From The Pulmonary Center, Boston University School
of Medicine, Boston, Massachusetts 02118 and
The Laboratory
of Epithelial Cell Biology, Department of Medicine, Renal/Electrolyte
Division, University of Pittsburgh School of Medicine, Pittsburgh,
Pennsylvania 15213
Received for publication, May 19, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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Although use of multiple alternative first exons
generates unique noncoding 5'-ends for The entire intron/exon structure of the mouse
Several human GGT cDNAs also exhibit unique 5'-ends and encode a
protein that is processed in a similar fashion and shares 79% amino
acid identity with that of the mouse (3). In addition, an alternatively
processed human GGT cDNA has been described that contains an
insertion of 22 bases within the coding domain. The extra nucleotide
bases introduce a frameshift and a premature stop codon so that the
predicted polypeptide would be a truncated GGT isoform. The protein
product has never been characterized, but the elimination of the small
subunit suggests that it would lack However, while characterizing the site of a point mutation in
the GGTenu1 mouse (5), we identified four
previously unknown alternative splicing events involving coding exons
in the normal mouse GGT gene (see Fig. 1). We studied these
events to determine whether GGT protein isoforms are being generated
through alternative processing of mouse GGT mRNA, to identify if
these events are shared between mouse and human GGT, and finally to
explore a potential role for these new mouse GGT protein isoforms in
glutathione metabolism.
Source of Probes and Tissues--
Tri-Reagent (Molecular
Research Center, Inc., Cincinnati, OH) was used according to the
manufacturer's protocol to isolate total RNA from tissue or cells.
Mouse genomic DNA was isolated from the lung. Human genomic DNA was
provided by Dr. Qiang Yu from the Pulmonary Center of Boston
University. Electrophoresis grade agarose was from International
Biotechnologies, Inc. (New Haven, CT), and DNA standards were from Life
Technologies, Inc. Materials for protein electrophoresis were from
Bio-Rad (Richmond, CA), and protein standards were High-Range molecular
weight markers from Amersham Pharmacia Biotech (Piscataway, NJ). X-OMAT
and BioMax MR films were used for radiography and obtained from Eastman
Kodak Co. (Rochester, NY). [ Genomic PCR, RT-PCR, Subcloning, and Sequencing--
Total RNA
from various tissues was used for RT-PCR as described previously (5).
PCR primers were selected for primary and secondary PCR reactions, and
each PCR reaction was performed for 20 cycles on a MJ Research thermal
cycler. PCR products were analyzed by agarose gel electrophoresis, then
eluted, cloned into an Invitrogen TA vector (San Diego, CA), and
sequenced for verification at the DNA-Protein Core facility at Boston
University School of Medicine. RT-PCR primers used for
constitutive GGT cDNA amplification were M72,
5'-CCTTTCGGTTTGCCTATGCCAAGAGGAC (upstream); 11M,
5'-GCGCTCCCTCTGTCCCACCCA (nested downstream); and 12M,
5'-GGCTTCCCGCAGCTTGGCGGTGG (primary downstream). For intron 7 and
intron 9 insertions, upstream hemi-nested primers were 22U,
5'-GCCAGCTCTGGGGTCTCGGCAG and 24D, 5'-CCTGTCTCCTCCTATGGATCATAG, respectively. The primary primers used for genomic PCR and cloning of intron 7 were M71, 5'-AGGCACTGACGTATCACCGTATCGTG and 10.1M, 5'-CCTCCATCATCCTGAAGGTAGA. Primers M72 plus 10.1M were used in secondary PCR. Intron 9 was cloned using M811,
5'-ACCGCTCACCTGTCTGCGGTTTC as the upstream primer for both PCR
reactions together with downstream primers 12M and 11M for primary and
secondary PCR, respectively. The relative mRNA abundance of GGT Transcription/Translation in Vitro--
TnT T7 Quick-Coupled
Transcription/Translation System, a rabbit reticulocyte lysate system,
was purchased from Promega and used according to the manufacturer's
instructions. Mouse GGT constructs were generated by PCR using
upstream primers with
(CGGACCGGGCCCTACTGGAAGCAGACCATGAAGA) and without
(CGGACCGGGCCCTACTGGAAGACCATGAAGAATC) the CAG insertion 5 bases
upstream of the ATG initiation codon along with a common 3' primer
(CCGGAATTCCCGCTGAGTGGGGCACTGGGCACG). Template cDNA was reverse-transcribed from normal mouse kidney mRNA. The 5' primers contained an EcoRI and the 3' primer an ApaI site
that were used to clone the PCR products into pCR 2.1 (Invitrogen).
Plasmids were sequenced to ensure accuracy, and the T7 promoter was
used for in vitro transcription. The translated products
were labeled with [35S]methionine, separated on a 12%
polyacrylamide gel, and visualized by autoradiography. The primary
translation product was predicted to have a molecular mass of 35.2 kDa
with the terminal 47 amino acid residues encoded from the vector. A
secondary translation product of 27 kDa was predicted from utilization
of an internal translation initiation site.
Analysis of RNA--
Total RNA was analyzed as previously
described (5). RNA obtained from cell lines was quantitated by
spectrophotometry and electrophoresed on a 1.0% agarose gel with 2.2 M formaldehyde in 1× MOPS, transferred to a HyBond
membrane (Amersham Pharmacia Biotech, Arlington Heights, IL) overnight,
both at RT, then cross-linked with a Stratagene UV cross-linker. The
membrane was prehybridized with QuickHyb (Stratagene) at 68 °C for
15 min. Radiolabeled probe was then added and incubated for 2 h.
The filter was washed twice at room temperature with 2× SSC plus 0.1%
SDS, washed twice more with 1× SSC in 0.1% SDS at 60 °C, and
dried. An exposure of the filter was made on Kodak X-OMAT film, and the
film was developed.
Stable Expression of mGGT Isoforms in CHO Cells--
A
full-length mouse GGT cDNA (type 3) was kindly provided by Drs.
Z.-Z. Shi and M. W. Lieberman (Baylor College of Medicine, Houston, TX). This was cloned into the expression vector pCDNA3.1 (Invitrogen). To express the alternatively spliced GGT cDNAs, this
wild type GGT cDNA sequence was replaced with that of GGT
Plasmids encoding the mGGT isoforms for cell transfections were
prepared with the JETstar 2.0 plasmid purification kit (Genomed, PGC
Scientifics, Frederick, MD). Stable clonal cell lines were obtained by
transfection of Chinese hamster ovary cells (CHO) using LipofectAMINE
(1:3 ratio of DNA:lipid; Life Technologies Inc., Gaithersburg, MD)
followed by selection in media containing G418 (0.5 mg/ml).
Transient Expression of mGGT Isoforms in CHO
Cells--
Transient expression of the mGGT isoforms was obtained in
CHO cells using a cowpox/bacteriophage T7 (vT7CP) expression system as
described previously (6, 7). Confluent cultures of CHO cells were grown
in 35-mm plastic dishes with a 1:1 mixture of DMEM and Ham's F-12
(1:1) supplemented with 3% FBS (normal culture media). Cells were
washed with 1 ml of serum-free medium and infected with vT7CP
(multiplicity of infection ~30) in 0.3 ml of the same media for 30 min. The media containing vT7CP was removed from the cells before
transfection with a mixture of plasmid DNA (pCDNA3.1 with a T7
promoter) and LipofectAMINE (Life Technologies) at a 1:3 ratio in 1 ml
of the same serum-free media for 3 h. The lipid and DNA mixture
was them removed, and the cells were washed with 1 ml of DMEM media
lacking methionine (Met) and cysteine (Cys) (ICN, Costa Mesa, CA) and
returned to culture for 15 min in the same media prior to metabolic labeling.
Metabolic Labeling of mGGT Isoforms in CHO Cells--
Either
stably transfected clonal cell lines expressing the mGGT isoforms or
CHO cells transiently expressing the mGGT isoforms (described above)
were starved for Met and Cys for 15 min in 1 ml of DMEM media lacking
Met and Cys before addition of 50-100 µCi of
[35S]Met/Cys for the times indicated in each experiment.
Cells were chased in normal culture medium as indicated. Surface levels
of the mGGT isoforms were determined by moving the dishes of cells to
ice for biotinylation (8) as previously described (12). Briefly, cells
were washed four times with 1 ml of PBS++ (137 mM NaCl, 2.6 mM KCl, 15.2 mM
Na2HPO4, 1.47 mM
KH2PO4, 0.5 mM MgCl2
and 0.7 mM CaCl2) then incubated with 0.5 mg/ml
sulfo-NHS-SS-biotin (Pierce, Rockford, IL) in triethanolamine-buffered
saline (10 mM triethanolamine, pH 7.6, 137 mM
NaCl, and 1 mM CaCl2) for 10 min. The reaction
was quenched by three washes of the cells with normal culture media.
Cells were solubilized as described above, and the supernatants were
rotated end over end overnight at 4 °C after addition of protein G
immobilized on Sepharose 4B (Sigma Chemical Co., St. Louis, MO) and 1 µl of goat anti-rat GGT antisera. Immunoprecipitates were recovered
by brief centrifugation and washed once with 0.5 ml each of 1% Triton
X-100 (Roche Molecular Biochemicals Corp., Indianapolis, IN) in HBS (10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl), 0.01% SDS
in HBS and HBS alone. Biotinylated mGGT was recovered by eluting the
immunoprecipitates for 2 min at 90 °C in 80 µl of 1% SDS in HBS
and further incubation with 30 µl of ImmunoPure Immobilized Avidin
(Pierce, Rockford, IL) after addition of 0.8 ml of HBS. After overnight
rotation at 4 °C, the avidin-conjugated beads were washed with 1 ml
each of 1% Triton X-100 in HBS, 0.01% SDS in HBS, and HBS alone. The
biotinylated mGGT isoforms were eluted by heating for 3.5 min at
90 °C in 50 µl of Laemmli SDS-sample buffer containing fresh 0.14 M Endoglycosidase H Treatment of
Immunoprecipitates--
Radioactive immunoprecipitates were
resuspended in 80 µl of 10 mM citrate buffer, pH 5.0, and
0.5% SDS by heating beads at 90 °C for 2 min (9). The eluted sample
was divided in half and incubated overnight at 37 °C with or without
1 milliunit (mU) of endoglycosidase H (endo H, Roche Molecular
Biochemicals) in the presence of protease inhibitors (Protease
Inhibitor Mixture Set III, Calbiochem, La Jolla, CA).
Immunofluorescence Microscopy--
COS cells were grown on glass
coverslips in 35-mm dishes, transiently transfected with the cDNAs
for the mGGT isoforms using LipofectAMINE, and analyzed by
immunofluorescence 2 days later. All steps were carried out at room
temperature. Cells were washed once with PBS++ for 5 min
prior to fixation with 3% paraformaldehyde in PBS++ for 12 min. After washing with 10 mM glycine in PBS++
(PBS-Gly), cells were permeabilized by incubation for 4 min in 0.5%
Triton X-100 in PBS-Gly and blocked with 5% goat serum in PBS-Gly for
5 min. Cells were incubated with rabbit anti-rat GGT antisera (diluted
1/600) for 45 min and then CY3-conjugated goat anti-rabbit IgG (diluted
1/2500; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for
30 min, both in 1% goat serum in PBS-Gly. Cells were incubated for 5 min with 0.5 µg/ml 4'-6-diamidino-2-phenylindole (Molecular Probes,
Eugene, OR) in PBS-Gly to stain nuclei and then washed twice for 5 min
with PBS-Gly before mounting on slides with 1 M
n-propylgallate in glycerol and viewing with a Nikon Optiphot-2 microscope. Calnexin was stained as a positive control for
localization in the endoplasmic reticulum (ER) using an antibody directed against the cytosolic tail (StressGen Biotechnologies Corp.,
Victoria, Canada).
Enzyme Activity--
Specific GGT enzyme activity was measured
at room temperature using the substrates Mouse GGT
To see if the proximity of this CAG insert to the ATG initiation site
could affect the site of translation initiation, we used a
transient VT7CP expression system to characterize the protein encoded
by the GGT
Similar data were obtained for the synthesis of GGT Mouse GGT
When CHO cells transiently transfected with the GGT
The persistence of the endo H sensitivity of the GGT Mouse GGT
We used the 22-base insert as one of the primers in a PCR reaction to
determine whether the GGT
To compare the relative abundance of GGT
When CHO cells transiently expressing the GGT Mouse GGT
To determine if
The elimination of the terminal four bases of exon 8 plus all of exon 9 together with the insertion of 24 bases from intron 9 removes 44 amino
acids residues from the C-terminal small subunit but adds 8 novel amino
acids proximal to active enzyme site residues (Fig. 8D).
When CHO cells transiently transfected with the GGT Stress Response Induction in the Endoplasmic
Reticulum--
Because GGT Herein we have characterized four new GGT protein isoforms that
are derived from alternative splicing events in mouse GGT cDNA. The
mouse and the human not only generate GGT protein isoforms by this
mechanism, but they share some of these splicing events in common. The
encoded GGT protein isoforms can be expressed as transferase active
heterodimeric glycoproteins on the cell surface or as transferase
inactive monomeric glycoproteins in the endoplasmic reticulum. These
latter findings suggest potentially novel functions for native GGT
protein and its protein isoforms within the endoplasmic reticulum in
addition to the known role of the native protein as a cell surface ectoenzyme.
Alternative pre-mRNA splicing of nascent eukaryotic mRNAs is a
post-transcriptional process that is known to be highly regulated and
widely utilized to generate multiple alternative products from a single
gene (14). A prime example of the power of alternative promoters to
generate such mRNA diversity can be found in the mouse
GGT gene itself. Six GGT cDNAs, each with a unique
5'-untranslated region, are generated from this single copy gene via
six alternative promoters (15). Alternative splicing events within the
open reading frame can generate protein isoforms by excluding specific exon sequences or including novel intron sequences in the mature mRNA transcript. However, prior to our study, only a constitutive splicing pattern had been identified in the mouse GGT gene,
which involves the invariant ligation of 12 coding exons, numbered by Arabic numerals 1 through 12, and one common
noncoding exon, numbered by Roman numeral I as
depicted in Fig. 1 (1).
The central difference between constitutive and alternative splicing
lies in the selection and ligation of specific pairs of donor/acceptor
splice sites. This is a complex process that is only partially
understood, but it is clear that certain cis RNA sequences
provide recognition sites for trans-splicing factors that
form the spliceosome apparatus. These cis RNA sequences
include the dinucleotides GT and AG located at the 5'- and the
3'-boundaries of an intron, respectively (16). These conserved
dinucleotide sequences are found at all of the intron boundaries in the
constitutively spliced mouse GGT gene (1). The factors
involved in the selection of these specific GT and AG residues from a
much large number of potential choices are not yet fully known for any
gene. However, additional consensus sequences surrounding these
dinucleotides as well as internal consensus sequences at the branch
point participate in the selection process. Selection of the AG residue
at the 3'-splice site appears to involve a scanning process initiated
from the branch point and a competition among various intervening AG
dinucleotides with the nucleotide preceding the AG having a profound
influence on its selection in the order CAG~TAG > AAG > GAG (16). In GGT In GGT GGT Our PCR data on the relative expression of GGT The dinucleotides delimiting the possible alternative 5'- and 3'-splice
sites in GGT Nonetheless, the expression of GGT-glutamyltransferase (GGT)
cDNAs in several species, we show here that alternative splicing
events also alter coding exons in mouse GGT to produce at least four protein isoforms. GGT
1 introduces CAG four bases upstream of the
primary ATG codon and encodes an active GGT heterodimeric ectoenzyme
identical to constitutive GGT cDNA but translational efficiency is
reduced 2-fold. GGT
2-5 deletes the last eight nucleotides of exon 2 through most of exon 5 in-frame, selectively eliminating residues
96-231 from the amphipathic N-terminal subunit, including four
N-glycan consensus sites, while leaving the C-terminal hydrophilic subunit intact. GGT
7 introduces 22 bases from intron 7 causing a
frameshift and a premature stop codon so a truncated polypeptide is
encoded terminating with 14 novel residues but retaining the first 339 residues of the native GGT protein. GGT
8-9 deletes the terminal
four nucleotides of exon 8 plus all of exon 9 and inserts 24 bases from
intron 9 in-frame so the C-terminal subunit of the encoded polypeptide
loses residues 401-444 but gains eight internal hydrophobic residues.
In contrast to the product of GGT
1, those derived from GGT
2-5,
7,
8-9 all lack transferase activity and persist as single-chain
glycoproteins retained largely in the endoplasmic reticulum as
determined by immunofluorescence microscopy and constitutive
endoglycosidase H sensitivity in metabolically labeled cells. The
developmental-stage plus tissue-specific regulation of the alternative
splicing events at GGT
7 and GGT
8-9 implies unique roles for
these GGT protein isoforms. The ability of the GGT
1 and GGT
7 to
mediate the induction of C/EBP homologous protein-10, CHOP-10, and
immunoglobulin heavy chain binding protein, BiP, implicates a specific
role for these two GGT protein isoforms in the endoplasmic reticulum
stress response.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glutamyltransferase gene (EC 2.3.2.2,
GGT)1 has been defined. This
single copy gene is regulated by multiple alternative promoters that
are coupled with alternative splicing mechanisms to generate several
GGT cDNAs each with a unique 5'-noncoding region. But all encode
the same protein, because coding exons appear to be spliced only in a
constitutive fashion (1). The protein product is synthesized as a
single-chain N-glycosylated propeptide, processed into an
N-terminal amphipathic subunit and a smaller C-terminal subunit, and
expressed on the cell surface where it functions as a key enzyme in
glutathione metabolism (2).
-glutamyltransferase activity.
The identification of this alternative transcript implies that human
GGT gene expression may be more complex than that of other
species (4).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]CTP, specific
activity 800 Ci/mmol, was obtained from ICN (Irvine, CA) and
[35S]Met/Cys, specific activity ~1000 Ci/mmol, was from
PerkinElmer Life Sciences (Wilmington, DE) as Easy Tag
Express-[35S]Protein Labeling Mixture. Primers were
synthesized at the DNA/Protein Core Molecular Biology Unit of
Boston University School of Medicine. The cDNA probes for CHOP and
BiP were generated by PCR after selecting primers based on the
published sequences in the GenBankTM. The probe for
-actin has been
used routinely in this laboratory. The cell lines hCAR (human coxsackie
and adenovirus cell surface receptor) and hMUC (human mucin 1) were
available in the laboratory of Dr. Rebecca Hughey (12).
7
versus constitutive GGT was determining by using primers 71M
plus 11M for the primary reaction, then 72M plus 810M,
5'-GAAACCGCAGACAGGTGAGCGGTGCCTCC, for the secondary reaction.
1,
2-5,
7 and
8-9. All plasmids were sequenced to ensure accuracy.
-mercaptoethanol. Samples were subjected to
SDS-PAGE on 3-15% polyacrylamide gradient gels, and radioactive
protein bands imaged and quantitated from the dried gel using a
PhosphorImager (Bio-Rad, Richmond, CA).
-glutamyl-para-nitroanilide
and glycylglycine as previously described (10). Protein was determined
by the method of Lowry.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1--
The majority of our PCR-derived GGT clones
generated from lung and kidney RNA of wild type and
GGTenu1 mice contained the trinucleotide
insert CAG five bases upstream of the ATG initiation codon (details in
Fig. 1). An examination of GGT cDNA
sequences in the GenBankTM revealed an absence of this CAG insert in
all mouse GGT cDNAs, as well as those from rat and pig, but
revealed its presence in most, but not all, human GGT cDNAs.
Certain human lung GGT cDNAs lacked this CAG insert (11). Because
this CAG insertion is located at an intron/exon junction, we compared
the intron sequences from the mouse, rat, and pig to determine whether
alternative splicing could account for its presence (Fig.
2A). All three introns contain
the highly conserved dinucleotides GT and AG at the 5'- and
3'-boundaries, respectively, and the AG is preceded by a cytosine
residue. However, the mouse intron contains two CAGs in tandem at its
3'-intron boundary, whereas the rat and the pig intron each contain a
single CAG. We then cloned and partially sequenced a corresponding
human GGT intron sequence. We confirmed that this intron is identical
in size to that of rat, mouse, and pig, ~0.5 kb, and contains two tandem CAG trinucleotides at the 3'-boundary like that of mouse.
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Fig. 1.
Alternative splicing events in mouse
GGT. This schematic depicts the constitutive (1) and
the alternative ( ) splicing events of mouse GGT mRNA
and the protein isoforms that result based on data in Table I and Figs.
2-8.
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Fig. 2.
Characterization of
GGT 1. A, comparison of
nucleotide sequences at the intron boundary between mouse, rat, pig,
and human GGT. Uppercase letters are cDNA, and
lowercase letters are intron sequences. Dinucleotides at
intron boundary are in boldface. B, an in
vitro transcription/translation assay as described under
"Experimental Procedures" using constructs which lack (
) or
contain (+) the CAG insertion as seen in constitutive mGGT or GGT
1,
respectively. Molecular weight markers (M.W.) are shown to
the left of the internal positive control (Ctl.).
The primary translation product is marked at 35 kDa. The translation
product from an internal initiation site is marked at 27 kDa.
1. Cow pox-infected CHO cells transfected with the GGT
1
plasmid were starved for Met and Cys and pulse-labeled in the same
media with [35S]Met/Cys, before a chase period of 0 or
2 h. SDS-PAGE analysis of GGT-specific immunoprecipitates from the
cell extracts revealed a single peptide of 83 kDa at t = 0, which was sensitive to treatment with endoglycosidase H and
produced a product of 59 kDa (Fig. 3),
consistent with expression of the full-length propeptide of 61 kDa
(Table I). Because endo H removes
all but one GlcNAc residue of high mannose N-glycans
(Mr ~ 3000) from glycoproteins, this difference in Mr (2400) is consistent
with N-linked glycosylation of the GGT
1 at all seven
consensus sites (Asn-X-Ser/Thr). After 2 h of chase,
only a trace of the 83-kDa propeptide was evident, whereas the two
expected subunits of the cleaved GGT propeptide were present at 50 and
23 kDa. Endo H treatment of this sample produced trace bands at 33 and
20 kDa, consistent with the presence of six N-glycans and
one N-glycan on the subunits, respectively. The more diffuse
gel pattern of the large subunit indicated that there is considerable
microheterogeneity in the processing of these N-glycans,
whereas the small subunit was more homogeneous despite its resistance
to endo H treatment, indicating that the single N-glycan is
minimally processed.
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Fig. 3.
Transient expression of mouse GGT isoforms in
CHO cells. Confluent cultures of CHO cells in 35-mm wells were
infected with cow pox for 30 min before transfection for 2 h with
18 µg of LipofectAMINE and either no plasmid (mock) or 6 µg of plasmid DNA encoding GGT 1, GGT
2-5, GGT
7, or
GGT
8-9. Cells were pulse-labeled for 15 min with
[35S]Met/Cys prior to chase periods of 0 or 2 h,
before extraction with octyl glucoside and immunoprecipitation of the
GGT-related peptides with a goat polyclonal antibody and subsequent
treatment overnight with (+) or without (
) endo H. Samples were
subjected to SDS-PAGE and PhosphorImager analysis. Numbers
to the right refer to the mobility of molecular mass
standards in kDa.
Characteristics of mouse GGT protein isoforms
1 in stable
transfectants of CHO cells (Fig. 4). When
these clonal CHO cells expressing GGT
1 were pulse labeled for 30 min
and chased for 2 h prior to cell surface biotinylation with the
membrane impermeant sulfo-NHS-SS-biotin, ~20% of the heterodimer was
recovered from the immunoprecipitates with avidin-conjugated beads.
This indicated that the GGT
1 protein did reach the plasma membrane (compare mock lanes in Fig. 4, A and
B). This cell surface localization of the GGT
1 was
confirmed by immunofluorescence analysis of COS cells 2 days after
transfection with the GGT
1 cDNA (Fig.
5A). A similar pattern of
immunofluorescence was observed in stably transfected CHO cells
expressing the GGT
1 (data not shown). In both cases nearly all the
GGT-specific immunofluorescence was found at the cell surface. An
equally important result is that the clonal CHO cells expressing
GGT
1 exhibited a much higher
-glutamyl transferase-specific
enzymatic activity (280 mU/mg) than nontransfected CHO cells (<1
mU/mg). And after overnight accumulation of GGT
1 in transfected cow
pox-infected CHO cells, this enzyme activity was greatly increased
(200-1000 mU/mg). Because this latter result indicated that the
GGT
1 protein must be relatively stable, the half-life was determined
for the radiolabeled GGT
1 during stable and transient expression by
pulse-labeling cells for 30 min and immunoprecipitating GGT
1 after
chase times of 3 and 18 h (Table I). Calculation of the half-life
from the percent loss of radiolabeled GGT between the two time points
indicated the half-life for the GGT
1 is similar in stable (18.7 h)
and transiently transfected (17.4 h) CHO cells. Thus GGT
1 encodes the normal mouse GGT protein. This protein exhibited normal synthesis, cell surface expression, stability, and enzymatic activity. Because the
expression of GGT
1 in cultured cells was indistinguishable from that
described previously for rat and human GGT, we next assessed whether
the CAG insert in the mouse GGT
1 could affect translational
efficiency using an in vitro transcription/translation assay. The synthesis of the primary translation product (35.2 kDa) from
the transcript containing the CAG insert was reduced ~2-fold when
compared with synthesis from a transcript lacking the CAG sequence,
whereas synthesis of an alternate protein from an internal ATG codon
(27 kDa) was unchanged (Fig. 2B). Thus, the CAG appears to
regulate translation of the GGT mRNA, not the protein product.
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Fig. 4.
Only GGT 1 is
expressed at the cell surface. Confluent cultures of clonal CHO
cells stably expressing the GGT
1 in 21-mm wells were infected with
cow pox for 30 min before transfection with 9 µg of plasmid DNA
encoding GGT
2-5, GGT
7, or GGT
8-9 (mock received
buffer and no DNA). The next day, cells were pulse-labeled for 30 min
with [35S]Met/Cys prior to a 2-h chase period, before
biotinylation of the cell surface on ice, extraction with octyl
glucoside, and immunoprecipitation of the GGT. A portion of the
immunoprecipitate (75%) was further incubated with avidin-conjugated
beads to obtain the biotinylated cell surface GGT. Both a portion of
the total immunoprecipitate (B, 25% of total) and the
biotinylated surface GGT (A) were subjected to SDS-PAGE and
PhosphorImager analysis. Numbers to the right
refer to the mobility of molecular mass standards in kDa. The mobility
of the large (L) and small (S) subunits of the
GGT
1 heterodimer are shown on the left, and the
asterisk indicates protein isoforms. The diffuse gel pattern
of the GGT
1 large subunit indicates that there is considerable
microheterogeneity in the processing of the N-glycans. This
band is less evident in B, because these lanes
represent only 25% of each immunoprecipitate.
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Fig. 5.
Immunofluorescence analysis. COS cells
were transfected with plasmids encoding GGT 1 (A),
2-5
(B),
7 (C), and
8-9 (D). The
steady-state localization of the expressed proteins were analyzed with
rabbit anti-rat GGT antiserum followed by fluorescein
isothiocyanate-labeled goat anti-rabbit IgG. COS cells (E)
were stained for calnexin as a positive control for an ER protein, and
primary antibody was omitted for a negative control (F).
Surface staining is observed only for mGGT
1. Although
2-5,
7,
and
8-9 predominantly show staining in the endoplasmic reticulum, a
low level of juxtanuclear Golgi-like staining is also seen for all the
proteins.
2-5--
This alternative splicing event produces an
in-frame deletion of the last eight nucleotides of exon 2, all of exons
3 and 4, and most of exon 5 (Fig.
6A). Nonconsensus 5'- and
3'-splice sites appear to have been utilized in exons 2 and 5, respectively. We could not identify a corresponding human GGT mRNA
transcript but did find at least two alternative rat GGT cDNAs
using mRNA from cultured rat alveolar type 2 cells that utilized
the same 5'-splice site (data not shown). The encoded protein loses
amino acid residues 96 through 231, including four N-glycan
consensus sites from the amphipathic N-terminal subunit, while
the C-terminal hydrophilic subunit remains intact (Fig.
6B).
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Fig. 6.
Characterization of
GGT- 2-5. A, an event resulting from
usage of 5'- and 3'-splice sites within exon 2 and exon 5, respectively. The last eight nucleotides of exon 2, all of exons 3 and
4, and most of exon 5 are eliminated. B, the encoded protein
loses amino acid residues 96 through 231, including four
N-glycan consensus sites from the amphipathic N-terminal
subunit, whereas the C-terminal hydrophilic subunit remains
intact.
2-5 were
pulse-labeled with [35S]Met/Cys for 15 min, a single
labeled protein of 47 kDa was immunoprecipitated after both 0- and 2-h
chases (Fig. 3). At both time points the protein was sensitive to endo
H treatment producing a protein of 38 kDa, consistent with the presence
of three N-glycans, but which is notably smaller than the
predicted sequence (46.7 kDa). Because the protein remains anchored to
the membrane (data not shown), this indicates that the GGT
2-5
either is cleaved after the N-glycan consensus site in the
small subunit or migrates anomalously on SDS gels.
2-5 protein
after the 2-h chase also suggests that the protein is retained in the
ER or early Golgi rather than moving to the cell surface. When this was
tested by transient expression of GGT
2-5 in CHO-GGT
1 cells, only
1.5% of the GGT
2-5 was biotinylated at the cell surface after a
2-h chase, whereas ~20% of the GGT
1 was biotinylated in the same
cells (Fig. 4). Localization in the ER was also evident by
immunofluorescence microscopy of COS cells transiently expressing the
GGT
2-5 (Fig. 5B). Determination of the half-life for the GGT
2-5 in both stable transfected (1.7 h) and transiently
transfected (2.1 h) CHO cells indicates that this isoform of the mouse
GGT is considerably less stable than the GGT
1 (Table I). Enzyme assays of these cell extracts reveal no increase in
-glutamyl- transferase activity (<1 mU/mg) above the control levels observed in
nontransfected CHO cells.
7--
This alternative splicing event introduces a
22-base insertion within the coding domain. This insertion mimics that
in humans and induces a frameshift and a premature stop codon within
the open reading frame (Fig.
7A). The encoded mouse protein
is a truncated GGT-like protein that retains only the first 339 amino
acids of the native GGT protein and gains 14 novel residues at the C
terminus, the first seven of which are identical to that in the human
(Fig. 7B). Because the intron/exon structure of mouse GGT is
known and the last 10 nucleotides at the 3' terminus of the insert
agree exactly with the intron 7 sequences in the literature (1), it
appears that the 22-base insertion results from an alternative splice
site at intron 7. To confirm this, we cloned and partially sequenced
the ~3 kb of mouse intron 7 (Fig. 7A). The remaining 10 nucleotides at the 5'-end of the insertion were identified as intron 7 sequences, and they were preceded by a CAG as a 3'-splice site. This
22-base mouse intron sequence is identical to the corresponding human
intron sequence (Fig. 7C).
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Fig. 7.
Characterization of
GGT 7. A, as a result of this
event, 22 bases are inserted (boldfaced) into the mouse
7
cDNA, and this alters the reading frame from that of mGGT cDNA.
B, a truncated mouse GGT protein (Mm) is encoded
with a new C terminus that is similar to that predicted from an
alternatively processed human GGT cDNA (Hs).
C, this 22-base insertion in mouse GGT cDNA is identical
to that described in human GGT cDNA and human lung-specific group
II GGT cDNAs (Gp II) and very similar to sequences of a
human GGT intronic promoter. D, PCR analysis as described
under "Experimental Procedures" with primer 22U shows that this
alternative splicing event is detectable in newborn mouse heart
(Ht) but not liver (Li) nor lung (Lu).
Adult lung (+) was amplified with constitutive primers to serve as the
positive control in this PCR reaction. In a separate PCR reaction for
adult tissues, GGT
7 is undetectable in heart (Ht) and
liver (Li) but evident in lung (Lu), kidney
(Ki), and thymus (Thy). E, a PCR
analysis was also performed as described under "Experimental
Procedures" to compare the relative abundance of GGT
7 (230-bp
product) versus constitutive mGGT (208-bp product) in heart
(Ht), liver (Li), lung (Lu), kidney
(Ki), and brain (Br).
7 splicing event is regulated in a
developmental or tissue-specific fashion. We analyzed heart, liver,
lung, kidney, and thymus RNA obtained from 1-day-old and adult mice
(Fig. 7D). We detected GGT
7 in the RNA from the heart but
not that from the liver or the lung during the neonatal period. RNA
from the adult lung served as a positive PCR control here. In adult
tissues, GGT
7 was present in RNA from the lung, the kidney, and the
thymus but absent from that of the heart and the liver. It was also
detectable in mRNA isolated from adult rat kidney and human
peripheral blood mononuclear cells (data not shown).
7 to constitutive mGGT, we
amplified the mRNAs from five different tissues using two sets of
PCR primers that were common to both transcripts with a fully nested
design (Fig. 7E). The 208-base pair signal amplified from
constitutive mGGT was evident in all samples. The 230-base pair signal
amplified from GGT
7 was only evident in lung, kidney, and brain and
was less abundant than that of the constitutive GGT signal.
7 were pulse-labeled
for 15 min with [35S]Met/Cys a single product of 44 kDa
was obtained after chase times of both 0 and 2 h (Fig. 3). Endo H
treatment produced a peptide of 32 kDa in both cases, which was
slightly smaller than the peptide size calculated from the predicted
sequence (38.2 kDa), but indicates that N-glycans are
present at four of the five consensus sites in the GGT
7. When the
cell surface of CHO-GGT
1 cells transiently expressing the GGT
7
were biotinylated after a 30-min pulse with [35S]Met/Cys
and a 2-h chase, only 0.5% of the GGT
7 was recovered with
avidin-conjugated beads while 37% of the GGT
1 was recovered from
the same cells (Fig. 4). Immunofluorescence microscopy of COS cells
transiently expressing the GGT
7 (Fig. 5C) revealed staining of the ER, which is consistent with both the persistence of
endo H sensitivity and the minimal cell surface expression of this
isoform. Enzyme assays for both CHO cells stably and transiently expressing GGT
7 revealed no increase in
-glutamyltransferase activity (<1 mU/mg) above the control levels observed in
nontransfected CHO cells. The half-life for the GGT
7 in these same
cells (6.5 and 10.2 h, respectively) was approximately half of
that observed for the GGT
1 (Table I).
8-9--
The
8-9 alternative splicing event
eliminates at least the last four nucleotides of exon 8 and all of exon
9 but introduces 24 novel bases (Fig.
8A). The last 10 of these 24 nucleotides are identical to the published sequence at the 3'-boundary
of intron 9 (1). This intron was cloned and sequenced, and the remaining 14 nucleotides were confirmed as intron 9 sequences, which
were preceded by a CAG sequence. If this CAG represents the 3'-intron
boundary, then the 5'-splice site lies within exon 8 but it is a
nonconsensus splice site. However, other nonconsensus dinucleotide
sequences could also border these boundaries as denoted in Fig.
8A, so the exact location of this splice site is unclear. This GGT cDNA has not been described in humans and we did
not detect any PCR product when this 24-base insert was used as a PCR
primer with human cDNA (data not shown). To confirm this, we cloned
and sequenced a corresponding human intron. The 67 nucleotides at the
3'-boundary of our clone matched perfectly with sequences in human
GGT genes 3, 6, and 11 (data not shown). However, comparison with mouse intron 9 revealed neither conserved sequences in this region
nor any conserved 3'-splice site sequences (Fig. 8B). Usage of the 24 corresponding human nucleotides also failed to produce a PCR
product. Hence, this particular GGT splicing event may be specific to
mouse.
View larger version (38K):
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Fig. 8.
Characterization of
GGT- 8-9. A, this exact site of the
splicing event for
8-9 is unclear but appears to involve
alternative nonconsensus 5'- and 3'-splice sites within exon 8 and
intron 9, respectively. The last four nucleotides of exon 8 are
eliminated, and 24 bases from intron 9 (boldfaced) are
inserted into the GGT
8-9 cDNA. B, comparison of the
terminal 25 nucleotides from mouse (Mm) intron 9 with those
from the corresponding human (Hs) intron reveals many
differences. C, PCR analysis for developmental and
tissue-specific expression of
8-9 was performed as in Fig. 7, but
the upstream primer in the secondary PCR reaction was 24D.
D, the amino acid residues that are eliminated from the
small C-terminal subunit of mGGT are boxed, and the eight
novel amino acids of
8-9 are boldfaced. These
hydrophobic residues are inserted proximal to the active enzyme site of
mGGT (underlined).
8-9 was expressed in a developmental or
tissue-specific fashion, we repeated the analysis outlined for GGT
7 but used the 24-base insert as one PCR primer. GGT
8-9 was not detected in RNA from the heart, the liver, or the lung during the
neonatal period, even though it was present in the adult lung-positive control. In adult tissues, it was evident in RNA from the lung and the
kidney but not that from the heart, the liver, or the thymus (Fig.
8C). Hence, GGT
8-9 exhibits developmental-stage and
tissue-specific regulation, but the patterns differ from that of
GGT
7.
8-9 were
pulse-labeled with [35S]Met/Cys for 15 min, a single
labeled protein of 52 kDa was immunoprecipitated after both 0- and 2-h
chases (Fig. 3). At both time points the band was sensitive to endo H
treatment producing a protein of 37 kDa, consistent with the presence
of five N-glycans on the protein. However, this is two less
N-glycans than would be predicted from the GGT
8-9
sequence. In addition, the protein size for the GGT
8-9 calculated
from the predicted sequence is considerably larger (57.6 kDa). The
cumulative data would be most consistent with cleavage of the
GGT
8-9 into an unstable heterodimer, immediate degradation of the
small subunit, and retention of the residual large subunit within the
ER. When the cell surface of CHO-GGT
1 cells transiently expressing
the GGT
8-9 were biotinylated after a 30-min pulse with
[35S]Met/Cys and a 2-h chase, only 2.2% of the
GGT
8-9 was recovered with avidin-conjugated beads while 35% of the
GGT
1 was recovered from the same cells (Fig. 4). Immunofluorescence
microscopy of COS cells transiently expressing the GGT
8-9 (Fig.
5D) revealed staining of the ER, which is consistent with
both the persistence of endo H sensitivity and the poor cell surface
expression of this isoform. The half-life of the GGT
8-9 in
transiently transfected cells was only 0.9 h, and there was no
measurable
-glutamyltransferase enzymatic activity. Although stably
transfected cells were not available for these studies, the similar
data obtained for the GGT
1, GGT
2-5, and GGT
7 between
transient and stably transfected cells (Table I) indicates that the
half-life and the enzyme activity data for GGT
8-9 in transiently
transfected cells are reliable.
1 is normally found at the cell surface,
the localization of the GGT
2-5,
7, and
8-9 in the ER could
simply indicate that these GGT isoforms are abnormally folded products and they are being retained for subsequent degradation. Alternatively, this subcellular localization could be consistent with a new previously undefined role for these proteins within the ER. The active GGT
1 enzyme is essential for turnover of glutathione at the cell surface. Although the other GGT isoforms lack this enzyme activity, they could
still bind substrate and act as sensors of the critical glutathione
redox levels found in ER. In support of this hypothesis, we found that
the phenotype of the stable CHO cells for GGT
1 and
7 changed
dramatically when the cell media was shifted from a 1:1 mixture of DMEM
(cystine) and Ham's F-12 (cysteine) with 3% FBS to DMEM alone with
3% FBS. Although the GGT
2-5 and
8-9 cells remained unchanged,
the GGT
1 and
7 cells rounded, lifted, and detached from the
culture plates within 12 h. The altered phenotype was reversed
upon return of the cells to the original media containing cysteine as
well as cystine. Because stress in the ER can be associated with this
dramatic change in phenotype, we probed the cellular RNA from each cell
line for the induction of CHOP-10 and BiP mRNAs, two markers of the
ER stress response. CHOP-10 mRNA was not detected by Northern blot
analysis in any of the cell lines before the change in media (data not
shown) but was dramatically induced in the GGT
1 and
7, but not
the GGT
2-5 and
8-9 cells, during the recovery period (Fig.
9). Actin mRNA was examined as a
control for RNA loading and integrity and was unchanged. To be sure
this induction of CHOP-10 was not due simply to overexpression of a
recombinant protein, two additional cell lines expressing high levels
of the recombinant glycoproteins hCAR or MUC1 (12) were similarly
characterized and found to not induce CHOP in response to the change in
media. Although hCAR is a 46-kDa glycoprotein cell surface receptor for
coxsackie and adenoviruses (13), MUC1 is a very heavily
O-glycosylated mucin-like transmembrane protein
(Mr > 220 kDa (12)). Finally, analysis of BiP
mRNA levels in all these cell lines indicated that its expression
paralleled that of CHOP-10.
View larger version (49K):
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Fig. 9.
Expression of GGT 1
and
7 mediate the ER stress response in CHO
cells. Control nontransfected CHO cells, and CHO expressing
GGT
1,
2-5,
7 or
8-9, or the recombinant control proteins
hCAR or hMUC1 were maintained in DMEM/Ham's F-12 with 3% FBS. The
media was changed to DMEM alone with 3% FBS for 12 h, and RNA was
extracted from cells after 7 days recovery in DMEM/Ham's F-12 with 3%
FBS. RNA was analyzed by filter hybridization as described under
"Experimental Procedures" using CHOP-1, BiP, and
-actin probes.
No signal for CHOP-10 was evident in cells at baseline.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and GGT
7 the alternative splicing event was
limited to the 3'-intron boundary and utilized an alternative AG
dinucleotide that was preceded by a cytosine residue. In GGT
2-5
and GGT
8-9 the alternative splicing events involved 5'- and
3'-splice sites as well as non-GT/AG dinucleotides.
1 two CAGs in tandem are present at the 3'-boundaries of the
corresponding introns in mouse and human GGT but absent from those of
the rat and the pig. Our results show that alternative utilization of
these sites can explain the presence or absence of a CAG trinucleotide
insert upstream of the ATG initiation codon specifically in mouse and
human GGT cDNAs. The scanning model predicts that the first CAG
should identify the 3'-boundary of the intron while the second should
be present in the cDNA. Our data agrees with this model, because
80% of the clones contained the CAG insertion. Despite the proximity
of these bases to the translation initiation site, our protein
expression studies clearly show that the heterodimer derived from
GGT
1 is a stable protein and identical to that described previously
in the rat and in humans (9, 17). Hence, the mouse and humans express
two GGT cDNAs that encode a GGT propeptide, which is heavily
glycosylated, especially on the large subunit, and processed into the
heterodimer before or after delivery to the cell surface. Our study now
represents the first detailed characterization of the GGT glycoprotein
in the mouse. The activity associated with this enzyme initiates the
hydrolysis of reduced or oxidized glutathione at the cell surface, a
process that is essential for recovery and uptake of cysteine/cystine.
However, this metabolism also appears to generate the pro-oxidant
hydrogen peroxide in the presence of iron (18). Hence, it is likely
that the level of GGT activity is regulated. Our in vitro
transcription/translation data shows that mouse and human GGT can use
the insertion of this CAG sequence to down-regulate GGT protein
production at the level of translation. The utilization of this same
alternative splicing strategy to alter mRNA translational efficiency has already been described in the gene for human
surfactant-associated protein A2, so it is not unique to GGT (19,
20).
7 also utilizes a nearby AG dinucleotide located only 24 bases
pairs upstream in intron 7 as the alternative 3'-intron boundary, and
this is preceded by a cytosine residue. Two factors could have selected
against this CAG as the constitutive 3'-splice site according to the
scanning hypothesis. The first is a distance of less than 12 nucleotides from the branch point; the second is a location within a
region of secondary mRNA structure such as stem loop. Our partial
sequence analysis of mouse intron 7 supports the first mechanism. We
found that there is a potential branch point consensus sequence that
would place this alternative CAG within this distance restriction.
There is also a second potential branch point sequence even further
upstream that would also place it as the first downstream CAG
trinucleotide (16). The factors that determine how these potential
branch points are utilized in the mouse GGT gene are not yet
known. The presence of these intron sequences in a human GGT
gene together with their regulation in a developmental-stage and
tissue-specific fashion in the mouse GGT gene suggests that
they serve a common role in GGT gene expression in these two
species. Determination of the degree of conservation will require
further examination of the GGT genes of additional species
such as the rat, the dog, the pig, and the cow. But these sequences are
not found in the GGT gene(s) of bacteria, yeast, flies, or
worms, suggesting that they are a relatively late addition to the
genomes of metazoans. Further studies will be required to fully
understand the complete role of these intron sequences for
GGT gene expression. However, we note that the identical
intron sequences are also found in the human lung-specific group II GGT cDNAs where they reside not in the coding domain but in the
5'-untranslated region (11). Similar, but not identical, sequences are
also present in a human intronic GGT promoter (21).
7 indicate that this
alternative splicing event is minor compared with the constitutive
event. However, we also found that the GGT
7 splicing variant was
detectable in tissues where constitutive GGT mRNA was highly
expressed, like the kidney, but not in tissues where GGT mRNA
abundance is very low, like the liver. Hence, the expression of
constitutive GGT and GGT
7 appears to be linked. An alternative splicing event like GGT
7 has previously been described in a human GGT mRNA, but three potential protein products were predicted based
on the presence of different open reading frames (4). Our study in the
mouse is the first to characterize the encoded protein product. Our
characterization of GGT
7 expression in stable and transiently
transfected CHO cells reveals that a single truncated form of GGT
protein is synthesized. It is a glycoprotein of 44 kDa and has 14 novel
residues at the C terminus. It is a relatively stable protein with a
half-life of ~8 h and is localized to the ER rather than the cell
surface. Because it lacks the C-terminal small subunit residues of the
active site, it predictably has no transferase activity. However, it
does retain the critical arginine 107 for substrate binding (23).
Because recent studies have established an essential role for the GGT
substrate glutathione in regulating the redox state of the ER (24), it
is possible that GGT
7 can bind glutathione and acts as a sensor for
glutathione levels within this compartment. Alternatively, the GGT
7
isoform may act as a chaperone for the synthesis of the native
enzymatically active GGT, blocking its activity within the ER where
degradation of glutathione would be unwanted. This would be supported
by finding that GGT
7 predominantly exists in tissues where high
levels of GGT are synthesized, and the low level of GGT
7 would
reflect that amount needed to balance the low transient levels of newly synthesized native GGT in the ER.
2-5 and GGT
8-9 differ from the consensus GT and AG
sequences. Hence, it is unclear exactly how these alternative events
were processed but there are certainly many examples in other genes of
splice sites that differ from the GT-AG consensus (25), including the
human GGT genes (22). The presence of such sites led to the
recent search and identification of an alternative intron subclass that
is bounded by AT-AC dinucleotides. Therefore, other intron subclasses
may also occur (26). Until further information becomes available in
this area, the mechanism for these alternative splicing events in mouse
GGT will remain uncertain. Nonetheless, the similarities between the
developmental-stage and tissue-specific expression of GGT
8-9 and
GGT
7 suggests that these GGT mRNA splicing events are highly
regulated, whereas the differences in tissue-specific expression
suggest they can be regulated independently. We were not able to find a
human correlate for GGT
8-9, so this event may be specific to the
mouse. The protein isoforms derived from GGT
2-5 and GGT
8-9 lack
-glutamyltransferase activity, like GGT
7. In each case, a residue
required for GGT activity, glutamic acid 108 (23) and aspartic acid 423 (27), respectively, is eliminated along with several other amino acids.
GGT
2-5 and
8-9 were also localized to the ER. Coexpression
studies failed to show any effect of these isoforms, including GGT
7,
on total endogenous GGT activity or delivery of active transferase to
the cell surface (data not shown). Hence, their exact function remains
obscure. Certainly the more rapid turnover rates for
2-5 and
8-9 could indicate that these glycoproteins are sensed as abnormal
GGT products within the ER and targeted for degradation.
1 and GGT
7 protein isoforms
appears to be able to impact the environment of the ER as suggested by
their ability to trigger an ER stress response with a change in cell
culture conditions. This stress was demonstrated by the parallel
induction of the mRNAs for CHOP, a nuclear protein that is
regulated by ER stress, and BiP, a chaperone whose expression during ER
stress is coordinately regulated with CHOP (28). The message for CHOP
is not normally expressed in cells but is strongly induced by ER
stress. CHOP protein then forms stable heterodimers with C/EBP family
members and binds to novel DNA target sequences to alter the pattern of
cellular gene expression in response to ER stress. When the ER stress
is severe, CHOP can activate a programmed cell death pathway. The
ability to trigger this response appears to be limited to the
full-length mouse GGT protein and the truncated GGT
7 protein isoform
under the conditions tested here. But the change in cellular phenotype
as well as the level of induction of CHOP mRNA was dramatic in
these two cell lines. CHOP can be strongly induced by deprivation of
nutrients such as glucose as well as the amino acids arginine, leucine,
lysine, methionine, phenylalanine, and threonine, but none of these
molecules were limiting in our conditions (29). However, reduced Cys is
found in Ham's F-12 and not in DMEM, which has only oxidized Cys, and this could directly impact the redox state of the ER. The lack of CHOP
induction in two other stably transfected CHO cells lines overexpressing human glycoproteins hCAR or hMUC1 suggests that this
triggering effect may be specific for these GGT isoforms. Further study
will be required to understand the basis for the ER stress and the
outcome of CHOP induction in these cell lines, but this will provide
new insight into a role for GGT and its isoforms in this intracellular
compartment. In addition, the GGTenu1 mouse, an animal
model of oxidant stress due to GGT deficiency, allows one to study how
the loss of GGT enzyme activity affects the pattern of GGT mRNA
splicing. In preliminary experiments, we found that the GGT
7
splicing event is increased in the lung of these animals, further
supporting a connection between GGT activity and the
7 splicing
event. The common expression of this event in human GGT suggests that
the results of further studies on mouse GGT will be directly relevant
to our understanding of human GGT gene expression and
ultimately the role of GGT in mammalian cellular physiology and
glutathione metabolism.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge Dr. Ora Weisz and Jennifer Henkel for assistance with immunofluorescence microscopy of the COS-transfected cells, and Carol L. Kinlough, Paul A. Poland, James B. Bruns, and Yue Liu for technical assistance.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants PO1HL47049 (to M. J. B.) and DK26012 (to R. P. H.) and by the Dialysis Clinic, Inc.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.
§ These authors contributed equally to this work.
¶ To whom correspondence should be addressed: Tel.: 617-638-4860; Fax: 617-536-8093; E-mail: mjbrady@lung.bumc.bu.edu.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M004352200
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ABBREVIATIONS |
---|
The abbreviations used are:
GGT, -glutamyltransferase;
mGGT, mouse GGT;
ER, endoplasmic reticulum;
endo H, endoglycosidase H;
HBS, HEPES-buffered saline;
CHO, Chinese
hamster ovary;
DMEM, Dulbecco's modified Eagle's medium;
Ham's F-12, nutrient mixture Ham's F-12;
FBS, fetal bovine serum;
CHOP, C/EBP
homologous protein-10;
BiP, immunoglobulin heavy chain binding protein;
hCAR, human coxsackie and adenovirus cell surface receptor;
hMUC, human
mucin 1;
RT, reverse transcription;
PCR, polymerase chain reaction;
MOPS, 4-morpholinepropanesulfonic acid;
PAGE, polyacrylamide gel
electrophoresis;
kb, kilobase(s);
PBS, phosphate-buffered saline.
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REFERENCES |
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