 35 cycles (Fig. 12C). Interestingly, in all samples apart from prostate tumor, the higher 500-bp amplification product could be detected. Representative amplifications with GAPDH primers are also shown (Fig. 12D).
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DISCUSSION |
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In this report we describe the cloning of two novel human
NAALADase peptidases that represent an expansion of the glutamate carboxypeptidase II gene family and which we have called NAALADase II
and NAALADase L. Both peptidases contain a single hydrophobic region
which is a likely membrane spanning a short intracellular domain and a
large globular extracellular domain, typical of type II integral
membrane proteins and common among membrane-bound hydrolases (23).
Carboxypeptidase activity of NAALADase I has been demonstrated against
two classes of substrate in vitro, -linked acidic
peptides such as NAAG or
-glutamylglutamate and
-linked peptides
such as folyl-poly-
-glutamate or
-glutamylglutamate. In brain
preparations, the QA-sensitive
-linked hydrolysis of NAAG was shown
to be increased by divalent cations and inhibited by divalent metal
chelators or general metalloprotease inhibitors (9, 24). In this study
we have transiently expressed NAALADase I, II, and L in COS cells, and
we have shown that NAALADase I and II but not NAALADase L confer
NAALADase activity to the cell homogenates comparable to that described
for lysed synaptosomal membranes (9). Alignment of NAALADase I with
zinc aminopeptidases from yeast and bacteria identified a domain of
conserved sequences involved in the coordination of two zinc ions
located in the catalytic site (12, 25). This catalytic site was
identified from the three-dimensional crystal structure of the
Aeromonas proteolytica zinc aminopeptidase, in which
residues His379, Asp389, Glu427,
Asp455, and His555 are thought to be involved
in the binding of two zinc ions, and Glu426 is proposed to
be a base residue important in catalysis (26, 27). Alignment of the
newly identified NAALADases with these aminopeptidases shows that the
putative zinc binding domains, including the five residues important in
Zn2+ binding, are highly conserved, suggesting that they
may share the same catalytic domain (Fig. 4 and Fig. 6). Shneider and
colleagues (16) have suggested that rat NAALADase L may also be a
member of the
/
hydrolase fold family because of its DPP IV and
acylaminoacylpeptidase-like activity, although sequence homology
alignments have shown these protein sequences to be clearly distinct.
However, an hypothesized catalytic site arrangement of nucleophilic,
acidic, and basic residues (Ser623, Asp663, and
His686) is conserved and found in NAALADase I, II, and L,
downstream of the predicted zinc binding domain (Fig. 4). In support of
this, transient transfection experiments with each NAALADase cDNA
did consistently confer enhanced DPP IV activity to cellular
homogenates as demonstrated by their ability to cleave the substrate
Gly-Pro-AMC. DPP IV activity is an abundantly expressed serine
peptidase activity. The physiological role of such an enzymatic
activity is not clear, but it may play a role in the regulation of
various biologically active peptides such as collagen, neuropeptide Y,
and growth hormone releasing factor both in the intestine and in other
organs (16). With abundant NAALADase expression in a number of
different tissues, it will be interesting to identify which of these or
other biologically important peptides can be cleaved by these peptidases.
The second -linked enzymatic activity of NAALADase I to peptides
such as pteroyl-
-glutamate (folate hydrolase activity) has been
found at high levels in the brush border of human small intestine (15,
28). In addition, carcinoma cells transfected with NAALADase I show
increased folate hydrolase activity and the ability to progressively
liberate glutamate from methotrexate triglutamate by hydrolysis of
-glutamyl linkages (15). A correlation has been observed between
increased pteroyl hydrolase activity and methotrexate resistance in
tumors (29), suggesting that modulating NAALADase activity may be
useful in developing improved or novel cancer treatments. It will be
interesting to see if human NAALADase II or L with their common
secondary structure have a dual peptidase activity similar to that of
NAALADase I.
Analysis of NAALADase L sequences using different PCR primers sets
revealed the presence of multiple splice variants and isoforms. Changes
in sequence due to alternative splicing may affect levels of
glycosylation and more importantly the conformation and activity of the
protein. Furthermore, inclusion of three different intronic sequences
identified in amplification reactions from numerous cDNAs results
in either the in-frame addition of a proline-rich (51-amino acid
residue) sequence close to the putative zinc binding domain or in
frameshifts and premature termination of the peptidase. These premature
terminations result in either partial deletion of the zinc binding
domain or in elimination of the predicted nucleophile acid base
arrangement of the putative /
hydrolase catalytic site (see Fig.
3). Finally, insertion of two intronic DNA sequences (at bp 1697 and
1870), resulting in frameshifts and premature protein termination, was
identified in all cDNAs studied suggesting that these are not
artifactual in nature due to contaminating genomic DNA, for example. It
is possible that expression of these sequences may be used to regulate
the levels of active protein. In addition, analysis of NAALADase II
tissue distribution by Northern hybridization revealed a number of
differently sized transcripts, suggesting the presence of multiple
isoforms. It remains to be determined whether these are due to
physiologically relevant smaller transcripts or due to problems with
the integrity of the mRNA. Future studies using different splice
variants will help us understand the structure/activity relationship of
NAALADase L and NAALADase II, and help to identify which domains are
important for enzymatic activity. Interestingly, a splice variant of
NAALADase I, lacking the first 40 amino acids including the membrane
spanning domain, has been observed at decreased ratios relative to its full-length transcript in malignant prostate tissues, suggesting that
expression of this alternative splice variant may correlate with tumor
progression (30). Further work will determine if NAALADase II or
NAALADase L is involved in oncogenesis or if alternative splicing of
these peptidases has any role to play in tumor progression.
Using FISH analysis to determine the chromosomal localization of NAALADase I, II, and L, we observed a signal on chromosome 11, at p11.21 for NAALADase I, between q14.3-q21 for NAALADase II and q12 for NAALADase L. In our studies, a NAALADase I probe revealed two hybridization signals, one hybridizing at 11p11.21 and another weakly at 11q14.3. This is similar to the results obtained by Leek et al. (31) who observed two hybridization signals at 11p11.2 and 11q13.5. Having localized NAALADase II to 11q14.3-q21, it is clear that the second signal is due to cross-hybridization of the NAALADase I probe with the NAALADase II locus. Chromosome 11 contains a number of genetic disease loci in these regions, including vitreoretinopathy (11q13-q23), xeroderma pigmentosa (11q12-q13), atopy (11q12-q13), and perhaps more interestingly, a tumor suppression locus (11p11.2-p11.13) involved in rat prostate carcinoma. Introduction of this portion of the chromosome into highly metastatic rat prostatic cells was able to suppress cancer metastases without suppression of the in vivo growth rate or tumorigenicity of the cells (32). Since it has been shown that NAALADase I expression increases with decreasing androgen levels, it is possible that current prostate cancer treatments involving androgen level reduction (e.g. orchidectomy) may work at least in part through alteration of NAALADase expression (33). The in vivo enzymatic activity of NAALADase I may be similar to that of NAALADase II and L, so it is conceivable that these enzymes may also have a role to play in tumor suppression. Interestingly, region 11q13-q23 has also been identified as a region with tumor suppressor activity using tumorigenic HeLa/fibroblast hybrids (34). In addition, in a systematic analysis of primary cervical carcinomas, region 11q22-q24 was shown to contain tumor suppressor activity (35). These latter two tumor-suppressing regions on the long arm of chromosome 11 cover the gene loci of NAALADase L and NAALADase II. It should be noted that mapping of these tumor-suppressing activities to these three chromosomal regions in no way establishes that the identified NAALADases are capable of having any tumor or metastasis-suppressing activity. Furthermore, it has been suggested that expression of NAALADase I is related to an increase in cancerous phenotype, suggesting an oncogenic rather than a tumor-suppressing role for NAALADase I (33). Given the fact that NAALADase II and NAALADase L also appear to be expressed in a number of prostate tumor cell lines as well as in prostate tumor tissue, it will be interesting to see if these two genes will be implicated in prostate tumor development.
In this study, NAALADases II has been shown to be able to hydrolyze [3H]NAAG to N-acetylaspartate and glutamate as has been previously shown with NAALADase I. Given the localization of these NAALADases in prostate and ovary, as well as other peripheral tissues, it is quite possible that these enzymes may modulate local extracellular glutamate levels in these tissues. For example it is known that substantial amounts of glutamate are present in seminal fluid. As endogenous levels of NAAG are high in brain, the catabolism of NAAG by NAALADases to glutamate and N-acetylaspartate may in theory be a rich source of glutamate. Whether in fact the inhibitory action of NAAG or its excitatory metabolite glutamate is the active species at particular synapses will be dependent upon the receptors present and factors regulating the expression of different NAALADases. However, aberrant catabolism of NAAG by NAALADases to release excessive levels of glutamate may well result in activation of numerous glutamate receptor subtypes, resulting in an excessive positive modulation of glutamatergic neurotransmission and excitotoxicity. The possible importance of careful regulation of NAALADase activity therefore becomes apparent if deleterious excitotoxic effects are to avoided. Indeed abnormal levels of NAAG or NAALADase activity have been suggested for a number of disorders including schizophrenia (36), ALS (37), Alzheimer's disease (38, 39), seizure disorders (40, 41), and stroke (42). It is clear from our studies that the NAALADase genes are differentially expressed within discrete brain areas at the mRNA level, but whether there are any disease-specific changes in NAALADase gene expression remains to be determined. In addition, molecules able to modulate the activity of specific NAALADases may be potentially useful in treating ischemia-induced neurodegeneration or other neurodegenerative disorders involving abnormalities in glutamate neurotransmission, such as Alzheimer's disease, schizophrenia, or amyotrophic lateral sclerosis (43). In vitro at least, the NAALADase (carboxypeptidase) inhibitor, 2-(phosphonomethyl)pentanedoic acid, inhibited toxicity induced by the carboxypeptidase cleavage of folic acid hexaglutamate (44).
In summary we have identified and characterized two novel human
peptidases NAALADase II and NAALADase L. With several biological roles
suggested for NAALADases, including regulation of glutamatergic neurotransmission and prostate tumor progression, it will be
interesting to see what role individual members of this newly expanded
protein family will play within these systems. Further work in the
understanding of the relative biological importance of each of these
proteins and elucidation of possible physiological substrates will help researchers to identify more specific, small molecule NAALADase inhibitors which may be of use in a number of clinically important disorders.
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ACKNOWLEDGEMENTS |
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We thank Professor R. C. A. Pearson for providing brain RNA samples and Dr. B.-J. Van Der Leede for providing prostate tumor RNA samples. We also thank Joerg Sprengel for valuable advice in bioinformatics and sequence analysis. Petra De Wilde and Nathalie Delcroix provided technical assistance for the sequencing reactions and Annemie Heylen helped with preparation of the figures.
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FOOTNOTES |
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* 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) HSA012370, AJ012370, HAS012371, AJ012371.
§ To whom correspondence should be addressed. SmithKline Beecham Pharmaceuticals, Neuropharmacology Research, Harlow, Essex CM19 5AW, U. K.; E-mail: menelas_n_pangalos{at}sbphrd.com.
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ABBREVIATIONS |
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The abbreviations used are:
NAAG, N-acetyl-L-aspartyl-L-(3,4)-glutamate;
EST, expressed sequence tag;
FISH, fluorescent in situ
hybridization;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
NAALADase, N-acetylated -linked acidic dipeptidase;
ORF, open reading frame;
DPP IV, dipeptidyl dipeptidase IV;
bp, base pair(s);
PCR, polymerase chain reaction;
PSM, prostate-specific
membrane antigen;
QA, quisqualate;
5'-RACE, 5'-rapid amplification of
cDNA ends;
RT-PCR, reverse transcription-polymerase chain reaction;
SDM, site-directed mutagenesis;
AMC, 7-amino-4-methylcoumarin;
mGluR, metabotropic glutamate receptor.
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REFERENCES |
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