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INTRODUCTION |
Cellular and secreted carboxypeptidases are important in the
generation, processing, and inactivation of different vertebrate neuropeptides (for recent reviews, see Refs. 1-7). In particular, an
important brain glutamate carboxypeptidase II (EC 3.4.17.21) also
called N-acetyl-aspartyl-
-glutamate carboxypeptidase
(NAALADase)1 (8, 9)
hydrolyzes the neuropeptide,
N-acetyl-L-aspartyl-L-glutamate, releasing glutamate, the dominant excitatory
neurotransmitter/neuromodulator of the mammalian central nervous system
(10, 11). NAALADase is also required for the intestinal uptake of
folate and is responsible for the resistance of some tumors to
methotrexate (12). Cloning of NAALADase (12-14) showed that it is
identical to the so-called prostate-specific membrane antigen (PSMA),
which is strongly expressed in prostate cancer and serves as a marker
for detection of prostatic cancer metastasis (reviewed in Ref. 15).
NAALADase is a 94-kDa transmembrane protein, homologous both to the
transferrin receptor and the bacterial cocatalytic zinc
metallopeptidases (16). Recent cloning of the human ileal membrane
dipeptidyl peptidase, I100 (17), the catalytic domain of which is
homologous to NAALADase/PSMA, revealed that in mammals, cocatalytic
zinc metallopeptidases may be represented by multiple enzymes with
different substrate specificity.
We describe a new human peptidase of the NAALADase/PSMA family, a
56-kDa blood plasma glycoprotein termed PGCP, which was affinity-co-purified with the lysosomal carboxypeptidase, cathepsin A
(CathA; EC 3.4.16.1). CathA forms a high molecular weight complex with
-galactosidase (EC 3.2.1.23), sialidase (neuraminidase; EC
3.2.1.18), and N-acetylgalactosamine-6-sulfate sulfatase (EC
3.1.6.4) that is essential for their function in the lysosome (18-20).
About two-thirds of lysosomal CathA is not complexed with
-galactosidase and sialidase (21) and can be excreted into plasma
from platelets (22) and lymphoid cells (23). Our recent studies on
CathA substrate specificity (24, 25) indicated that CathA is the major
carboxypeptidase in human tissues able to cleave hydrophobic amino acid
residues, including the amidated ones. In vitro, CathA can
inactivate regulatory peptides like endothelin, substance P,
bradykinin, and angiotensin I (22, 23, 26-29), suggesting that it can
be involved in the regulation of central neural and cardiovascular
functions. Our recent studies propose that CathA may be associated with
the metabolism of peptides implicated in memory consolidation (30).
For purification of CathA, we designed an affinity column by coupling a
specific substrate of CathA, Phe-Leu, to an epoxy-activated agarose
matrix. Analysis of the protein fraction purified from human tissues
using this column revealed a novel 56-kDa glycoprotein, PGCP. In this
paper, we report cloning of PGCP cDNA; characterization of its
primary structure, enzymatic activity, expression, and metabolism; and
its immunolocalization in human tissues and cells, which altogether
suggest that PGCP is a novel glutamate carboxypeptidase secreted into
the blood plasma.
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EXPERIMENTAL PROCEDURES |
Purification of PGCP from Human Placenta--
A crude
glycoprotein fraction was purified from 10 human placentas (~5 kg of
wet tissue weight) using a concanavalin A-Sepharose affinity column
(21). The preparation was passed through a
p-aminophenyl-
-D-thiogalactopyranoside-agarose affinity column to separate the
-galactosidase-CathA-sialidase complex and then applied onto a 20-ml Phe-Leu-agarose affinity column, which specifically binds CathA and cathepsin D (CathD) (21).
The column was washed with 20 volumes of 20 mM sodium acetate buffer, pH 4.75, containing 0.15 M NaCl, and eluted
with 5 volumes of 0.1 M Tris-HCl buffer, pH 7.5, containing
5 mM benzyloxycarbonyl-Phe-Leu. The eluate was concentrated
using an Amicone stirred cell with a PM-10 membrane, dialyzed against
10 mM Tris-HCl buffer, pH 7.5, centrifuged at 10,000 × g for 10 min, applied to an FPLC Mono Q column (Amersham
Pharmacia Biotech) equilibrated with the same buffer, and eluted with a
linear NaCl gradient from 0 to 0.4 M. The 0.075-0.1
M NaCl fractions, which contained cathepsin D and PGCP,
were pooled, concentrated, applied to a FPLC Superose 12 gel filtration
column (Amersham Pharmacia Biotech), and then eluted in 20 mM sodium acetate buffer, pH 5.2, containing 0.15 M NaCl and 0.02% (w/v) NaN3. Eluted fractions
were analyzed by SDS-PAGE. Fractions corresponding to the first protein
peak, containing only PGCP, were pooled, concentrated, and stored at
30 °C. The molecular masses of the eluted proteins were determined
using the following molecular mass standards (Amersham Pharmacia
Biotech): blue dextran (~2,000 kDa), ferritin (440 kDa), catalase
(232 kDa), aldolase (158 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa), and ribonuclease A (13.7 kDa). A similar procedure was used for the purification of PGCP from mouse liver.
N-terminal Amino Acid Sequencing of Proteins--
SDS-PAGE was
performed as described by Laemmli (31) on an 11% acrylamide gel.
Protein bands were stained with Coomassie Blue. For
NH2-terminal sequencing, the proteins were
electrotransferred to ImmobilonTM-P membrane (Millipore
Corp.) in a Trans-Blot cell (Bio-Rad) for 2.5 h at 4 °C in 10 mM CAPS buffer, pH 11, 10% (v/v) methanol at a current of
480 mA. After staining with Coomassie Blue R-250, the bands were
excised, and amino acid sequences were determined in an Applied
Biosystems 470 A gas phase sequencer. Sequences were analyzed using the
BLAST network service of the National Center for Biotechnology
Information (Bethesda, MD).
EST Clone of PGCP--
The dbEST was screened with the
NH2 terminal amino acid sequence of PGCP,
DVAKAIINLAVYGKAQ(N)RSYERLALLVDTVG, using the NCBI pBLAST search engine.
Clone 30142, which contained deduced amino acid sequence identical to
the NH2-terminal sequence of PGCP (GenBankTM
accession number R18560), was supplied by the IMAGE Consortium, Human
Genome Center (Livermore, CA). The 1.62-kilobase pair PGCP cDNA
insert was excised by HindIII/NotI digestion,
subcloned into pBluescript (Stratagene), and sequenced using the PRISM
Ready Reaction Dye Deoxy Terminator cycle sequencing kit on an Applied Biosystems 373A automated sequencer. Conditions for sequencing were 25 cycles at 96 °C for 30 s, 50 °C for 15 s, and 60 °C
for 4 min. Products were purified by size exclusion chromatography on
CENTRI SEP columns (Princeton Separations) and fractionated by
electrophoresis on a 6% denaturing (8.3 M urea)
polyacrylamide gel. Sequences were analyzed using the SeqED software.
cDNA Cloning of PGCP--
A 5'-PGCP cDNA subclone was
obtained by 5'-rapid amplification of cDNA ends polymerase chain
reaction from total human placenta RNA using a MarathonTM
cDNA amplification kit (CLONTECH) and an
oligonucleotide primer, 5'-CCAATCGCTCATAGGATCTGTTCTG-3', complementary
to the PGCP cDNA sequence (nucleotides 178-202). The product was
cloned into a pCRII vector (Invitrogen) and sequenced. Analysis of the
amplified sequence and its comparison with sequence of EST clone 30142 and several other EST clones containing fragments of the PGCP cDNA showed that clone 30142 contained a complete cDNA of PGCP with the
exception of the first nucleotide of the initiation ATG codon.
To complete a PGCP cDNA at 5', we amplified a 400-kilobase
pair 5'-end fragment using a
5'-CGATAAGCTTGCGGCCGCACGAGGCATGAA-3' primer,
which contains the PGCP initiation methionine codon (underlined) plus
25 nucleotides of upstream sequence with HindIII and
NotI sites (boldface type), a 5'-ACCCAGGATGGCTATCTTAT-3'
primer, and the pBluescript-PGCP vector as a template. The
fragment was digested with HindIII and EcoRI and
cloned into pBluescript-PGCP, from which the
HindIII-EcoRI cassette had been excised, and the
insert of the resulting construct (pBluescript-PGCPc) was completely sequenced on each strand as above.
RNA Isolation and Northern Blots--
Human skin fibroblasts
from normal controls were cultured to confluency in Eagle's minimal
essential medium (Mediatech, Washington, D. C.) supplemented with 10%
(v/v) of fetal calf serum (MultiCell) and antibiotics. Total RNA was
isolated from the cell pellet or from human placenta tissue by
ultracentrifugation in a CsCl2 gradient as described by
Maniatis et al. (32).
Purified RNA was analyzed by Northern blot as described (32). A
HindIII/NotI PGCP cDNA fragment from clone
31259 was labeled with [32P]dCTP using the Oligolabeling
Kit (Amersham Pharmacia Biotech) and used as a hybridization probe.
Antibodies--
Rabbit polyclonal antibodies against PGCP were
prepared as follows. A 577-kilobase pair fragment of PGCP cDNA
spanning codons 127-320 was obtained by
EcoRI/BglII digestion. After blunting with Klenow
fragment DNA polymerase, the fragment was inserted into the
SmaI site of pGEX-2T vector (Amersham Pharmacia Biotech). The resulting plasmid was expressed in Escherichia coli
801-C to produce PGCP-glutathione S-transferase fusion
protein. The fusion protein was purified from the bacteria homogenate
by affinity chromatography on glutathione-Sepharose (Amersham Pharmacia
Biotech) as described by the manufacturer. Purified fusion protein (1 mg), homogeneous by SDS-PAGE analysis, was used to immunize a rabbit. IgG fraction purified from the antiserum by ammonium sulfate
fractionation was passed through a recombinant glutathione
S-transferase-Sepharose column to absorb the
anti-glutathione S-transferase-specific antibodies. The
resulting antibody preparation was used in a dilution of 1:10,000 or
1:15,000 for Western blotting, 1:500 for immunoelectron, and 1:200 for
immunofluorescent microscopy.
Western Blotting--
Cellular or tissue homogenates,
concentrated cellular medium, blood serum, or purified preparation of
PGCP, was subjected to SDS-PAGE and electrotransferred to a NITRO ME
nitrocellulose membrane (Micron Separations Inc., Westboro, MA). The
detection of protein bands cross-reacting with anti-PGCP antibodies was performed using the BM Chemiluminescence kit (Boehringer Mannheim) in
accordance with the manufacturer's protocol.
The deglycosylation of proteins was performed by treatment of 20-µg
samples with 2 units of endoglycosidase F (Sigma) in 20 mM
sodium phosphate buffer, pH 7.0, containing 0.1% (w/v) SDS, 0.2%
(w/v) dithiothreitol, and 0.5% (v/v) Triton X-100 for 17 h at
37 °C.
Expression of PGCP in COS-1 Cells--
The full-length PGCP
cDNA was obtained by NotI restriction of
pBluescript-PGCPc and cloned into the NotI site of pCMV
expression vector (33), kindly provided by Dr. S. G. MacGregor and
Dr. T. Caskey (Baylor College of Medicine, Houston).
COS-1 cells were transfected with pCMV-PGCP expression vector using
Lipofectamine (Life Technologies Inc.) in accordance with the
manufacturer's protocol. 24, 48, and 72 h after transfection, different peptidase activities and control
-hexosaminidase activity were assayed in cell homogenates and medium.
Immunofluorescent Microscopy--
Cultured human skin
fibroblasts of normal controls were fixed on the glass slides with
acetone/methanol (4:1) at
20 °C, washed in phosphate-buffered
saline, blocked for 1 h with 2% (w/v) bovine serum albumin in
phosphate-buffered saline, incubated with anti-PGCP antibodies at a
final dilution of 1:200 for 1 h at room temperature, washed with
phosphate-buffered saline, and further incubated for 30 min with
rhodamine-conjugated goat anti-rabbit IgG at a dilution of 1:100.
Epifluorescent microscopy was performed using a Zeiss Axioskop microscope.
Immunoelectron Microscopy--
Cultured human skin fibroblasts
were detached from the culture dishes with a rubber policeman, washed
with Hanks' balanced salt solution, and fixed in 4% paraformaldehyde,
0.5% glutaraldehyde in 50 mM phosphate buffer, pH 7.5. The
cell pellets were dehydrated in methanol and embedded in Lowicryl K4
M as described previously (34, 35).
Ultrathin Lowicryl sections were mounted on 300-mesh Formvar-coated
nickel grids (Polysciences, Inc., Warrington, PA). Each section was
incubated for 15 min in 20 mM Tris-HCl-buffered saline (TBS), containing 0.1% (v/v) Tween 20 as well as 15% (v/v) goat serum
and for 30 min with anti-PGCP antibodies diluted 1:500 in TBS. The
sections were then washed four times with TBS containing 0.05% (v/v)
Tween 20, incubated for 15 min in TBS containing 15% (v/v) goat serum,
and for 30 min with colloidal gold (10 nm)-conjugated goat anti-rabbit
IgG (Zollinger Inc., Montréal, Québec, Canada). The
sections were washed two times with TBS containing 0.05% (v/v) Tween
20 and two times with distilled water and counterstained with uranyl
acetate followed by lead citrate (34, 35). Normal rabbit serum was used
as a control. Electron micrographs were taken using a Philips 400 electron microscope (Philips Electronics, Toronto, Ontario, Canada).
Metabolic Labeling--
48 h after transfection with pCMV-PGCP
expression vector, COS-1 cells grown to confluency in
75-cm2 culture flasks (~106 cells) were
washed twice with Hanks' balanced salt solution and then incubated for
2 h in methionine-free Dulbecco's modified Eagle's medium (Life
Technologies) supplemented with L-glutamine and sodium
pyruvate, and for 40 min in 5 ml of the same medium supplemented with
[35S]methionine (DuPont), 0.1 mCi/ml. The radioactive
medium was then removed, and the cells were washed twice with Hanks'
balanced salt solution and chased at 37 °C in Eagle's minimal
essential medium supplemented with 20% (v/v) fetal calf serum.
At the times indicated in the figures, the cells were placed on ice,
washed twice with ice-cold phosphate-buffered saline, and then lysed
for 30 min on ice in 1 ml of radioimmunoprecipitation assay buffer,
containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 5 µg/ml leupeptin, and 0.1 mM
-toluenesulfonyl
fluoride. The lysate was collected and centrifuged at 13,000 × g for 10 min to remove the cell debris.
Immunoprecipitation, Electrophoresis, and Detection of
PGCP--
1.0 ml of lysate was incubated for 4 h with preimmune
serum at a final dilution of 1:20. Then the pellet obtained from 300 µl of Pansorbin cells (Calbiochem) was added, and the resulting suspension was incubated for 2 h at 4 °C, followed by
centrifugation for 10 min at 13,000 × g. Supernatants
were incubated overnight with the anti-PGCP antibodies in a 1:100 final
dilution and then for 2 h at 4 °C with the pellet from 100 µl
of Pansorbin cells and precipitated as above. The pellet was washed
three times with 1 ml of radioimmunoprecipitation assay buffer. The
antigens were eluted from the pellet by the addition of 100 µl of a
buffer containing 0.1 M Tris-HCl, pH 6.8, 4% (w/v) SDS,
20% (v/v) glycerol, 0.2 M dithiothreitol, and 0.02% (w/v)
bromphenol blue. The proteins were denatured by boiling for 5 min, and
50 µl of each sample were subjected to SDS-PAGE according to Laemmli
(31). The molecular weights were determined with
14C-labeled protein markers (Amersham Pharmacia Biotech).
The gels were fixed in acetic acid/isopropyl alcohol/water
(10/50/40), soaked for 30 min in AmplifyTM solution
(Amersham Pharmacia Biotech), vacuum-dried at 60 °C, and
analyzed by autoradiography.
Immunoaffinity Column Purification of PGCP--
Immunoaffinity
gel was prepared by coupling of 25 mg of anti-PGCP rabbit antibodies to
3 ml of BrCN-activated Sepharose 4B (Sigma) using the manufacturer's
protocol. The PGCP-containing fraction of human plasma proteins
obtained by ammonium sulfate precipitation (33-50% saturation) from
30 ml of serum was loaded on the immunoaffinity column. The column was
washed by 10 ml of 50 mM sodium phosphate buffer, pH 7.5, containing 0.4 M NaCl, and eluted with 5 ml of 0.2 M glycine buffer, pH 2.3. The pH of collected 1-ml
fractions was immediately adjusted to 7.5 by 1 M Tris-HCl buffer.
Enzyme Activity Assays--
CathA activity was measured as
described by Pshezhetsky et al. (24) with
benzyloxycarbonyl-Phe-Leu as a substrate; CathD activity was measured
according to the method of Barret (36) with bovine hemoglobin (Sigma)
as a substrate; and
-hexosaminidase activity was measured using
4-methylumbelliferyl-glucosaminide (37). Aminopeptidase, dipeptidyl
peptidase, endopeptidase, carboxypeptidase, and esterase activities
were assayed fluorometrically or spectrophotometrically as described
previously using the following substrates (all Bachem or Sigma):
H-Ala-AMC, H-Arg-AMC, H-Glu-AMC, H-Leu-AMC, H-Lys-AMC, H-Phe-AMC,
H-Gly-Phe-
NA, Z-Arg-Arg-
NA,
benzoyl-DL-Arg-pNA,
Z-Gly-Gly-Leu-pNA, Suc-Phe-Leu-Phe-pNA,
Suc-Leu-Tyr-AMC, Z-Phe-pNA, FA-Ple-Ala, FA-Phe-Arg, FA-Phe-Glu, FA-Phe-Gly, FA-Phe-Phe, and FA-Glu-Tyr. Glutamate carboxypeptidase activity was measured with Ac-Asp-Glu (Sigma) substrate as follows. The 100 µl of incubation mixture contained 1 mM Ac-Asp-Glu and 1-5 µg of sample protein in 100 mM Tris-HCl buffer, pH 7.5, containing 1 mM
ZnCl2. After 1-15 h of incubation at 37 °C, samples
were incubated for 3 min at 100 °C and mixed with 0.9 ml of 100 mM Tris-HCl buffer, pH 8.0, containing 1 mM NAD+ (Sigma) and 0.25 units/ml of bovine glutamate
dehydrogenase (Boehringer Mannheim). After a 1-h incubation at
37 °C, the concentration of glutamate was measured
spectrophotometrically at 340 nm using the calibration curve obtained
with 1-200 nmol of glutamate. One unit of enzyme activity corresponds
to a conversion of 1 µmol of substrate/min. Proteins were assayed
according to Bradford (38) with bovine serum albumin (Sigma) as a standard.
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RESULTS AND DISCUSSION |
Purification and NH2-terminal Sequencing of
PGCP--
The fraction of crude glycoproteins of human placenta
retained by the Phe-Leu-agarose affinity column contained four major proteins. We separated these proteins by FPLC ion exchange
chromatography using Mono Q column (Fig.
1) and identified three of them by
NH2-terminal sequencing as cathepsin D (CathD) (peak I
eluted at NaCl concentration between 0.057 and 0.1 M),
CathA (peak II, 0.1-0.17 M NaCl), and a so-called IP-30
(39), a 30-kDa glycoprotein induced in hematopoietic cells in response
to
-interferon, the biological function of which is unclear (peak
III, 0.27-0.35 M NaCl). Peak I in addition to 31- and
15-kDa subunits of CathD also contained a 56-kDa glycoprotein, further
termed PGCP. NH2-terminal amino acid sequence of PGCP, (G)DVAKAIINLAVYGKAQ(N)RSYERLALLVDTVG didnot match any of proteins in the Non-Redundant GenBankTM data base (all nonredundant
GenBankTM cDNA sequence translations, plus all entries from
the Protein Data Bak, Swiss-Prot, and PIR data bases).

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Fig. 1.
FPLC anion-exchange chromatography on Mono Q
column (a) and SDS-PAGE analysis (b)
of the proteins purified from human placenta using concanavalin
A-Sepharose and Phe-Leu-agarose affinity columns.
Dashed line, absorbance at 280 nm; , CathA
activity; , CathD activity; dotted line, NaCl
concentration. The indicated fractions were analyzed by SDS-PAGE, as
described. The protein bands are identified on the right of
the gel: 30- and 20-kDa polypeptide chains of CathA (CathA30 and
CathA20); 31- and 14-kDa polypeptide chains of CathD (CathD31 and
CathD14), -interferon inducible protein, IP-30, and PGCP.
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PGCP and CathD were resolved on a Superose 12 FPLC gel filtration
column. Eluted fractions were analyzed for CathD activity and by
SDS-PAGE (Fig. 2). Using a Superose 12 column, the molecular mass of PGCP (peak I at Fig. 2) was estimated as
120 kDa, suggesting that PGCP may form homodimers of 56-kDa
subunits.

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Fig. 2.
FPLC gel filtration on Superose 12 HR column
(a) and SDS-PAGE analysis (b) of the
FPLC Mono Q chromatography fractions corresponding to peak
I on Fig. 1a and containing PGCP and
CathD. Dashed line, absorbance at 280 nm;
, CathD activity. The positions of the elution peaks of the
molecular mass standards and void volume (V0)
are shown by arrows. The indicated gel filtration fractions
were analyzed by SDS-PAGE as described. The protein bands are
identified on the right of the gel: 31-kDa polypeptide chain
of CathD (CathD31) and PGCP.
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cDNA and mRNA of PGCP and Its Chromosomal
Localization--
A search in the dbEST data base showed that the
NH2-terminal amino acid sequence of PGCP had a 100%
identity with a deduced amino acid sequence of an EST clone 30142 (GenBankTM accession number R18560). From this cDNA
fragment, which we extended by 5' rapid amplification of cDNA ends
polymerase chain reaction, we derived a cDNA that we predict
contains the entire PGCP coding sequence (see "Experimental
Procedures"). This cDNA, confirmed by sequencing of several
independent clones, contains a 1623-base pair open reading frame
starting from a potential ATG initiation codon and 147 base pairs of
3'-untranslated region containing a polyadenylation site (Fig.
3). On Northern blots of mRNA from
human placenta and fibroblasts, the PGCP cDNA hybridized with a
single ~1.7-kilobase pair transcript, indicating that the acquired
cDNA was near full-length (Fig.
4).

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Fig. 3.
Nucleotide sequence of PGCP cDNA and
predicted amino acid sequence. The putative signal peptide is
boxed, and the NH2-terminal amino acid sequence
of the purified protein is underlined. Potential
glycosylation sites are marked with black dots,
and the terminal stop codon is marked with an
asterisk.
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Fig. 4.
Northern blot analysis of PGCP mRNA.
20 µg of total RNA from cultured human skin fibroblasts
(lane 1) and placenta (lane
2) were used. The membrane was hybridized with
32P-labeled PGCP cDNA probe as described under
"Experimental Procedures."
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Analysis of the deduced 541-amino acid sequence of PGCP using a SignalP
program (40) predicted that the first 24 amino acids may represent the
signal peptide (boxed in Fig. 3), with a typical positively charged
n-region (amino acids 1-3), a central h-region represented by a hydrophobic
-helix (amino acids 4-18), and a polar
c-region with a predicted cleavage site between
Cys24 and Lys25 (41). The
NH2-terminal sequence of the purified protein
(underlined in Fig. 3) starts at Asp45,
suggesting that amino acids 25-45 may represent a propeptide not
retained in mature PGCP. The sequence contains 5 potential N-glycosylation sites (indicated by black dots in
Fig. 3), which is consistent with the binding of PGCP to concanavalin
A-Sepharose.
Screening of the dbSTS library at GenBankTM with an
accession number R18560 for the EST clone revealed the identity of PGCP with an unidentified STS transcript WI-6164 (similar to Cda01e07, or
D8S1377E) encoded on human chromosome 8 between D8S257 and D8S270
markers (8q22.2 locus).
Expression and Metabolic Labeling of PGCP--
To study the
synthesis and processing of PGCP, we cloned its full-length cDNA in
the pCMV expression vector (33) and expressed it in COS-1 cells.
48 h after transfection, the total cell homogenate and
concentrated cell medium were studied by Western blot using antibodies
against a recombinant PGCP-glutathione S-transferase fusion
protein. We detected a 56-kDa protein that cross-reacted with anti-PGCP
antibodies in the homogenates of COS-1 cells transfected with pCMV-PGCP
expression vector but not in untransfected control cells or cells
transfected with pCMV-
-galactosidase vector (Fig. 5a). The molecular size of the
expressed protein was similar to that of PGCP purified from human
placenta (Fig. 5a, lane 1), suggesting that in COS-1 cells PGCP is properly processed and glycosylated. Western blot analysis of the culture medium (Fig. 5b) showed
that the majority of expressed PGCP is secreted. The treatment of the cells with 10 mM NH4Cl (19) did not increase
the secretion of PGCP, suggesting that mannose 6-phosphate receptors
are not involved in the trafficking of this protein.

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Fig. 5.
Expression and biogenesis of PGCP in COS-1
cells. a, Western blot of homogenates of COS-1 cells
harvested 48 h after transfection with pCMV-PGCP (lanes
2 and 3) or with pCMV- -galactosidase
(lanes 4 and 5) or of homogenates of
nontransfected cells (lanes 6 and 7).
Lane 1 contained 0.5 µg of purified PGCP.
Protein samples (20 µg for lanes 2,
4, and 6 and 10 µg for lanes
3, 5, and 7) were subjected to
SDS-PAGE, transferred to a nitrocellulose membrane, and stained with
rabbit anti-PGCP antibodies as described. b, Western blot of
culture medium of COS-1 cells 48 h after transfection with
pCMV-PGCP. Cells were cultured in the presence (lane
1) or in the absence (lane 2) of
mannose 6-phosphate. 20 µg of total protein were applied on each
lane. c, fluorographs of pulse labeling and chase of PGCP in
COS-1 cells 48 h after transfection with pCMV-PGCP expression
vector. Lane 1, pulse; lanes
2-5, chase for 1, 3, 6, and 22 h, respectively.
Molecular mass values of PGCP precursor (62 kDa) and its mature form
(56 kDa) are shown.
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30-h pulse-chase experiments (Fig. 5c) demonstrated that
PGCP is initially synthesized as a 62-kDa polypeptide, with a molecular weight consistent with that of the PGCP precursor. During a 6-h chase,
the precursor is completely processed to a 56-kDa mature form similar
to that detected by Western blotting in the cell lysates and in culture
medium (Fig. 5, a and b).
Tissue and Cellular Distribution of PGCP--
Western blot
analysis showed that the protein is unequally expressed in human
tissues (Fig. 6a). PGCP was
abundant in placenta and kidney; low in muscles, liver, and skin
fibroblasts; and undetectable in brain or white blood cells. Since the
highest level of PGCP was found in total blood, we performed a Ficoll
fractionation of blood and found that the protein is localized
exclusively in plasma (Fig. 6a).

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Fig. 6.
Detection of PGCP in human tissues and blood
fractions. a, Western blots of total homogenates of
placenta (lane 1), total brain (lane
2), kidney (lane 3), muscles
(lane 4), white blood cells (lane
5), fibroblasts (lane 6), liver
(lane 7), total blood from fetal umbilical cord
(lane 8), total venous blood (lane
9), blood plasma (lane 10), and red
blood cells (lane 11). 20 µg of protein were
applied on each lane. b, PGCP purified from human placenta
or blood plasma before ( ) or after (+) treatment with endoglycosidase
F.
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To characterize PGCP detected in blood plasma, we developed its rapid
purification procedure using an immunoaffinity column. The fraction of
blood serum proteins obtained by ammonium sulfate precipitation
(33-50% saturation) was loaded on the immunoaffinity column
containing anti-PGCP antibodies coupled to BrCN-activated Sepharose 4B.
The preparation eluted from the column with 0.2 M glycine
buffer, pH 2.3, contained only a 56-kDa protein that strongly
cross-reacted with anti-PGCP antibody and had an affinity to the
Phe-Leu-agarose column. Endoglycosidase F treatment of PGCP
preparations purified from human placenta using a Phe-Leu-agarose column and from human blood using an immunoaffinity column equally reduced their molecular mass to 52 kDa (Fig. 6b), close to
that predicted for the mature deglycosylated PGCP, suggesting that the
processing and glycosylation of PGCP secreted into blood serum is
similar to that of PGCP purified from placenta.
The intracellular distribution of PGCP was studied in skin cultured
fibroblasts by both immunohistochemistry and immunoelectron microscopy.
Fluorescent microscopy of fibroblasts revealed a peripheral punctate
intracellular staining consistent with localization of PGCP in a
visicular compartment (Fig. 7,
upper panel). Indeed, immunoelectron microscopy
of fibroblasts with anti-PGCP antibody (Fig. 7, lower
panel), revealed heavily labeled electron lucent vesicles of
approximately 150 nm in diameter that are located close to the plasma
membrane. Taking into the account that PGCP is found in blood plasma
and that most of PGCP from transfected COS-1 cells was secreted into
the culture medium, the results of the immunofluorescent and
immunoelectron microscopy can be interpreted as the localization of
PGCP in secretory granules.

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Fig. 7.
Localization of PGCP within cultured human
skin fibroblasts by immunofluorescence (upper
panel) and immunoelectron microscopy (lower
panel). Upper panel, fibroblasts
were fixed with cold acetone/methanol (4:1) and stained with anti-PGCP
rabbit antibodies, followed by staining with rhodamine-conjugated goat
anti-rabbit IgG antibodies. Magnification was × 400. Lower panel, a, electron luscent
vesicles close to the plasma membrane are heavily labeled with 15-nm
gold particles after incubation with anti-PGCP antibodies.
b, the same vesicles are unreactive with normal rabbit serum
(control). Magnification was × 24,700. Bar, 100 nm.
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Amino Acid Sequence Homology of PGCP to Amino- and
Carboxypeptidases--
The homology search in GenBankTM
Non-Redundant data base and SwissProt data bases using the complete
amino acid sequence of PGCP revealed significant homology with human,
rat, and mouse NAALADase/PSMA as well as with bacterial zinc
aminopeptidases from Aeromonas proteolytica (Vibrio
proteolyticus, VIBR), Vibrio cholerae, and
Streptomyces griseus (SGAP). Lower homology was found with
X-Hex-dipeptidase (carnosinase) from Lactobacillus delbueckii subsp. lactis and a carboxypeptidase G2
(folate hydrolase G2) from Pseudomonas sp. (CPG2). Since the
tertiary structures of VIBR and SGAP are resolved by x-ray
crystallography (42-44) and the structure of human NAALADase/PSMA was
recently modeled (16), we performed the alignment of the PGCP amino
acid sequence with the sequences of these enzymes to study conservation
of the residues that comprise the active site regions (Fig.
8). PGCP shows similarity to
NAALADase/PSMA all through its sequence; the overall amino acid
identity with human and rat PSMA is 27.1 and 25.0%, respectively
(Table I). The lowest local identity
score between PGCP and PSMA is observed for the sequence fragments
suggested to be a NH2-terminal cytoplasmic "tail"
(amino acids 1-18) and a transmembrane domain (amino acids 19-42) of
PSMA protein. This is consistent with our finding that, unlike PSMA,
PGCP is a soluble enzyme. The highest identity score was observed
between the "catalytic domain" of PSMA (residues 279-587 in human
and 276-589 in rat protein) and the aligning residues of PGCP (Table I
and Fig. 8), suggesting that PGCP may have a similar catalytic domain
spanning residues Gly218-Leu451. This fragment
of PGCP also has the highest local identity with the VIBR and SGAP
sequences (Table I).

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Fig. 8.
Amino acid sequence alignment of PGCP with
homologous peptidases: zinc aminopeptidases from A. proteolytica
(V. proteolyticus, VIBR) and S. griseus
(SGAP) or NAADAse/prostate-specific membrane antigen from humans
(PSMAHUM) and rat (PSMARAT). The
figure shows the central part of the deduced "catalytic
domain" of PGCP (amino acids 255-428, ~30% of the total sequence)
and aligning residues of PCMAHUM, PCMARAT, VIBR, and SGAP that
demonstrated the highest identity score as is shown in Table I.
Alignment was performed using the Lipman-Pearson algorithm and a
BLOSUM62 matrix by ProteinManagerTM software package for
sequence analysis (ACD Inc.). Identical amino acids are
colored. Hyphens represent gaps introduced to
optimize the alignment. Active site residues are indicated with the
following symbols: , nucleophyl; , zinc-binding residues,
×, residues of the substrate-binding pocket.
Numbers refer to the positions of amino acids.
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Table I
Percentage of identical amino acids in sequences of PGCP and homologous
cocatalytic metallopeptidases as aligned in Fig. 8
The values shown are percentages of identical amino acids in the whole
sequences (upper right) or in the deduced "peptidase domains"
(lower left) that included the following residues:
Gly218-Leu451 of plasma glutamate carboxypeptidase
(PGCP), Ala274-Leu587 of human and
Ala276-Leu589 of rat NAADase/prostate-specific
membrane antigen (PSMAHUM and PSMARAT, respectively),
Ile110-Leu446 of zinc aminopeptidase from
A. proteolytica (VIBR), and the whole sequence of zinc
aminopeptidase from S. griseus (SGAP).
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VIBR, SGAP, CPG2, and NAALADase/PSMA all belong to peptidase family
M28, including amino- and carboxypeptidases, that are called
cocatalytic because these peptidases contain two zinc atoms closely
located in their catalytic site (16, 45). The first zinc atom is
coordinated with Asp, Glu, and His, and the second is coordinated with
His and two Asp residues. Since these residues are topologically
conserved in all aligned proteins, we suggest that PGCP also contains
two cocatalytic zinc atoms ligated with Asp302,
Glu337, and His433 and with His290,
Asp302, and Asn353, respectively (marked by
triangles in Fig. 8). As in VIBR, SGAP, and NAALADase/PSMA,
Asp302 can be a ligand for both zinc atoms. Catalytic site
nucleophyl, Glu336, is also topologically conserved in PGCP
and may form a cis-peptide bond with Glu337 as
in the case of other members of the M28 family. In VIBR and SGAP
aminopeptidases, the specificity pockets are formed by markedly hydrophobic residues (for example, Met280,
Ile299, Cys329, Tyr331,
Cys333, Met348, Phe350,
Phe354, Tyr357, and Ile361 in
VIBR). In contrast, only two of the corresponding positions are
occupied by hydrophobic residues in PGCP (Ile254 and
Leu269). Similarly to NAALADase, PGCP contains basic
residues (Arg382, Lys428) in the substrate
binding pocket that may contribute to the binding of substrates with
negatively charged amino acids.
Carboxy- and Endopeptidase Activity of PGCP--
In order to
verify if PGCP has a peptidase activity as predicted both by its amino
acid homology with amino- and carboxypeptidases and its affinity to
Phe-Leu-agarose, we tested the preparations of PGCP purified from human
placenta, mouse liver, and blood plasma for the enzymatic activity
against the number of substrates including those of aminopeptidase
(H-Ala-AMC, H-Arg-AMC, H-Glu-AMC, H-Leu-AMC, H-Lys-AMC, and H-Phe-AMC),
dipeptidyl peptidase (H-Gly-Phe-
NA and Z-Arg-Arg-
NA),
endopeptidase and esterase (benzoyl-DL-Arg-pNA, Suc-Phe-Leu-Phe-pNA, Z-Gly-Gly-Leu-pNA,
Suc-Leu-Tyr-AMC, and Z-Phe-pNA), and carboxypeptidase
(FA-Phe-Ala, FA-Phe-Arg, FA-Phe-Glu, FA-Phe-Gly, FA-Phe-Phe,
FA-Glu-Tyr, and Ac-Asp-Glu). The results of these experiments are shown
in Fig. 9a). We observed a
significant glutamate carboxypeptidase activity with Ac-Asp-Glu
substrate (2.5-4.5 nmol/min/mg of protein) but also a low
chymotrypsin-like activity with endopeptidase substrate,
Suc-Leu-Tyr-AMC (0.04-0.07 nmol/min/mg of protein) and esterase
substrate, Z-Phe-pNA (~0.02 nmol/min/mg of protein). We
have chosen these three substrates to assay the activity in the
homogenate and culture medium of COS-1 cells transfected with pCMV-PGCP
expression vector. The results (Fig. 9b) showed a 6-8-fold increase of glutamate carboxypeptidase activity, assayed with Ac-Asp-Glu; a 1.5-2-fold increase of esterase activity, assayed with
Z-Phe-pNA; and a 7-9-fold increase of the endopeptidase
activity assayed with Suc-Leu-Tyr-AMC in cells transfected with
pCMV-PGCP as compared with untransfected cells or with
pCMV-
-galactosidase-transfected cells.

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Fig. 9.
Peptidase activities of purified preparations
of PGCP and in transiently transfected COS-1 cells. a,
PGCP purified from mouse liver, human placenta, or human blood plasma
showed detectable endopeptidase activity toward Suc-Leu-Tyr-AMC
(A, left axis),
Suc-Phe-Leu-Phe-pNA (C, left
axis), and Z-Gly-Gly-Leu-pNA (D,
left axis); esterase activity toward
Z-Phe-pNA (B, left axis);
and glutamate carboxypeptidase activity toward Ac-Asp-Glu
(E, right axis). Activity values
measured with other substrates listed under "Experimental
Procedures" were below the detection level (less then 1 pmol/min/mg).
b, enzymatic activities toward Suc-Leu-Tyr-AMC
(A, left axis), Z-Phe-pNA
(B, left axis), and Ac-Asp-Glu
(E, right axis) substrates were
increased in the homogenates and in the culture medium of COS-1 cells
48 h after transfection with pCMV-PGCP expression vector
(PGCP) as compared with those of control COS-1 cells
(C) or cells 48 h after transfection with
pCMV- -galactosidase (GAL). Cells were transfected, and
peptidase activities were assayed as described under "Experimental
Procedures." Values represent means ± S.D. of triplicate
experiments.
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In conclusion, we have cloned a new 56-kDa human blood plasma
glycoprotein. Enzymatic analysis of the protein showed that it has a
glutamate carboxypeptidase activity, consistent with its amino acid
sequence homology with zinc cocatalytic metalloprotease, glutamate
carboxypeptidase II (also known as
N-acetyl-aspartyl-
-glutamate carboxypeptidase and PSMA).
In contrast to glutamate carboxypeptidase II, PGCP also showed
endopeptidase activity with substrates containing hydrophobic amino
acid residues at the P1 and P1' positions similar to that of
chymotrypsin or calpain. Although understanding the exact biological
function of this new peptidase requires further characterization of its
physiological substrates, the high level of PGCP observed in blood
serum suggests that the protein may play an important role in the
hydrolysis of circulating peptides.