(Received for publication, January 26, 1996; and in revised form, February 14, 1996)
From the
Purified -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.
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
-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.
A rat hepatoma cDNA library was obtained from Stratagene
(Palo Alto, CA). A total of 10
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.)
cDNA inserts were excised
from the 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.
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.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-CH
PteGlu
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-CH
PteGlu
(methotrexate). Replacement of anti-GH with protein A-purified prebleed
rabbit serum gave the same results as in lane
1.
Figure 2:
Nucleotide sequence of the insert
containing the cDNA encoding -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.
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].