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(Received for publication, January 26, 1996; and in revised form, February 14, 1996) From the
Purified
The cDNA
prepared from H35 mRNA was synthesized by Moloney murine leukemia
virus-reverse transcriptase and ligated to the Marathon cDNA adaptor
(Clontech, Palo Alto, CA). RACE-ready cDNA was amplified with nested
A rat hepatoma cDNA library was obtained from Stratagene
(Palo Alto, CA). A total of 10 cDNA inserts were excised
from the
Figure 1:
A, SDS,
12.5% polyacrylamide gel electrophoresis of purified GH. GH from peaks
1 (lane 1), peak 2 (lane 2), and peak 3 (lane
3) from TSK-gel Toyopearl butyl-650S were purified to homogeneity
on Matrex gel green A as described under ``Experimental
Procedures.'' A portion of the purified peak (0.5 µg) was
applied to each lane. B, analysis of native and
deglycosylated GH. Purified pooled peaks 1 and 2 (1 µg of protein)
were utilized without further treatment in lanes 1 and 3. Samples (lane 2, 0.5 µg; lane 4,
0.125 µg) were incubated with PNGase F (10 and 5 µg,
respectively) in 0.1% SDS and 0.1% Triton X-100 for 20 h at 30 °C.
Following electrophoresis, the gel was stained with 0.05% Coomassie
Brilliant Blue (lanes 1 and 2) or evaluated by
Western blot using a 1:500,000 dilution of rabbit anti-GH (lanes 3 and 4). Although PNGase F also migrates at approximately
33 kDa, it did not stain because of the low amount of protein used in
the incubation.
Figure 2:
Nucleotide sequence of the insert
containing the cDNA encoding
The availability of the cDNA and amino acid sequence for
GH and a polyclonal antibody to the protein offers the possibility of
investigating a number of questions concerning this enzyme. The role of
GH in the cellular metabolism of folylpolyglutamate coenzymes and in
the cytotoxic activity of antifolates can be evaluated in detail. GH
activity is known to be altered by a number of factors including
insulin(18) , estrogen(19) , and selection for
resistance with 5,10-didiazatetrahydrofolate in rat (17, 21) and human (29) cell lines. With the
availability of the molecular and immunological probes described in
this report, the mechanism of alterations in GH activity can be
investigated. The cellular trafficking of the glycoprotein can be
approached with an emphasis on the mechanism and significance of
secretion. Studies are under way to determine if the sequences of the
rat and human cDNAs are homologous and the cross-reactivity of the
anti-rat GH antibody. If feasible, these probes will be used to
evaluate the abundant secretion of GH by human breast cancer cell lines
in culture(22) , which is potentially related to the high
levels of GH in serum of metastatic breast cancer
patients(30) . In addition, relatively large amounts of enzyme
should become available for the first time when an appropriate
expression system is established, and this will allow detailed analysis
of the structure and mechanism of GH.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
U38379[GenBank].
Volume 271,
Number 15,
Issue of April 12, 1996 pp. 8525-8528
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
-Glutamyl
Hydrolase (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-glutamyl hydrolase secreted from rat H35 hepatoma
cells has been characterized as a diffuse band of 55 kDa on
SDS-polyacrylamide gel electrophoresis that is converted to bands of 35
and 33 kDa after enzymatic removal of N-linked carbohydrate.
Polyclonal antibodies against 55-kDa -glutamyl hydrolase captured
the enzyme activity and recognized the glycosylated and both
deglycosylated forms of -glutamyl hydrolase. A complete cDNA
sequence of -glutamyl hydrolase was obtained using degenerate
oligonucleotides derived from peptide sequences, screening of a rat
hepatoma cDNA library, and reverse transcription polymerase chain
reaction. Based upon the deduced amino acid sequence the peptide
component of -glutamyl hydrolase had a molecular weight of 33,400.
The results of amino acid analysis of the purified protein agreed with
the deduced amino acid sequence in which there are seven potential
asparagine-containing glycosylation sites.
-Glutamyl hydrolase (EC 3.4.22.12) catalyzes the hydrolysis
of the polyglutamate side chain of folyl polyglutamates and anti-folyl
polyglutamates(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) .
-Glutamyl hydrolase (GH) (
)has been characterized from
a number of sources, and it exhibits either endo- or exopeptidase
activity, depending upon the tissue of
origin(1, 2, 4, 5, 6, 7, 8, 9, 10, 11) .
In many tissues the enzyme is lysosomal with an acidic pH
optimum(12) . In addition, the enzyme is often sulfhydryl- and
Zn
-dependent (3, 4, 5, 7, 12, 13, 14, 16) ,
appears to be a glycoprotein in many cases(4, 16) ,
and has a reported molecular mass of 50-150
kDa(2, 3, 4, 9, 12, 14, 15, 16) .
A thorough analysis of the mechanism of this unique peptidase that
cleaves only -glutamyl linkages is not available, and a knowledge
of the detailed structure of the protein and the gene encoding it is
not yet delineated. It has been shown that resistance to the
anti-folate 5,10-didiazatetrahydrofolate can be acquired by enhancement
of this enzyme activity in rat H35 hepatoma cells(17) . In
addition, the enzyme is hormonally controlled with both insulin and
estrogen altering its activity in responsive cell lines and tissues (16, 18, 19) . For many years it was thought
that GH was a lysosomal enzyme, but recent studies from this laboratory
using cell culture systems have shown that while its intracellular
location is primarily the lysosome, most of the enzyme activity is
secreted, a feature that appears thus far to be universal in neoplastic
cells(20, 21, 22) . In order to be able to
study this enzyme and its synthesis and regulation in detail, we have
cloned a cDNA coding for this enzyme and evaluated some of the
properties of the glycoprotein itself. These studies used the H35 rat
hepatoma
system(16, 17, 18, 20, 21, 22) ;
this is the first report of a cDNA sequence and corresponding deduced
amino acid sequence for GH.
Materials
Cell Culture
H35 hepatoma cells were cultured
and transferred to serum-free medium as described
previously(16) . The pooled medium was stored at -70
°C until used.Purification of
On the
basis of the earlier study of Lin et al. (23), the individual
peaks from TSK-gel Toyopearl butyl-650S (16) were further
purified by chromatography on Matrex gel green A (Amicon Division, W.
R. Grace & Co.). Up to 200 µg of protein was applied to a
column (1 -Glutamyl Hydrolase
10 cm) of Matrex gel green A that had been
equilibrated with 10 mM sodium acetate (pH 6.0) containing 0.1
mM zinc acetate and 50 mM 
-mercaptoethanol. The
column was washed with 40 ml of equilibration buffer and 40 ml of the
same buffer containing 0.15 M sodium chloride. The enzyme was
eluted with equilibration buffer containing 0.4 M sodium
chloride and 10 mM octyl -glucoside. The specific
activity of five independent preparations was 107,000 nmol
min
mg with a S.D. of
±16%. The specific activity of each of the individual TSK-gel
Toyopearl butyl 650S peaks fell within that range following
purification on Matrex gel green A. GH was assayed as described
previously (16) using
4-NH
-10-CH
PteGlu as substrate.Methods
Microsequencing of Intact GH and Cyanogen Bromide
Fragments
Purified enzyme was analyzed by SDS, 10%
polyacrylamide gel electrophoresis using a Tris-Tricine buffer
containing 0.3 mM thioglycolic acid in a Bio-Rad Mini Protean
system. The gel was electroblotted (Bio-Rad Mini Trans-Blot apparatus)
onto a polyvinylidene difluoride membrane (Bio-Rad) using 10 mM CAPS buffer, pH 11, containing 10% methanol and 0.4 mM dithioerythritol. The membrane was stained with 0.01% Coomassie
Blue and the bands of interest excised and analyzed on an Applied
Biosystems 477A sequencer. For internal sequencing a single crystal of
cyanogen bromide was added to a solution of purified enzyme
(30-40 µg) in 70% trifluoroacetic acid and the solution
heated at 45 °C for 45 min. The solution was lyophilized and
fragments sequenced as above using a SDS, 13% polyacrylamide gel.Preparation of a Polyclonal Antibody to GH
A
single rabbit was immunized with purified GH (an initial immunization
of 50 µg, followed by three boosts of 50 µg each) using a
standard protocol by Biodesign International, Kennebunk, ME. The
studies in this report were done with an early test bleed, purified on
Protein A-agarose, which had a 1 to 500,000 titer against 50 ng of
purified -GH in an enzyme-linked immunosorbent assay.Cloning and Sequencing of GH
The sequence of the
open reading frame corresponding to -glutamyl hydrolase was
determined using a combination of RT-PCR, RACE, and screening of a
commercial cDNA library. Total cellular RNA was prepared from rat
hepatoma H35 cells using TRI REAGENT (Molecular Research Center, Inc.),
according to the manufacturer's instructions. Poly(A)
mRNA was isolated from total RNA by affinity chromatography using
a spin column of microcrystalline oligo(dT)-cellulose (New England
Biolabs). Poly(A) mRNA eluate was precipitated with
ethanol and stored at -70 °C for later use. The
poly(A) mRNA from H35 cells was reverse transcribed
into cDNA using Moloney murine leukemia virus-reverse transcriptase and
an oligo(dT) primer (Stratagene). The cDNA was subjected to PCR
amplification using degenerate primers derived from the amino acid
sequence of the N terminus and of CNBr-generated fragments. The primers
used for RT-PCR were modified by carrying deoxyinosine residues at
positions corresponding to ambiguous nucleotides(24) . The
RT-PCR generated a 605-base fragment denoted pGH-1.-GH-specific primers in combination with Marathon adaptor primers,
AP1 and AP2, respectively. For sequencing, PCR products generated with Taq polymerase were cloned into a PCR II vector in the TA
cloning system, as the 3` A overhangs are not removed (Invitrogen, San
Diego, CA). The amount of PCR product needed to ligate with 50 ng of
PCR II vector was estimated according to the manufacturer's
directions.
ZAP II recombinant
phage plaques on E. coli strain XL1-blue cells were screened.
Phage plaques were lifted twice onto nitrocellulose membranes,
denatured in 0.5 N NaOH/l.5 M NaCl, and neutralized in 1.5 M NaCl/1.0 M Tris HCl, pH 7.5. The membranes were
baked for 2 h at 80 °C in a vacuum oven, and then prehybridized 16
h at 37 °C in prehybridization solution containing 5X SSC, 5X
Denhardt's, 0.1% SDS, 50% formamide, and 0.2 mg/ml salmon sperm
DNA. This was followed by hybridization for 20 h in the above buffer
with 
P-randomly-labeled pGH-1. Three partial cDNA's
were obtained (pGH-3, pGH-4, and pGH-5.)ZAP II cDNA library and subcloned into pBluescript
according to the in vivo excision procedure described by
Stratagene. Plasmid DNA was prepared with a Wizard miniprep DNA
purification system and used as a sequencing template. Initial
sequencing for pGH-1 and pGH-2 was done using M13 forward and M13
reverse primers on the PCR II vector (Invitrogen). For the clones of
pGH3, -4 and -5, T3 and T7 primers on pBluescript were used for
determining the sequences at both ends of the insert. The
dideoxynucleotide chain termination method of Sanger et al. (25) was used with Sequenase (U. S. Biochemical Corp.). Primers
were synthesized by the Molecular Genetic Core at the Wadsworth Center.
Purification of GH
Rat GH was previously shown
to elute in three peaks of activity from TSK-gel Toyopearl butyl
650S(16) . These three peaks (peaks 1, 2, and 3) have now been
individually purified to homogeneity by chromatography on Matrex gel
green A. Each peak when purified appeared as a single diffuse band of M
55,000 on SDS-polyacrylamide gel
electrophoresis (Fig. 1A). The broad appearance of
these bands and the binding of the enzyme to lentil lectin affinity
columns (16) indicated that GH was a glycoprotein. Because of
the limited amounts of enzyme available and the difficulty in
eliminating impurities consistently from peak 3, peaks 1 and 2 were
routinely pooled, purified by Matrex gel green A, chromatographed, and
utilized experimentally (Fig. 1, B and C).
After treatment with the enzyme PNGase F, which cleaves Asn-linked
carbohydrate from glycoproteins(26) , the enzyme appeared as
two discrete bands with molecular masses of approximately 33 and 35 kDa (Fig. 1B, lane 2), suggesting that the protein
is highly glycosylated, primarily at Asn. Both deglycosylated forms
were identified as GH because their N-terminal sequences were identical
to that of the 55-kDa band. ()A polyclonal antibody raised
in rabbits to combined purified peaks 1 and 2 detected the 55-kDa form
in Western blots (Fig. 1B, lane 3) and the two
deglycosylated forms of GH (Fig. 1B, lane 4).
The antibody captured the enzyme activity in a sandwich-type assay (Fig. 1C) verifying its specificity. Although combined
peaks 1 and 2 were used in the study described in Fig. 1,
replication of the experiment described in Fig. 1with each of
the purified individual peaks gave similar results. The
reason for the two bands upon deglycosylation is not presently
understood. It may be due to some residual PNGase F-resistant
carbohydrate or O-linked carbohydrate. It is also possible
that differences occur in other posttranslational modifications or that
some variation in the protein structure exists. Further studies are
under way to resolve this question and to determine whether the
deglycosylated enzyme retains catalytic activity.
C, capture assay of GH. Plates
were incubated with goat anti-rabbit IgG (1:1000, 100 µl,
Tagoimmunologicals) overnight at 4 °C, washed with
phosphate-buffered saline (7 mM sodium phosphate buffer, pH
7.0, containing 140 mM sodium chloride, 3 200 µl),
and incubated with 1% bovine serum albumin for 2 h at 23 °C. The
plates were then incubated with 100 µl of rabbit anti-GH IgG
(1:5000) for 5 h at 23 °C, followed by washing with
phosphate-buffered saline (3 200 µl). GH (200 ng in 100
µl) was added and incubated overnight at 4 °C. Following
washing as above, the reaction was initiated by the addition of 10
µM 4-NH-10-CHPteGlu and the enzyme activity assay (2 h) conducted as described under
``Experimental Procedures.'' 1, assay mixture
lacking anti-GH antibody; 2, complete reaction; 3,
lacking GH. The results are presented as the percent of the total
substrate converted to 4-NH-10-CHPteGlu
(methotrexate). Replacement of anti-GH with protein A-purified prebleed
rabbit serum gave the same results as in lane
1.
Sequencing of Intact GH and Cyanogen Bromide Fragments of
GH
The N-terminal sequences of the Matrex gel green A purified
combined peaks 1 and 2 and purified peak 3 were found to be identical
(GSYERGSKRPIIGII). Peak 3 was purified separately because it yielded
one or two contaminating proteins in many of its preparations. Cyanogen
bromide digestion of either combined peaks 1 and 2 or peak 3 yielded
two fragments (designated band 3 and band 4) with molecular masses less
than 10 kDa. The N-terminal sequence of band 3 from either combined
peaks 1 and 2 or peak 3 was the same (FRNLPEELLN). The N-terminal
sequence of band 4 from purified peak 3 was EGYDYPIYAV, while the same
band from combined peaks 1 and 2 had E instead of D at position 4.
There was some ambiguity assigning the residue at position 4 because
significant amounts of lysine were also determined at this position.
The identical N-terminal and internal structural sequences of combined
peaks 1 and 2 and peak 3 along with the amino acid analysis (Table 1) and immunoreactivity (Fig. 1) suggested
extensive amino acid sequence similarity of the three peaks from
TSK-gel Toyopearl butyl 650S. Analysis of GH from rat hepatocyte and
human cell lines (MCF7, HL60, and HEPG2) by hydrophobic chromatography
resulted in a single peak of enzyme activity. These results suggest
that the apparent heterogeneity of GH from H35 cells on hydrophobic
chromatography may be unique and related to its extensive
glycosylation.
Cloning and Sequencing Strategy for a cDNA Coding for
GH
Portions of the N-terminal sequence and one internal sequence
(band 4) from purified peak 3 were used to construct primers for RT-PCR (Fig. 2). This produced a cDNA fragment (pGH1) composed of bases
100-705. This fragment contained a nucleotide sequence encoding
the protein sequence of band 3. Screening of a commercial cDNA library
using this RT-PCR product identified three clones containing cDNA
consisting of bases 347-1096 (pGH3) and bases 1-902 (pGH4/5).
Using the technique of 3`-RACE (see ``Methods''), a cDNA
fragment was obtained that contained the complete 3` end of the coding
region. A full-length cDNA for the GH coding region was prepared by PCR
amplification using poly(A) mRNA from H35 cells and
primers that flanked the coding region (Fig. 2). The insert
consisted of 1,204 nucleotides. The first 9 nucleotides of the 5` end
of the sequence correspond to part of a consensus sequence for the
initiation of translation in vertebrates(27) .
-glutamyl hydrolase and the
translated amino acid sequence. Determined amino acid sequences are
shown in blue. The primers used for the initial RT-PCR are
shown as white letters against a red background. The
seven consensus sequences for Asn-linked glycosylation are shown in white against a magenta background. The three Cys
residues are in black against a green background. The
primer sequences used to construct a full-length insert are shown in white against a green background.
Characteristics of the Encoded GH
The cDNA coded
for a protein of 317 amino acids. Based on the N-terminal sequence of
the purified enzyme, there is a leader sequence (residues 1-24)
with Gly-25 as the N terminus of the mature enzyme. The calculated
molecular weight of the mature protein was 33.4 kDa, which is within
the range of the deglycosylated proteins (Fig. 1). The
calculated amino acid analysis of the protein was consistent with the
determined amino acid composition of the purified enzyme (Table 1). The deduced amino acid sequence (Fig. 2)
contained seven potential N-linked glycosylation
sites(28) , supporting the observation that the protein is
highly glycosylated. There are three Cys residues in the sequence (Fig. 2). The finding that the enzyme activity of GH is enhanced
by the presence of sulfhydryl-containing compounds (16) and the
observed inhibition of GH by iodoacetic acid suggest that
the enzyme may contain an active site or structurally critical
cysteine.
)-glutamyl hydrolase;
4-NH-10-CHPteGlu,
4-amino-10-methylpteroylglutamyl -glutamate; PNGase F,
peptide-N
-(N-acetyl--D-glucosaminyl)
asparagine amidase; RACE, rapid amplification of cDNA ends; RT-PCR,
reverse transcription polymerase chain reaction; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
CAPS, 3-(cyclohexylamino)propanesulfonic acid.)
We acknowledge the invaluable suggestions of Drs.
Daniel Rosen, Paul Masters, Erasmus Schneider, and Li-Ming Changchen
during the course of this study. The molecular genetics and protein
sequence cores of the Wadsworth Center are thanked for the synthesis of
oligonucleotides and peptide sequence analysis.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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