From the Jejunal folylpoly- Dietary folates, a heterogeneous mixture of
folylpoly- Attempts at molecular characterization of pig jejunal FGCP were
facilitated by the recent and serendipitous descriptions of the
molecular properties of two other proteins, human prostate-specific membrane antigen (PSM) and rat brain N-acetylated The goals of the present study were to characterize the molecular
structure of pig jejunal FGCP while exploring its potential genetic and
biological similarities to human PSM and rat NAALADase. We found
extensive molecular homology and overlapping catalytic capabilities
among pig FGCP, human PSM, and rat NAALADase, consistent with the
concept that the three proteins represent varied expressions of the
same gene in different species and tissues. The original discovery of
the pig FGCP gene provides a molecular framework for future studies on
the biological relationships among these proteins and on the
integration of jejunal folate hydrolysis within the overall process of
the intestinal absorption of dietary folates.
Reagents--
The SuperScript preamplification system was
purchased from Life Technologies, Inc. Taq DNA polymerase
was purchased from Sigma. [ Animal and Human Tissues--
Fresh jejunal and ileal mucosal
scrapings were obtained from market pigs within 5 min of killing at the
University of California (Davis, CA) slaughterhouse and were
immediately washed in ice-cold saline, frozen in liquid nitrogen, and
stored at Cell Lines--
Tumor cell lines were obtained from the American
Type Culture Collection (Rockville, MD). PC3 cells were grown in MEM
supplemented with 2 mM glutamine, 10% fetal bovine serum,
50 units/ml penicillin G, and 50 µg/ml streptomycin; LNCaP cells were
cultured in RPMI supplemented with nonessential amino acids, 5% fetal
bovine serum, 50 units/ml penicillin G, and 50 µg/ml streptomycin.
All media reagents were obtained from Life Technologies.
Peptide Microsequencing--
As described previously, FGCP was
purified from pig jejunal brush-border membranes, and the major subunit
protein was identified at 120 kDa by denaturing 6% polyacrylamide gel
electrophoresis and immunoblot with Mab-3 monoclonal antibody (5). A
parallel gel was stained with Coomassie Blue, and the single 120-kDa
band was electroeluted using the Amicon Centrilutor system (12). A
peptide digest was prepared by overnight incubation of the eluate with
a 50-fold molar excess of cyanogen bromide in 70% formic acid. The
resultant peptide fragments were separated on a 7.5% Tricine gel and
blotted to ProBlott membranes (Applied Biosystems, Foster City, CA).
Peptide sequencing followed the Edman reaction, and amino acids were
identified by high performance liquid chromatography (12).
Department of Internal Medicine,
Center for Engineering of Plants for Resistance
against Pathogens, University of California, Davis, California 95616 and the ¶ Department of Psychiatry, Harvard Medical School,
Boston, Massachusetts 02115
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-glutamate carboxypeptidase
hydrolyzes dietary folates prior to their intestinal absorption. The
complete folylpoly-
-glutamate carboxypeptidase cDNA was isolated
from a pig jejunal cDNA library using an amplified homologous probe incorporating primer sequences from prostate-specific membrane antigen,
a protein capable of folate hydrolysis. The cDNA encodes a
751-amino acid polypeptide homologous to prostate-specific membrane antigen and rat brain N-acetylated
-linked acidic
dipeptidase. PC3 transfectant membranes exhibited activities of
folylpoly-
-carboxypeptidase and N-acetylated
-linked
acidic dipeptidase, while immunoblots using monoclonal antibody to
native folylpoly-
-glutamate carboxypeptidase identified a
glycoprotein at 120 kDa and a polypeptide at 84 kDa. The kinetics of
native folylpoly-
-carboxypeptidase were expressed in membranes of
PC3 cells transfected with either pig folylpoly-
-carboxypeptidase or
human prostate-specific membrane antigen.
Folylpoly-
-carboxypeptidase transcripts were identified at 2.8 kilobase pairs in human and pig jejunum, human and rat brain, and human
prostate cancer LNCaP cells. Thus, pig folylpoly-
-carboxypeptidase,
rat N-acetylated
-linked acidic dipeptidase, and human
prostate-specific membrane antigen appear to represent varied
expressions of the same gene in different species and tissues.
The discovery of the jejunal folylpoly-
-carboxypeptidase gene
provides a framework for future studies on relationships among these
proteins and on the molecular regulation of intestinal folate
absorption.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-glutamates, are absorbed by a two-stage process of
progressive hydrolysis at the jejunal brush border membrane followed by
transport of monoglutamyl folate derivatives across the intestinal
mucosa (1). Previously, our laboratory (2) purified
folylpoly-
-glutamate carboxypeptidase
(FGCP)1 from human jejunal
brush-border membranes as a zinc-activated exopeptidase that releases
terminal glutamates sequentially and is stable at pH greater than 6.5. We identified a separate intracellular lysosomal carboxypeptidase in
human jejunal mucosa that cleaves folylpoly-
-glutamates with an
endopeptidase mode of action at a pH optimum of 4.5 and that is
distinguished from membranous FGCP by its complete inhibition by
p-hydroxymercuribenzoate (3). Subsequent experiments
detected the two separate folate hydrolases in intracellular and
brush-border membrane fractions of pig jejunal mucosa, each with
properties identical to those found in human jejunum (4). A monoclonal
antibody Mab-3 to the purified pig jejunal brush-border FGCP detected a
120-kDa subunit protein that was localized by immunoreactivity to the
jejunal brush-border site of in vivo hydrolysis of
folylpoly-
-glutamates (5).
-linked
acidic dipeptidase (NAALADase). The cDNAs encoding these two
proteins demonstrate 87% nucleotide and 85% amino acid sequence
identity (6-8) and appear to be homologues of the same enzyme.
Previously, we (8, 9) showed that PC3 cells transfected with either of
these cDNAs exhibit N-acetylaspartylglutamate
(NAAG)-hydrolyzing activity characteristic of NAALADase. Others found
that PC3 cells transfected with the human PSM cDNA are capable of
hydrolysis of folylpoly-
-glutamate (10) with an exopeptidase
activity mechanism similar to that previously described for human
jejunal FGCP (2). The discovery that the hydrolysis of both NAAG and folylpoly-
-glutamate can be attributed to the same molecule (PSM) led to the recommendation that human PSM and rat brain NAALADase be
identified under a single IUBMB-approved name (11), subsequently designated glutamate carboxypeptidase II (GCP II; EC 3.4.17.21).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-32P]dCTP (3000 mCi/mmol)
and [
-35S]dATP (1000 mCi/mmol) were purchased from
Amersham Pharmacia Biotech. A cDNA probe for human actin was
obtained from CLONTECH (Palo Alto, CA).
N-Acetylaspartyl-[3,4-3H]glutamate (41.8 Ci/mmol) and
-[32P]dATP (6000 Ci/mmol) were obtained
from NEN Life Science Products. AG 1-X8 anion exchange resin
(200-400-mesh, formate form) was purchased from Bio-Rad.
2-(Phosphonomethyl)pentanedioic acid was a gift of Dr. Barbara Slusher,
Guilford Pharmaceuticals (Baltimore, MD).
Folyl-
-Glu-
-[14C]Glu was available as a prior gift
from Dr. C. Krumdieck (University of Alabama Birmingham). Purified
native pig jejunal FGCP and its monoclonal antibody Mab-3 were
available at
70 °C from our previous experiment (5).
Peptide-N-glycosidase F was purchased from Oxford Glyco
Sciences (Bedford, MA). All other reagents were obtained from Sigma,
Fisher, and various other commercial sources.
70 °C. They were then used for the preparation of
brush-border membranes that were purified >20-fold according to
appropriate marker enzymes and our previously described procedure (5).
For subsequent RNA and poly(A+) RNA preparations, portions
of pig liver; renal cortex; and duodenal, jejunal, and ileal mucosa
were frozen in liquid nitrogen and stored at
70 °C. Human jejunal
segments of ~2-cm length were obtained fresh in the operating room
from obese patients undergoing elective gastric bypass surgery with
gastrojejunal anastomosis, according to acceptable use exemption from
the University of California Davis Human Subjects Committee. Segments
were opened longitudinally and were washed immediately in ice-cold 4 M guanidium thiocyanate prior to freezing in liquid
nitrogen and storage at
70 °C.
Pig Jejunal cDNA Library Construction and
Screening--
Approximately 10 µg of poly(A+) RNA was
prepared from pig jejunal mucosal RNA by the FastTrak 2.0 poly(A+) RNA isolation system (Invitrogen, Carlsbad, CA)
(16) and was used for custom construction of a pig jejunal mucosal
cDNA library in ZAP by Stratagene Cloning Systems, with a yield
of 1.1 × 1010 plaque-forming units/ml. The cDNA
library was probed with the amplified 853-bp cDNA fragment using
established screening methods (17), and positive plaques were purified
by secondary and tertiary screening. Following in vivo
excision and agarose gel electrophoresis, six purified cDNA clones
of different sizes between 1.6 and 2.5 kb were identified by Southern
analysis using the 853-bp cDNA probe.
cDNA Sequence Analysis--
Both strands from each clone
were sequenced completely by the dideoxy chain termination reaction
using the T3 or T7 polymerase vector primer sequences (15) and by
primer walking using gene-specific oligonucleotide primers that were
constructed from bases 8 to
5, 203-223, 590-605, 822-836,
948-962, 1237-1251, 1526-1540, 1847-1861, and 2078-2092 (sense)
and from bases 284-303, 544-558, 786-800, 1110-1115, 1456-1470,
1645-1660, 1988-2001, and 2237-2245 (antisense). The full cDNA
sequence was confirmed independently by cycle sequencing of each clone
using the LI-COR 4200 automated sequencer (LI-COR, Lincoln, NE). Clone
7 incorporated all sequences represented in the others, except for an
additional 46 bp in the 5'-untranslated region of clone 10 and 25 bp in
the 3'-untranslated region of clone 4. No additional sequences were
detected in the 5'-untranslated region by rapid amplification of
cDNA ends (18). Nucleotide and amino acid sequence identities among
pig FGCP, human PSM (6), rat NAALADase (7, 8), and other relevant proteins were analyzed by the BESTFIT and PILEUP programs of version 9.1 of the Genetics Computer Group sequence analysis software package
(Madison, WI).
Preparation and Expression of the Cloned Enzyme-- A construct of the cDNA of FGCP was prepared by HindIII and XbaI excision from the vector, followed by ligation into the mammalian expression vector pcDNA3 (Invitrogen). One hundred-mm dishes of PC3 cells were transfected with 25 µg of supercoiled plasmid DNA containing the cDNA of pig FGCP or human PSM (construct PSMA2) (9) using the calcium phosphate-mediated method in 50 mM Hepes buffer, pH 7.05 (19). Mock transfected PC3 cells served as controls. Cells were harvested 72 h post-transfection for enzymatic assays by scraping them into 50 mM Tris-HCl buffer (pH 7.4 at 37 °C). Membranes were prepared from the transfected and control PC3 cells by brief sonication followed by centrifugation (35,000 × g) for 30 min. The membrane pellets were then solubilized by sonication into 50 mM Tris-HCl plus 0.5% Triton X-100. The protein concentration of the solubilized membrane was determined using the enhanced protocol BCA assay (Pierce) or Bio-Rad kit.
Enzyme Activities-- The hydrolysis of NAAG was measured in purified pig jejunal and ileal brush-border membranes and in transfected and mock transfected PC3 cell membranes by radioenzymatic assay, whereby hydrolysis was quantitated via scintillation spectrometry of [3H]glutamate produced from radiolabeled substrate after separation of substrate and product by ion exchange chromatography (20). Assays were initiated by the addition of labeled NAAG at a concentration of 2.5 nM.
Folate hydrolysis was measured in membranes from PSM and FGCP transfectants and mock transfected PC3 cells using substrate folyl-Immunoblots-- Membranes from the PC3 cells that were transfected with the cDNA of either human PSM or pig FGCP or that were mock transfected were solubilized in 0.1% Triton X-100. Membrane proteins from the FGCP transfectant were deglycosylated under denaturing conditions using peptide-N-glycosidase F according to the manufacturer's protocol. Solubilized membrane proteins and a sample of purified native pig jejunal brush-border FGCP (5) were electrophoresed in parallel on 8% SDS-polyacrylamide gels (22), followed by transfer to polyvinylidene difluoride membranes (Millipore Corp., Marlborough, MA). Protein bands were identified using the monoclonal antibody Mab-3 to the purified native pig FGCP (5) followed by a secondary goat anti-mouse antibody conjugated with alkaline phosphatase (Bio-Rad). The authenticity of Mab-3 immunoreactivity was proven previously by its ability to immunoprecipitate the 120-kDa subunit of FGCP from solubilized pig jejunal brush-border membranes and to localize FGCP in pig intestine immunohistochemically (5).
Northern Blots-- Total RNA was extracted from rat brain, LNCaP cells, and pig and human jejunal mucosa (13). Poly(A+) RNA was prepared from pig liver and kidney and duodenal, jejunal, and ileal mucosa (16). Human brain poly(A+) RNA was obtained from CLONTECH Inc. (Palo Alto, CA). A 2.4-kb EagI-NdeI fragment of FGCP was purified and 32P-labeled for subsequent probing of Northern blots. Pig tissue samples were also probed with a 32P-labeled fragment of human actin cDNA as a positive internal control. After electrophoretic separation in 1.2% agarose, 2.2 M formaldehyde gels and transfer to nylon membranes (Schleicher & Schuell), RNA species were identified by hybridization to cDNA probes as detected autoradiographically (23).
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RESULTS |
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Molecular Sequence of Pig Jejunal FGCP-- The complete nucleotide and deduced amino acid sequences of the cDNA of pig FGCP are shown in Fig. 1. The deduced amino acid sequences KILIARYGKIF and MYSLVYNLTKELQ correspond with 100 and 85% identities to two amino acid sequences, KILIARYGKIF and MYILVYGLTKELQ, that were identified in the peptide digest of the native purified enzyme. The complete cDNA of FGCP is composed of 2532 bases: 146 in the 5'-untranslated region, 2253 in the open reading frame that encode 751 amino acids, and 133 in the 3'-untranslated region. The nucleotide and deduced amino acid sequences of pig FGCP were compared with those of human PSM (6) and rat NAALADase (7, 8). Within the open reading frame, the nucleotide identities between pig FGCP and human PSM and rat brain NAALADase were 88 and 83% respectively, while there was very little similarity in the 5'-untranslated region. The amino acid sequence of pig FGCP was 92% similar and 91% identical to that of human PSM and was 87% similar and 83% identical to that of rat NAALADase (Table I). Structural comparisons followed the recent Rawlings and Barrett analysis of human PSM and rat NAALADase (11). The Kyte and Doolittle hydropathy plot (24) of pig jejunal FGCP was identical to those of human PSM and rat NAALADase and typifies a type II protein that conserves a short N-terminal cytoplasmic region and a single hydrophobic transmembrane between residues Trp20 and Ile43. Like human PSM and rat NAALADase, pig FGCP lacks an N-terminal signal sequence but contains positively charged residues at the N-terminal side of the transmembrane domain that are characteristic of type II membrane proteins (25), while the remainder of the molecule containing the catalytic domain occupies an extracellular site. The putative catalytic domain of human PSM and rat NAALADase is conserved in FGCP between residues 275 and 588. Twelve NX(S/T) potential glycosylation sites occur at Asn positions 51, 77, 122, 141, 154, 196, 337, 460, 477, 614, 639, and 646, of which 10 are conserved by human PSM and nine by rat NAALADase. Five putative catalytic zinc binding residues are conserved at positions His378, Asp388, Glu426, Asp454, and His554. Within the proposed specificity pocket, four positively charged residues are conserved at Arg464, Lys501, Arg537, and Lys546.
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Homologies with Other Relevant Proteins--
The BESTFIT computer
program was used to analyze regional amino acid sequence homologies
between pig FGCP and selected structurally and functionally related
proteins (Table I). In addition to extensive sequence similarities and
identities among FGCP, PSM, and NAALADase, FGCP exhibited similarities
with three other M28 family members: human transferrin receptor (26)
and aminopeptidases from Vibrio proteolyticus (27) and
Streptomyces griseus (28). Rat I100, a recently
characterized ileal peptidase with type II structure (29), also shares
extensive amino acid similarity with FGCP, whereas there was less
sequence similarity between FGCP and human dipeptidyl peptidase IV, an
enzyme that appears to be functionally related to I100 (30). The PILEUP
program was used to clarify amino acid alignments within the putative
catalytic regions of FGCP, rat ileal I100 (29), and human dipeptidyl
peptidase IV (30). All five putative catalytic zinc binding residues
(11) were conserved between pig jejunal FGCP and rat ileal I100 at His378, Asp388, Glu426,
Asp454, and His554, while only one zinc binding
residue at Glu426 was conserved in dipeptidyl peptidase IV.
Among the putative substrate binding basic amino acids (11) that were
conserved in FGCP, PSM, and NAALADase, only Arg464 was
conserved in I100, and only Arg537 was conserved in
dipeptidyl peptidase IV. Several amino acids typical of a serine
carboxypeptidase mechanism (29) were conserved further downstream,
including Ser632 in all three proteins and
Asp667 and His690 in FGCP and I100. Structural
similarities between FGCP and selected other proteins relevant to
folate hydrolysis and transport were also investigated. Human glutamate
hydrolase (an intracellular peptidase capable of
folylpoly--glutamate hydrolysis (31)) and two proteins involved in
the transport of monoglutamyl folates (the mouse reduced folate carrier
protein (RFC) (32) and pig folate-binding protein (FBP) (33)) showed
only weak similarities to short regions at the N- or C-terminal ends
outside of the catalytic region of FGCP.
Enzyme Activities-- As depicted in Fig. 2, NAALADase-specific activity was 16-fold greater in pig jejunal brush-border membranes than in ileal brush-border membranes. NAALADase was abundant in membranes from PC3 cells transfected with the cDNA of pig jejunal FGCP but was absent from control PC3 cells. Previously characterized inhibitors (9, 20) nearly eliminated NAALADase activity in jejunal brush-border membranes and in FGCP transfectant membranes but had minimal effect on NAALADase activity in ileal brush-border membranes.
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Immunoblots-- Fig. 4 compares the immunoreactivities of the monoclonal antibody Mab-3 (5) with purified native pig FGCP, with pig FGCP transfectant membranes before and after treatment with peptide-N-deglycosidase F, and with human PSM transfectant membranes. Mab-3 detected the native pig FGCP and the pig FGCP transfectant glycoprotein at the identical size of 120 kDa and detected the deglycosylated polypeptide at 84 kDa but did not react with the human PSM transfectant membranes or with mock transfected control membranes.
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Northern Blots-- The cDNA of pig FGCP showed a strong hybridization signal at 2.8 kb in pig duodenum and jejunum and a faint signal in pig kidney, while no signal was detected in pig liver or ileum (Fig. 5). A band of similar size was identified in RNA extracts from pig and human jejunal mucosa. A positive actin signal was present in all samples. Several bands of hybridization appeared in RNA samples from rat and human brain and the LNCaP prostate carcinoma cell line (Fig. 6). Bands of roughly equal intensity were observed in rat brain at approximately 3.9, 2.95, and 2.8 kb, while a predominant species of 2.8 kb was found in human brain and in the human LNCaP prostate cancer cell line.
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DISCUSSION |
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The present study has achieved the original molecular characterization of FGCP from pig jejunal mucosa. The authenticity of the pig FGCP cDNA sequence and its specific functional expression was established by (a) the incorporation of two native peptide sequences into the deduced amino acid sequence (Fig. 1), (b) the reproduction of the activity profile and kinetics of native pig FGCP (2, 4) in FGCP transfectant membranes (Fig. 3), (c) the immunoblot identification of the FGCP transcript by monoclonal antibody to native pig FGCP at the identical 120-kDa molecular size of the purified native enzyme (Ref. 5; Fig. 4) and identification of the deglycosylated polypeptide at the 84-kDa molecular size predicted by the amino acid sequence (Fig. 1), and (d) the identification of FGCP transcripts at 2.8 kb in pig jejunal mucosa and their absence in pig ileal mucosa (Fig. 5), consistent with the established intestinal distribution of the activity and immunoreactivity of the native enzyme (5). The additional presence of similar FGCP transcripts in pig and human jejunal mucosa (Fig. 5) suggests that the same gene expresses FGCP in human and pig jejunal brush-border membranes (2, 5).
The present experiments complete a circle of evidence for extensive
molecular homologies among pig FGCP, human PSM, and rat NAALADase. The
findings of 83-91% amino acid sequence identities between pig FGCP
and each of the other sequences (Fig. 1; Table I) is in keeping with
prior reports on the extensive amino acid identities between human PSM
and rat NAALADase (6-9, 11) and is consistent with the concept that
all three proteins represent species-specific homologues of the same
gene. While the amino acid sequence of each protein predicts a
polypeptide molecular size of 84 kDa (Fig. 1; Refs. 6-8), the presence
of 12 glycosylation sites accounts for the greater 120-kDa molecular
size of native (5) or transfectant FGCP (Fig. 4) compared with the
reported molecular sizes of 100 kDa for PSM with 10 glycosylation sites (6) and of 94 kDa for NAALADase with nine glycosylation sites (7, 8,
34). While the epitope for our monoclonal antibody to native pig FGCP
is unknown, incomplete amino acid sequence identities and differences
in glycosylation between pig FGCP and human PSM could account for the
lack of antibody cross-reactivity with PSM in transfectant membranes
(Fig. 4). Prior findings of NAALADase transcripts at 2.8 kb in rat
kidney (7, 8) are extended by the detection of a weak FGCP
hybridization signal at 2.8 kb in pig kidney poly(A+) RNA
(Fig. 5), while the prior findings of PSM-like transcripts and
immunoreactivity in human small intestine (35-37) are complemented by
the detection of the FGCP hybridization signal at 2.8 kb in pig
duodenal and jejunal poly(A+) RNA and in human jejunal RNA
(Fig. 5). The tissue distribution and predominant size of FGCP-like
transcripts in rat and human brain and LNCaP cells (Fig. 6) is similar
to other descriptions of the distribution and sizes of PSM and
NAALADase transcripts in these tissues (6-9, 38). The previous finding
of NAALADase activity in membranes of LNCaP cells and PSM transfectants
(9) is complemented by finding NAALADase activity in pig jejunal
brush-border membranes and in FGCP transfectant membranes (Fig. 2). The
observation that membranes of LNCaP cells or PSM transfectants were
capable of progressive hydrolysis of folylpoly--glutamates (10) is confirmed and extended by finding nearly identical kinetic properties of purified native FGCP in FGCP or PSM transfectant membranes (Fig. 3;
Table II).
A recent analysis classified human prostate PSM and rat brain NAALADase as GCP II, a single type II glycoprotein member of the M28 family of peptidases (11) (EC 3.4.17.21). The extensive amino acid identities, common structural motifs, and conservation of the identical five co-catalytic zinc-binding amino acids and four putative substrate binding basic amino acids suggest that FGCP derives from the pig homologue of the GCP II gene (Fig. 1). GCP II and two prototypical bacterial aminopeptidases V. proteolyticus (27) and S. griseus (28) are members of the M28 peptidase family by virtue of homologous catalytic domains, which appear to bind two co-catalytic zinc atoms (11, 39). The three-dimensional structural analysis of V. proteolyticus aminopeptidase suggested the location of a substrate specificity pocket, which is composed of basic amino acids in PSM and NAALADase (11, 27). The loci of the human PSM gene and a second similar sequence have been found on human chromosome 11 (40, 41). Others recently identified another type II ileal brush-border membrane protein, I100, that shares 60 and 59% sequence identities with rat NAALADase and human PSM (29), of which the human homologue might comprise the second locus on chromosome 11. I100 exhibits activity similar to human dipeptidyl peptidase IV, another peptidase associated with the apical brush border of intestinal epithelial cells (29, 30). These relationships prompted our evaluation of potential structural similarities among FGCP, I100, and dipeptidyl peptidase IV. The conservation of all five zinc-binding residues suggests that FGCP and I100 share the same catalytic mechanism. On the other hand, an alternative potential serine carboxypeptidase mechanism (29) is suggested by conservation of Ser632 in all three sequences.
While pig FGCP, rat NAALADase, and human PSM may represent different
species-specific expressions of same GCP II gene, their functions
appear to differ according to the tissue in which the gene is
expressed. Thus, GCP II may function as FGCP in the jejunum by cleaving
-linked glutamyl residues sequentially from dietary folylpoly-
-glutamates prior to the intestinal transport of folic acid (1, 2, 4, 5) and as NAALADase in the brain to release
-linked
glutamate from NAAG to regulate subsequent neurotransmission (8, 9).
These different functions may reflect tissue differences in available
substrate, since NAAG is concentrated at neuronal synapses (8), while
folylpoly-
-glutamates are concentrated as dietary components at the
brush-border surface of the proximal small intestine (1).
The present study offers molecular clarity to the mechanism of folate
absorption at the intestinal brush-border membrane. Our original
studies identified an initial stage of jejunal hydrolysis of dietary
folylpoly--glutamates that precedes the intestinal uptake of the
folic acid product (1). We identified and characterized FGCP as a
zinc-dependent exopeptidase that is active at a neutral pH
optimum in human and pig jejunal brush-border membrane fractions (2, 4)
and that was localized in the pig to the jejunal brush-border membrane
and was excluded from the ileal brush-border membrane by the monoclonal
antibody Mab-3 to the purified enzyme (5). These observations are
extended by the present molecular characterization of FGCP as a type II
protein of the M28 peptidase family with a zinc-binding motif, for
which the transcripts are expressed in proximal but not distal pig
small intestine (Fig. 5). The finding of a different activity profile
of folate hydrolysis by mock transfected PC3 cells including an acid pH
optimum and complete p-hydroxymercuribenzoate inhibition
(Fig. 3) is consistent with our prior definition of the characteristics
of a separate lysosomal endopeptidase that provides intracellular
folate hydrolysis in human and pig jejunal mucosa (3, 4). The recently
described PSM' splice variant (42) cannot provide the separate profile of folate hydrolysis found in mock transfected PC3 cells (Fig. 3),
since no genetically similar species is expressed in native PC3 cells
(6, 9). Alternatively, the second folate hydrolyzing activity in mock
translated PC3 cell membranes (Fig. 3) and in the lysosomal fraction of
jejunal mucosa (3) may be attributed to the recently described and
genetically dissimilar glutamate hydrolase (EC 3.4.19.9) (Table I; Ref.
31).
The present studies provide a molecular framework for future studies on
the regulation of FGCP by conditions known to affect intestinal folate
absorption and on the relationship of FGCP to RFC and FBP, two proteins
involved in membrane transport of monoglutamyl folates (Table I). The
cDNA sequences of mouse and human RFC have been defined, and its
intestinal transcription and functional capability for transport of
monoglutamyl folate in cell transfectants has been proven (32, 43, 44).
The alternate receptor FBP has been characterized at the molecular
level in pig liver, but its transcripts and activity are absent from
the jejunum (33).2 The
present study shows that FGCP is genetically distinct from both RFC and
FBP, since their amino acid sequences are minimally represented in FGCP
(Table I). In summary, the available data indicate that the intestinal
absorption of dietary folylpoly--glutamates is achieved by a
two-step process of progressive hydrolysis of
-linked glutamyl
residues by FGCP at the jejunal brush-border membrane, releasing folic
acid and other monoglutamyl folate derivatives for subsequent membrane
transport by genetically distinct RFC. The integration of folate
hydrolysis by jejunal FGCP and folic acid transport by intestinal RFC
in the overall process of folate absorption has yet to be defined.
These studies are now feasible due to the molecular identification of
FGCP.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK-35747, DK-45301, and MH-572901.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) AF050502.
§ To whom correspondence should be addressed: TB 156, School of Medicine University of California, Davis, CA 95616. Tel.: 530-752-6778; Fax: 530-752-3470; E-mail: chhalsted{at}ucdavis.edu.
The abbreviations used are:
FGCP, folylpoly--glutamate carboxypeptidase; NAALADase, N-acetylated
-linked acidic dipeptidasePSM, prostate-specific membrane antigenNAAG, N-acetylated
aspartylglutamateGCP II, glutamate carboxypeptidase III100, ileal
100-kDa proteinDPP IV, dipeptidyl peptidase IVGH, glutamate
hydrolaseRFC, reduced folate carrier proteinFBP, folate-binding
proteinTricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinebp, base
pair(s)kb, kilobase pair(s).
2 J. A. Villanueva and C. H. Halsted, unpublished data.
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REFERENCES |
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