The Critical Role of the Stem Region as a Functional Domain
Responsible for the Oligomerization and Golgi Localization of
N-Acetylglucosaminyltransferase V
THE INVOLVEMENT OF A DOMAIN HOMOPHILIC INTERACTION*
Ken
Sasai
§,
Yoshitaka
Ikeda
,
Takeo
Tsuda
,
Hideyuki
Ihara
,
Hiroaki
Korekane
,
Kunio
Shiota§, and
Naoyuki
Taniguchi
¶
From the
Department of Biochemistry, Osaka University
Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan and the
§ Laboratory of Cellular Biochemistry, Department of Animal
Resource Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan
Received for publication, June 8, 2000, and in revised form, October 6, 2000
 |
ABSTRACT |
We demonstrated that a region in the stem of
N-acetylglucosaminyltransferase V (GnT-V), a Golgi resident
protein, is not required for enzyme activity but serves as functional
domain, responsible for intracellular localization. Deletion of the
domain led to complete retention of the kinetic properties but resulted
in the cell surface localization of the enzyme as well as its efficient secretion into the medium. The lack of this domain concomitantly abolished the disulfide-mediated oligomerization of GnT-V, which appears to confer the Golgi retention. When the domain was inserted into the stem region of a cell surface-localized type II membrane protein, the resulting chimeric protein was substantially oligomerized and predominantly localized in the intracellular organelle.
Furthermore, it was found that the presence of this domain is
exclusively responsible for homo-oligomer formation. This homophilic
interaction appears to involve a hydrophobic cluster of residues in the
-helix of the domain, as indicated by secondary structure
predictions. These findings suggest that the domain specifically
participates in the Golgi retention of GnT-V, probably via inducing
homo-oligomer formation, and would also provide a possible mechanism
for the oligomerization, which is critical for localization in the Golgi.
 |
INTRODUCTION |
1,6-N-Acetylglucosaminyltransferase V (EC 2.4.1.155,
GnT-V),1 a type II membrane
protein that is localized in the Golgi apparatus, is involved in the
biosynthesis of Asn-linked oligosaccharides. The enzyme catalyzes the
transfer of a GlcNAc residue from UDP-GlcNAc to an
1,6-linked
mannose moiety of Asn-linked oligosaccharides via a
1,6-linkage (1,
2). It is known that the levels of products of GnT-V, which are
1,6-branched N-glycans, are frequently increased in many
malignant tumors and that this is closely correlated with tumor
progression (3-5). Several studies have suggested that the
1,6-branched N-glycans directly contribute to the tumor growth and metastasis (6-8). The GnT-V protein appears to consist of
an N-terminal cytoplasmic tail, a transmembrane region, a stem region,
and a large C-terminal catalytic domain, as suggested by the primary
structure (9, 10), and thus shares a domain structure that is common to
many glycosyltransferases (11).
Oligosaccharides of glycoconjugates such as glycoproteins and
glycolipids are synthesized and processed via catalysis by a variety of
glycosyltransferases in the endoplasmic reticulum and the Golgi
apparatus. Such enzymes, which are involved in oligosaccharide biosyntheses, must be localized to the appropriate intracellular destination to permit a properly ordered processing of the
oligosaccharides. It is generally thought that many
glycosyltransferases contain a structural region that is responsible
for localization in the Golgi. Such a portion is often referred to as a
Golgi retention signal. However, a common motif for serving as such a
retention signal has not been found to date, probably because of the
lack of homologous sequences among the Golgi-localized
glycosyltransferases (11). It is believed that GnT-V is localized to
the Golgi apparatus and participates in N-linked
oligosaccharide processing therein. Nevertheless, the mechanism of the
retention of the enzyme in the Golgi apparatus has not been explored,
and a structural region responsible for this localization has not been identified.
Two models, the bilayer thickness model and the kin recognition model,
have been proposed to date for the mechanism of the Golgi retention of
glycosyltransferases (11). The bilayer thickness model, also known as
the lipid sorting model, postulates that the length of the
transmembrane domain of glycosyltransferases mediates Golgi retention
(12, 13). Alternatively, in the case of the kin recognition model, it
is proposed that a homo-/hetero-oligomerization of glycosyltransferases
prevents the delivery of the enzymes to secretary vesicles and ongoing
transport to the plasma membrane, and, as a result, this process
functions as a Golgi retention signal (14, 15). However, except for a
few exceptions, a clear and discrete structural factor that plays a
primary role in the Golgi retention has not yet been defined in glycosyltransferases.
To better understand the mechanism of Golgi retention, additional
experimental data will be required and further studies on a variety of
Golgi resident proteins, including many glycosyltransferases, will be
highly desirable. The mechanism that allows proteins to be localized in
the Golgi have been substantially established by many studies using
glycosyltransferases, and in fact it appears that glycosyltransferases
provide a typical model for the Golgi retention of type II membrane
proteins. In the present study, we expressed various deletion mutants
of GnT-V and its chimeric proteins in COS cells and analyzed their
intracellular localization by immunofluorescence microscopy. As a
result, a region in the stem of GnT-V was identified as a functional
domain that is specifically responsible for intracellular localization
and that is not associated with the activity of the enzyme. Functional
characterization of the domain and related studies suggests that
homo-oligomer formation of GnT-V takes place and that this process is
mediated by the domain that is responsible for intracellular
localization. These conclusions were further confirmed by conversion of
a cell surface-localized protein into an intracellularly localized form
by insertion of the domain.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Restriction endonucleases and DNA-modifying
enzymes were purchased from Takara, Toyobo, or New England Biolabs.
UDP-GlcNAc and GlcNAc were obtained from Sigma. Oligonucleotide primers
were synthesized by Greiner Japan. Antibodies were obtained from the following sources: anti-human GnT-V monoclonal antibody (a gift from
FUJIREBIO Inc.); anti-FLAG epitope antibody (M5, Sigma); anti-His tag
antibody (Tetra-His antibody, Qiagen); anti-GST antibody (Amersham
Pharmacia Biotech); horseradish peroxidase-conjugated anti-mouse IgG
antibody (Promega); horseradish peroxidase-conjugated anti-goat IgG
(DAKO); and rhodamine isothiocyanate-labeled anti-mouse IgG antibody
and rhodamine isothiocyanate-labeled anti-goat IgG antibody (Santa Cruz
Biotechnology). Other common chemicals were obtained from Wako pure
chemicals, Nacalai Tesque, and Sigma.
Expression Plasmids for a Human GnT-III and Human GnT-V--
An
EcoRI fragment, which contained the entire coding sequence
of human GnT-III (hGnT-III) (16), and a KpnI-XbaI
fragment, which encodes human GnT-V (hGnT-V) (10), were subcloned into an SV40-based expression vector, pSVK3 (Amersham Pharmacia Biotech), and were used for the expression of their wild type proteins.
Construction of Chimeric GnT-V Proteins--
In this study,
three types of the chimeric proteins, GnVd73, GnVd187, and GnVd242,
were used in which the GnT-III N-terminal sequences including the
cytoplasmic tail, transmembrane domain, and portions of the stem region
were fused to the luminal regions of GnT-V. For the preparation of the
GnVd73 protein, a BspEI site was created by a site-directed
mutagenesis according to the method of Kunkel (17), as described
previously (18), to fuse the hGnT-V sequence (10) following Ala-74 to
the N-terminal 62 residues of rat GnT-III (rGnT-III) (19). An
oligonucleotide primer (5'-GTGGTGGATGGTCCGGACGCTGGAGTC-3') was used for
this mutation with a uracil-substituted single stranded template of the
hGnT-V sequence, which was prepared using Escherichia coli
CJ236 (dut
, ung
). The
2.0-kilobase fragment that encodes Ala-74 to Leu-741 (the C-terminal
residue) of hGnT-V was obtained by digestion with BspEI and
XbaI and was inserted into the EcoRI and
XbaI sites of the pSVK3 along with an
EcoRI-BspEI fragment for Met-1-Pro-62 of
rGnT-III (19). When the other two chimeric proteins were constructed, the nucleotide sequences corresponding to Tyr-188-Leu-741 and Arg-243-Leu-741 were amplified by the polymerase chain reaction (PCR)
to yield the fragments flanked by KpnI and XbaI
sites for 5' and 3' ends, respectively. A primer set used for the
amplification was a combination of 5'-CAAGGTACCGGCGAATGGCTG-3' for
Tyr-188-Leu-741 or 5'-CCTGGTACCTCAGTGAGGTTG-3' for Arg-243-Leu741 and
a common antisense primer 5'-GAGCTCTAGAGGCAGTCTTTGC-3'. The amplified
DNA fragments were digested by KpnI and XbaI and
were used to replace the hGnT-III nucleotide sequence encompassing
Tyr-149 to the C terminus in the hGnT-III/pSVK3.
Deletion Mutant Constructs--
A DNA fragment encoding
Met-1-Ser-44 of hGnT-V was amplified by PCR using the following
primers: 5'-CAGGTACCATGGCTCTCTTCAC-3' and
5'-CGAGGTACCCGGAGCTGCTTTCAG-3'. A 0.13-kilobase
KpnI fragment resulting from the digestion of the amplified
DNA was inserted, in an appropriate orientation, into the
KpnI site in the plasmid in which the 1.7-kilobase
KpnI-XbaI fragment for Tyr-188-Leu-741 had been
ligated, thereby leading to the hGnT-V sequence lacking Met-45-Ile-187. This deletion mutant was designated GnV
1. Another deletion mutant, GnV
2, was prepared by the removal of the region covering Asp-136-Ser-435 from the expression plasmid carrying the wild
type hGnT-V by EcoRV digestion followed by
self-ligation.
FLAG Epitope-tagged Constructs for the Wild Type and the Deletion
Mutants of GnT-V--
To add the FLAG epitope at the N terminus,
EcoRI-XbaI fragments of the complete sequences
for the wild type and GnV
1 and GnV
2 mutants, excised from their
respective expression plasmids constructed using the pSVK3, were
ligated into a pME18FLAG vector (20), a generous gift from Dr. Takaomi
Ishida (Institute of Medical Science, University of Tokyo). These
FLAG-tagged constructs were designated FWT for the wild type, F
1 for
GnV
1, and F
2 for GnV
2. The FN235 mutant was also prepared by
ligating the PCR-amplified fragment encoding Met-1-Phe-235, in which
both ends were flanked by EcoRI, into the EcoRI
site of the pME18FLAG. For the construction of the FCStem mutant, in
which Met-45-Ser-184 was fused to C terminus of F
1 mutant, two DNA
fragments were amplified by PCR using the following primer sets:
5'-CAGAATTCATGGCTCTCTTCACTC-3' and 5'-CCAACTCGAGTAGGCAGTCTTTGCAG-3' for
a full length of F
1 mutant and 5'-ACATCTCGAGATGCTGCGCGAGCAGATC-3'
and 5'-TATCTCGAGCTAAGAGCAGGTGGATCC-3' for Met-45-Ser-184. The
amplified DNA fragments were ligated into EcoRI and
XhoI sites of a pME18FLAG vector.
Insertion of the GnT-V Sequence of Glu-41-Ser-184 into
-Glutamyl Transpeptidase--
The DNA fragment corresponding to the
Glu-41-Ser-184 of hGnT-V was amplified by a PCR with primers,
5'-TTGGCCGCGGAAAGCAGCTCCATGCT-3' and 5'-TGTCCGCGGCAAAAGAGCAGGTGGA-3'
and then digested by SacII. The resulting fragment was
inserted into the SacII site in the human GGT/pSVK (21),
whose position corresponds to amino acid residues 44-46, which is
located adjacent to the membrane anchoring domain in the luminal region
of GGT.
Cell Culture--
COS-1 cells and CHO-K1 cells were grown and
maintained at 37 °C in Dulbecco's modified Eagle medium and Ham's
F-12 medium, respectively, supplemented with 10% fetal calf serum, 50 units/ml penicillin G, and 50 mg/ml streptomycin under a humidified
atmosphere of 95% air and 5% CO2.
DNA Transfection--
Expression plasmids were transfected into
cells by electroporation (22) using a Gene Pulser (Bio-Rad), as
described previously (21). The cells were washed with Hepes-buffered
saline and resuspended. Plasmids (20 µg), purified by CsCl gradient
ultracentrifugation, were added to the cell suspension (1 × 107), followed by electrification. The transfected cells
were subjected to biochemical and histological analyses, 48 h
after transfection.
Subcellular Fractionation--
Cells were homogenized in
phosphate-buffered saline (PBS) and centrifugation at 10,000 × g for 10 min at 4 °C to remove cellular debris and
nuclei. The resulting supernatants were further centrifuged at
100,000 × g for 1 h at 4 °C. The pellets were
resuspended in a volume of PBS containing 1% Triton X-100, which was
equal to that of the supernatants.
SDS-PAGE, Immunoblotting, and Lectin Blot Analysis--
SDS-PAGE
was carried out according to Laemmli (23). The separated proteins were
electrophoretically transferred onto a nitrocellulose membrane
(Schleicher & Schuell, PROTORAN), followed by blocking with 5% skim
milk or 2% bovine serum albumin. The resulting membrane was incubated
with the first antibody or biotinylated lectin. After washing, the
membrane was reacted with an appropriate second antibody that was
conjugated to horseradish peroxidase for immunoblotting or with an
avidin-biotin complex with the peroxidase for the lectin blot. The
reactive (glyco)protein bands were visualized by chemiluminescence using an ECL system (Amersham Pharmacia Biotech).
GnT-V Activity Assay and Kinetic Analysis--
The GnT-V
activity was assayed using a fluorescence-labeled oligosaccharide
acceptor, as described previously (24). Cell homogenates (5-20 µg of
proteins) were incubated at 37 °C for 2 h with 20 µM pyridylaminated agalacto biantennary oligosaccharide as an acceptor and 40 mM UDP-GlcNAc as a donor in 125 mM MES-NaOH (pH 6.25) containing 200 mM GlcNAc,
0.5% Triton X-100, and 10 mM EDTA. The reaction was
terminated by heating the mixture at 100 °C for 3 min, and the
sample was then centrifuged at 15,000 rpm for 5 min in a
microcentrifuge. The resulting supernatant was analyzed by reversed
phase high performance liquid chromatography (Shimazu) using a TSKgel
ODS-80TM (4.6 × 150, TOSOH). The solvent used was a 20 mM, pH 4.0, ammonium acetate buffer, and the substrate and
the product were isocratically separated. Fluorescence was detected
with a fluorescence detector (RF-10AXL, Shimazu) at excitation and
emission wavelengths of 320 and 400 nm, respectively. In the kinetic
analysis, the activity was assayed with various concentrations of
UDP-GlcNAc in the presence of 20 µM acceptor to determine
the apparent parameters for the donor. To obtain the parameters for the
acceptor, various concentrations of the acceptor were used with a fixed
concentration of the donor, namely, 40 mM UDP-GlcNAc. Other assay conditions were identical to those described above.
Immunofluorescence Microscopy--
The transfected COS-1 cells
(1 × 104) were seeded into individual wells of an
8-well chamber slide (Lab-Tek) and grown for 48 h, as described
for the cell cultures. The cells were fixed with 3.7% formaldehyde in
PBS and then permeabilized by treatment with 0.2% Triton X-100 in PBS
for 15 min. The resulting cells were blocked by 2% bovine serum
albumin in PBS for 60 min at room temperature and then incubated
sequentially with a primary antibody for 60 min and an appropriate
rhodamine isothiocyanate-conjugated second antibody for 20 min. The
stained cells were then examined by fluorescence microscopy (Olympus)
and photographed.
Assay for Protein Interaction by an Yeast Two-hybrid
System--
A DNA fragment encoding Met-45-Ser-184 was amplified by
PCR using GnT-V cDNA as a template and a primer set of
5'-CATGAATTCATGCTGCGCGAGCAGATC-3' and 5'-TATCTCGAGCTAAGAGCAGGTGGATCC-3'
to introduce EcoRI and XhoI sites at the 5' and
3' ends, respectively. A 1.6-kilobase EcoRI-XhoI fragment that encodes the region of Glu-234-Leu-741 was excised from
the human GnT-V/pSVK plasmid. To perform a yeast two-hybrid assay, a
LexA two-hybrid system (CLONTECH) was employed in
this study, and the assay was carried out according to the
manufacturer's recommended protocol. The DNA fragments were subcloned
into the EcoRI and XhoI sites of pLexA-B.D. and
pB42-A.D. plasmids, and the resulting respective plasmids enabled the
expression of LexA and B42 fusion proteins with these GnT-V sequences.
Saccharomyces cerevisiae EGY48[p8op-lacZ] was used as host
strain. This strain had been transformed with a reporter plasmid,
p8op-lacZ, conferring LEU2 and lacZ expression under the control of
LexA operators. The host cells were transformed with the LexA and B42
plasmids and grown at 30 °C for 3 days on the SD plates, which
contain 2% dextrose and an amino acid mixture that lacks histidine,
tryptophan, and uracil. The colonies, when grown, were then transferred
to the X-gal plates, which contain 2% galactose instead of dextrose, 1% raffinose, a mixture of amino acids that lacks in leucine as well
as histidine, tryptophan, and uracil. These colonies were allowed to
grow at 30 °C for a further 3 days.
Glutathione S-Transferase Pull-down Assay--
The GnT-V
EcoRI-XhoI fragments, prepared for the yeast
two-hybrid assay, were ligated into pGex4T1 (Amersham Pharmacia
Biotech) and pET32a (Invitrogen) to express GST fusion proteins and
polyhistidine-tagged thioredoxin fusion proteins, respectively, in
E. coli. In the cells transformed with these plasmids,
expression of both proteins was induced with 0.4 mM
isopropyl-
-D-thiogalactopyranoside. GST was also
expressed similarly in the cells transformed with an empty pGex4T1. The
cells were lyzed by sonication, and the proteins were extracted. The
GST fusion protein and nonfused GST were immobilized on glutathione
Sepharose beads (Amersham Pharmacia Biotech) and were then incubated
with the thioredoxin fusion protein at 4 °C for 3 h. After
washing three times with PBS, the precipitated samples were subjected
to SDS-PAGE followed by immunoblot analysis using anti-GST and
anti-polyhistidine antibodies.
Protein Determination--
Protein concentrations were
determined according to the method of Bradford (25) using bovine serum
albumin as a standard.
 |
RESULTS |
It is known that the GnT-V molecule contains a larger number of
amino acids than many other glycosyltransferases, and, as a result, it
is a reasonable assumption that the protein contains several
functionally separable domains, each of which contributes to the
overall function of the glycosyltransferase, but in a different manner
(for example, catalytic activity versus the Golgi
retention). The present study was initiated to identify a functional
domain that is different from the catalytic domain and to investigate its specific role. Such a study would potentially lead to elucidation of the "functional domain architecture" in GnT-V and possibly in
other glycosyltransferases, and the detailed functional
characterization of the specific domain might provide a clue for the
issue of, for example, the mechanism of Golgi retention.
To identify the structural region that is not associated with enzyme
activity, chimeric GnT-V proteins, which contain the GnT-V sequences
truncated using several restriction enzyme sites, were expressed in COS
cells, and the enzyme activities of these products were examined. These
chimeric proteins were prepared by fusion of the GnT-V regions of
Ala-74-Leu-741, Tyr-188-Leu-741, and Arg-243-Leu-741 to the GnT-III
N-terminal 62 residues for Ala-74-Leu-741 or 149 residues for the
others, and were designated as GnVd73, GnVd187, and GnVd242,
respectively (Fig. 1A). An
assay of GnT-V activity using the homogenates of the transfected COS-1 cells indicated that GnVd73 and GnVd187 were enzymatically active, whereas GnVd242 exhibited no detectable activity, even though its
protein expression level was comparable with other chimeric enzymes, as
shown in Fig. 1 (B and C). Thus, it can be
concluded that the N-terminal 187 amino acid residues are not required
for GnT-V activity but that the loss of additional 55 residues
abolishes all activity.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1.
Enzyme activity of the wild type GnT-V and
chimeric proteins expressed in COS-1 cells. A,
schematic representation of the chimeric proteins used in this study.
Regions indicated by filled and open boxes were
derived from GnT-III and GnT-V, respectively. B, detection
of chimeric proteins by immunoblotting using anti-GnT-V antibody.
C, specific activities in cell homogenates. N.D.,
not detected; WT, wild type; IB,
immunoblotting.
|
|
To further confirm that the region corresponding to residues 45-187 is
not associated with enzyme activity, deletion mutants of the GnT-V, in
which residues 45-187 and residues 136-435 were deleted, were also
prepared and designated as GnV
1 and GnV
2, respectively (Fig.
2A). Consistent with the
results obtained for the chimeric proteins, the GnV
1 mutant was
found to be active, whereas the mutation made in GnV
2 led to a
complete loss of activity (Fig. 2, B and C). The
kinetic properties of the GnV
1 mutant was nearly indistinguishable
from those of the wild type, as revealed by kinetic analysis using cell
homogenates; the apparent Km for the donor and
acceptor were 13 mM and 77 µM, respectively, in the wild type, and the respective values for the mutant were 9.1 mM and 67 µM. These results clearly indicate
that the region corresponding to residues 45-187 is not involved in
enzyme activity.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 2.
GnT-V activity of the deletion mutants.
A, schematic representation of the GnT-V deletion mutants,
GnV 1 and GnV 2. B, immunoblot analysis of the
expression of the deletion mutants. C, specific activity of
the deletion mutants in the transfected COS-1 cells. N.D.,
not detected; WT, wild type; IB,
immunoblotting.
|
|
Despite the fact that no difference in enzymatic properties was
observed, when the enzyme activity in the culture medium was assayed
and compared with the cell-associated activity, it was found that the
GnV
1 mutant is much more efficiently released from the cells,
compared with the wild type (Fig.
3A). Furthermore, even in the
cell-associated activity of the mutant, a major fraction (
50%) was
found in the soluble fraction, as evidenced by subcellular fractionation by ultracentrifugation (Fig. 3B). In the case
of the wild type, on the other hand, the enzyme was nearly exclusively located in the membrane fraction, which is consistent with the fact
that many glycosyltransferases are generally anchored to the Golgi
membrane. This distribution profile for the wild type is nearly the
same as that of another glycosyltransferase, GnT-III, which is also
known to be localized in the Golgi apparatus (26). These results show
that the loss of the region corresponding to residues 45-187 causes a
dramatic alteration in the cellular distribution of GnT-V.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 3.
Characterization of
GnV 1 mutant. A, comparison of
secreted (open bars) and cell-associated activities
(filled bar) in the wild type and the mutant. B,
subcellular distribution of the enzyme activities. The activity in the
S-100 (filled bar) and P-100 (open bar) fractions
are shown. C, lectin blot analysis of the CHO-K1 cells
transfected with the GnV 1 mutant. Detailed experimental conditions
are described under "Experimental Procedures."
|
|
Although the significant activity of the GnV
1 mutant remained
associated with the membrane, it appeared that
1,6-branched sugar
chains, which are formed by the action of GnT-V, were not increased in
the case of the GnV
1-transfected CHO-K1 cells, as shown by lectin
blot analysis using leukoagglutinating phytohaemagglutinin, which
preferentially binds to a
1,6-branched oligosaccharide (Fig.
3C). In this experiment, CHO-K1 cells were used instead of
COS-1 cells because the reactivity of the lectin is not substantially increased, even by the overexpression of the wild type GnT-V in COS-1
cells, probably the result of relatively high background signals. The
deletion of region 45-187 resulted in the inability of the protein to
modify cellular oligosaccharides even though this deletion mutant is
sufficiently enzymatically active. It thus appears that the mutant is
not properly localized within the cells. This suggests that the region
that had been deleted could serve as a functional domain that could
play a role in appropriate intracellular localization.
To investigate the intracellular localization of the wild type and
GnV
1 mutant of GnT-V in more detail, the cellular distribution of
these proteins were analyzed by immunofluorescence microscopy. Because
the anti-GnT-V antibody used in the immunoblot analyses did not appear
to react with the nondenatured proteins, N-terminally FLAG-tagged
forms of the wild type and the mutants were constructed (Fig.
4A) and probed with an
anti-FLAG epitope antibody. An FN235 mutant, an N-terminal 235-amino
acid fragment that contains the stem region but lacks the majority of
the large catalytic domain was also prepared to explore the requirement
of this domain in the intracellular localization of GnT-V. As shown in
Fig. 4B, the wild type displayed a typical Golgi-staining
pattern, as reported (27), whereas the F
1 mutant was not localized
in the Golgi apparatus but, rather, was diffusely distributed
throughout the cells, probably the result of being expressed on the
cell surface. These results indicate that the absence of the region
corresponding to residues 45-187 (which is tentatively referred to as
the "stem domain" to discriminate from "stem region") perturbs
the normal Golgi localization of the GnT-V, suggesting that this domain
is involved in the retention of the enzyme in Golgi. Because other mutants, F
2 and FN235, are also localized in the Golgi apparatus, as
was the full-length wild type protein (Fig. 4B), the
localization of GnT-V in Golgi apparatus appears to be primarily
conferred by the stem domain, particularly the region encompassed by
residues 45-135. As shown by the analysis of an additional mutant
(Fig. 4A, FCStem), however, relocation of the stem domain to
the C terminus led to different intracellular localization (Fig.
4B). This result suggests that the position of the domain is
important, possibly because of collaboration with the transmembrane
domain in the vicinity.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 4.
Subcellular localization of the wild type
GnT-V and its deletion mutants. A, schematic
representation of FLAG epitope-tagged constructs of the wild type
(FWT) and mutants (F 1,
F 2, FN235, and FCStem).
FLAG epitope tag and the region of Met-45-Ser-184 are shown by
hatched and filled boxes, respectively.
B, localization of FLAG epitope-tagged GnT-V proteins, as
probed by an anti-FLAG antibody.
|
|
If the function of the stem domain is exclusively as a "Golgi
retention signal" for GnT-V and is independent of other structural regions such as the transmembrane domain and the catalytic domain, it
would be expected that the insertion of the domain would be sufficient
to convert a plasma membrane-localizing type II membrane protein, which
is unrelated to GnT-V, into a Golgi localized form.
-Glutamyl
transpeptidase (GGT) represents a typical type II membrane glycoprotein
that is of critical importance to glutathione metabolism and is known
to be localized on the cell surface (28, 29). Hence, this protein was
used as a model protein to examine the effect of the domain on the
localization of such a type II membrane protein. An amino acid sequence
nearly identical to the domain comprised by residues 41-184 was
inserted into almost the equivalent position in GGT (Fig.
5A), and the resulting
chimeric protein was expressed in COS cells. As revealed by
immunofluorescence microscopic analysis using anti-GGT antibody (30),
the insertion of the domain had a dramatic effect on the localization
of the protein: the chimeric protein was localized in the intracellular organelles, predominantly the Golgi apparatus, in contrast to the cell
surface expression of the nonchimeric GGT (Fig. 5B). This
altered intracellular distribution did not appear to be precisely identical to that of GnT-V, and thus this slight difference could be
due to the subtle interference of the GGT transmembrane region, the
length and amino acid composition of which are distinct from those of
glycosyltransferases. However, such a dramatic conversion into an
intracellularly localized form clearly indicates that the stem domain
from GnT-V is fully capable of altering the localization of the cell
surface-localized type II membrane protein. It thus appears likely that
the mechanism of the intracellular localization of GnT-V is dependent,
almost exclusively, on the nature of the stem domain but does not
necessarily involve its intrinsic transmembrane domain nor any other
structural regions. It also appears that the intracellular localization
of GnT-V does not essentially depend on lipid sorting, a process for
which the transmembrane domain plays a critical role.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 5.
Subcellular localization of GGT and its
chimeric protein. A, a construct of chimeric GGT
protein, in which the region of Glu-41-Ser-184 of hGnT-V was inserted.
A GnT-V portion is indicated by a filled box. B,
localization of the nonchimeric GGT and the chimeric GGT/GV proteins in
the transfected COS-1 cells.
|
|
When the homogenates from cells that express GnT-V were analyzed by
immunoblot analysis following SDS-PAGE, performed under nonreducing
conditions, it was found that the wild type GnT-V is highly
oligomerized via disulfide linkages (Fig.
6A). This feature appears to
be frequently observed in the glycosyltransferases whose intracellular
localization is determined by the oligomerization-based or kin
recognition mechanisms (14, 31, 32). The F
2 mutant, which was found
to be localized in the Golgi (Fig. 4B), was also significantly oligomerized via disulfide linkages, as was the wild
type. On the other hand, oligomer formation was substantially decreased
in the cell-associated forms of the F
1 mutant, intracellular localization of which is impaired because of the lack of the stem domain (Fig. 6A). Moreover, essentially no oligomer
formation was observed in the secreted enzyme, even the wild type, as
well as the GnV
1 mutant (Fig. 6B). These secreted GnT-Vs,
both wild type and
1 mutant, were smaller by about 5 kDa than those
of intracellular forms, as estimated by SDS-PAGE. Thus, the secreted species of the wild type appeared to still contain the stem domain, but
both secreted proteins appeared to lack the cytoplasmic tail and
transmembrane domain. It seemed that their secretion results from
proteolytic cleavage at the site(s) immediately close to the
transmembrane domain. The disulfide bridge-mediated oligomerization appeared to fail in some fractions of the translated GnT-V protein. Considering the lack of disulfide linkages in the GnV
1, it is conceivable that the consequence of this failure may be the result of
the release of the enzyme from the cells even in the case of the wild
type. Therefore, it is likely that a GnT-V fraction that remained in
the nonoligomeric state because of absence of disulfide linkages could
no longer be retained in the Golgi and might subsequently be released.
Oligomer formation such as this was also observed in the case of a
significant fraction of the chimeric GGT, in which the stem domain of
GnT-V was inserted into GGT, whereas the nonchimeric GGT, which is
localized on the cell surface, failed to form any detectable
disulfide-linked oligomer (data not shown). The results showing that
the absence of disulfide-linked oligomer formation is concomitant with
the release of protein from the cells and impaired localization are
consistent with the conclusion that the domain identified in this study
serves to localize GnT-V in the Golgi apparatus by facilitating the
formation of oligomers. Because the FN235 mutant oligomerized via
disulfide bridges to a lesser extent, the formation of the covalent
linkages in GnT-V may involve not only the stem domain but also other
regions.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 6.
Disulfide-linked oligomerization of the wild
type GnT-V and the deletion mutants. A, the FLAG
epitope-tagged wild type and mutants expressed in COS-1 cells were
analyzed by immunoblotting using an anti-FLAG antibody, under both
reducing and nonreducing conditions. B, the GnT-V proteins
secreted from the COS-1 cells transfected with wild type GnT-V and
GnV 1 were analyzed by immunoblotting using an anti-GnT-V antibody,
under both reducing and nonreducing conditions. WT, wild
type.
|
|
As shown in Fig. 6A, immunoblot analysis of the FN235 mutant
gave a series of the immunoreactive bands corresponding to dimer, trimer, tetramer, and higher oligomer, suggesting homo-oligomer formation. This result and the substantial dependence of the oligomer formation of GnT-V on the stem domain strongly suggest that the domain
is exclusively capable of forming a homo-oligomer via intrinsic homophilic interactions. Thus, such a homophilic interaction was examined using a yeast two-hybrid system, and, as the result, specific
homophilic binding of the domain was observed, but interaction with the
C-terminal catalytic domain was not (Fig.
7A). This homophilic interaction was also confirmed by a GST pull-down assay. The His-tagged thioredoxin fusion protein bound to the GST fusion protein but not to
nonfused GST (Fig. 7B). In both of these two assays,
interaction or binding was detected only in the combination of
fragments that contained residues 45-184 but not in any other
combination. These results suggest that the domains mutually interact
in a homophilic manner. Thus, it is more likely that oligomerization of
GnT-V is primarily mediated via the homophilic interaction of the
domain, thus further supporting the critical role of the stem domain in the Golgi localization of GnT-V, which appears to be accomplished by an
oligomerization-based mechanism.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 7.
Homophilic interaction of the region of
Met-45-Ser-184 in GnT-V. Detection of the homophilic interaction
by a yeast two-hybrid system (A) and GST pull-down assay
(B). Detailed assay conditions are described under
"Experimental Procedures." IB, immunoblotting.
|
|
 |
DISCUSSION |
In this study, we report the identification of the region
comprised by ~45-184 amino acid residues in the stem region as the functional domain that is responsible for the Golgi localization but is
not associated with enzymatic activity, thus demonstrating that GnT-V
consists of the separable functional domains that confer either
catalytic activity, intracellular localization, or membrane anchoring.
The absence of the domain identified in the stem region of GnT-V led to
a loss of disulfide linkage-mediated oligomer formation in conjunction
with a considerably diminished Golgi retention, whereas the enzymatic
properties remained nearly completely intact. Furthermore, it was found
that even if the protein contains the domain, i.e. a
full-length wild type GnT-V, a failure in forming this covalently
linked oligomer may also lead to an impaired retention of protein in
the Golgi, leading to its subsequent release from the cells. It was
also shown that the domain that was identified as being essential for
retention is capable of forming its homo-oligomer by the homophilic
interaction. These findings suggest that the intracellular localization
of GnT-V is due, nearly exclusively, to this domain and that the
mechanism for the intracellular localization involves homo-oligomer
formation, triggered by the domain, but not substantially affected by
the other regions that are intrinsic to GnT-V, such as the
transmembrane domain and the catalytic domain.
The mechanism for the retention of GnT-V in Golgi is consistent with
kin recognition or a similar mechanism and is most probably based on
homo-oligomerization (11). Formation of the disulfide-linked homo-oligomerization would represent a critical step for the retention of this glycosyltransferase. Intermolecular covalent linkages appear to
be efficiently formed, not only in the stem domain, in which 4 cysteine
residues are present, but also in the other regions, probably because
the entire luminal region of GnT-V contains as many as 20 cysteine
residues (10). In fact, although F
2 lacks those 4 cysteine residues
in the stem domain, this mutant forms the disulfide-linked oligomer and
is localized in the Golgi apparatus. The initial event leading to such
covalent homo-oligomer formation would involve a clustering of newly
biosynthesized GnT-V molecules, in which the covalent linkages have not
yet been formed, and, subsequently, the clustered proteins might be
covalently linked by enzymatic or nonenzymatic oxidation in the Golgi
or more proximal subcellular compartments. Because the domain is absolutely required but not necessarily sufficient for the formation of
covalent linkages, as suggested by the absence of intermolecular disulfides in the secreted form of the wild type GnT-V, the primary function of the domain would involve triggering the initial clustering via its homophilic interactions. The domain might also be able to
facilitate intermolecular disulfide formation by increasing the
probability of intermolecular contact, although a possible noncovalently associated homo-oligomer, formed only via homophilic interactions of this domain, is unstable to retain the GnT-V in the
Golgi apparatus.
All secondary structural predictions, which are based on the methods of
Garnier and co-workers (33), Eisenberg and co-workers (34), and Chou
and Fasman (35), suggest an
-helical structure for the region
encompassed by residues Lys-84-Val-108 in the stem domain, as shown in
Fig. 8. The helical wheel model of the
region reveals the presence of a one-sided hydrophobic amino acid
cluster in the helix. It is known that such a structural feature is
frequently involved in an intermolecular or intermolecular hydrophobic
contact formed by multiple helices, as found, e.g. in gp41,
a transmembrane subunit of HIV-1 envelope glycoprotein complex,
gp120-gp41 complex (36, 37). The putative gp41 amphipathic
-helix,
Leu-550-Leu-582, was demonstrated to be essential for
hetero-oligomeric formation with gp120, indicating that hydrophobic
contacts contribute to this oligomeric structure (37, 38). Therefore,
it may also be possible that such a hydrophobic contact could enable
the stem domain to display a homophilic interaction, which plays a role in the formation of the initial clustering of the GnT-V protein.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 8.
A prediction of secondary structure of the
region Lys-84-Val-108 of GnT-V. A helical wheel model based on
the method of Chou and Fasman (35). Hydrophobic amino acids
(I, L, and V) are highlighted.
|
|
In the kin recognition via hetero-oligomeric interaction or
homo-oligomer-based mechanism for the retention of various proteins in
Golgi, it is generally thought that the formation of a sufficiently large molecule prevents the delivery of proteins from the Golgi to more
distal compartments, such as the plasma membrane (11). Although
oligomerization is a critical event that is common to these Golgi
resident proteins, as suggested by many studies, a causal process seems
to be different and divergent among individual proteins. The
elucidation of the mechanistic and structural bases for the formation
of an oligomer would be desired, to better understand the mechanism for
the retention of glycosyltransferases and other Golgi proteins. The
present study has clearly identified and characterized the domain
responsible for the retention of GnT-V and thereby provides a possible
mechanism for conferring Golgi retention, namely, the formation of oligomers.
 |
ACKNOWLEDGEMENTS |
We thank the Japanese Cancer Research Bank
for providing us with COS-1 cells and CHO-K1 cells. We thank Dr.
Takaomi Ishida (Institute of Medical Science, University of Tokyo) for
providing us with pME18FLAG expression vector.
 |
FOOTNOTES |
*
This work was supported in part by a Grant-in-Aid for
Scientific Research on Priority Areas 10178104 from the Ministry of Education, Science, Sports and Culture of Japan.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.
¶
To whom correspondence should be addressed: Dept. of
Biochemistry, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3420; Fax:
81-6-6879-3429; E-mail: proftani@biochem.med.osaka-u.ac.jp.
Published, JBC Papers in Press, October 9, 2000, DOI 10.1074/jbc.M004972200
 |
ABBREVIATIONS |
The abbreviations used are:
GnT-V,
1,6-N-acetylglucosaminyltransferase V;
GnT-III,
1,4-N-acetylglucosaminyltransferase III;
GGT,
-glutamyl transpeptidase;
PAGE, polyacrylamide gel electrophoresis;
PBS, phosphate-buffered saline;
PCR, polymerase chain reaction;
GST, glutathione S-transferase;
MES, 4-morpholineethanesulfonic acid;
X-gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside;
HIV-1, human immunodeficiency virus type 1.
 |
REFERENCES |
1.
|
Schachter, H.
(1986)
Biochem. Cell Biol.
64,
163-181[Medline]
[Order article via Infotrieve]
|
2.
|
Pierce, M.,
Arango, J.,
Tahir, S. H.,
and Hinds Gaul, O.
(1987)
Biochem. Cell Biol. Commun.
146,
679-684
|
3.
|
Dennis, J. W.,
and Laferte, S.
(1989)
Cancer Res.
49,
945-950[Abstract]
|
4.
|
Pierce, M.,
Buckhaults, P.,
Chen, L.,
and Fregien, N.
(1997)
Glycoconj. J.
14,
623-630[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Taniguchi, N.,
Miyoshi, E.,
Ko, J. H.,
Ikeda, Y.,
and Ihara, Y.
(1999)
Biochim. Biophys. Acta
1455,
287-300[Medline]
[Order article via Infotrieve]
|
6.
|
Demetriou, M.,
Nabi, I. R.,
Coppolino, M.,
Dedhar, S.,
and Dennis, J. W.
(1995)
J. Cell Biol.
130,
383-392[Abstract]
|
7.
|
Seberger, P. J.,
and Chaney, W. G.
(1999)
Glycobiology
9,
235-241[Abstract/Free Full Text]
|
8.
|
Granovsky, M.,
Fata, J.,
Pawling, J.,
Muller, W. J.,
Khokha, R.,
and Dennis, J. W.
(2000)
Nat. Med.
6,
306-312[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Shoreibah, M.,
Perng, G. S.,
Adler, B.,
Weinstein, J.,
Basu, R.,
Cupples, R.,
Wen, D.,
Browne, J. K.,
Buckhaults, P.,
Fregien, N.,
and Pierce, M.
(1993)
J. Biol. Chem.
268,
15381-15385[Abstract/Free Full Text]
|
10.
|
Saito, H.,
Nishikawa, A.,
Gu, J.,
Ihara, Y.,
Soejima, H.,
Wada, Y.,
Sekiya, C.,
Niikawa, N.,
and Taniguchi, N.
(1994)
Biochem. Cell Biol. Commun.
198,
318-327
|
11.
|
Colley, K. J.
(1997)
Glycobiology
7,
1-13[Abstract]
|
12.
|
Masibay, A. S.,
Balaji, P. V.,
Boeggeman, E. E.,
and Qasba, P. K.
(1993)
J. Biol. Chem.
268,
9908-9916[Abstract/Free Full Text]
|
13.
|
Munro, S.
(1998)
Trends. Cell Biol.
8,
11-15[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Nilsson, T.,
Hoe, M. H.,
Slusarewicz, P.,
Rabouille, C.,
Watson, R.,
Hunte, F.,
Watzele, G.,
Berger, E. G.,
and Warren, G.
(1994)
EMBO J.
13,
562-574[Abstract]
|
15.
|
Nilsson, T.,
Rabouille, C.,
Hui, N.,
Watson, R.,
and Warren, G.
(1996)
J. Cell Sci.
109,
1975-1989[Abstract/Free Full Text]
|
16.
|
Ihara, Y.,
Nishikawa, A.,
Tohma, T.,
Soejima, H.,
Niikawa, N.,
and Taniguchi, N.
(1993)
J. Biochem. (Tokyo)
113,
692-698[Abstract]
|
17.
|
Kunkel, T. A.
(1985)
Proc. Natl. Acad. Sci. U. S. A.
82,
488-492[Abstract]
|
18.
|
Ikeda, Y.,
Fujii, J.,
Anderson, M.,
Taniguchi, N.,
and Meister, A.
(1995)
J. Biol. Chem.
270,
22223-22228[Abstract/Free Full Text]
|
19.
|
Nishikawa, A.,
Ihara, Y.,
Hatakeyama, M.,
Kangawa, K.,
and Taniguchi, N.
(1992)
J. Biol. Chem.
267,
18199-18204[Abstract/Free Full Text]
|
20.
|
Ishida, T.,
Tojo, T.,
Aoki, T.,
Kobayashi, N.,
Ohishi, T.,
Watanabe, T.,
Yamamoto, T.,
and Inoue, J.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
9437-9442[Abstract/Free Full Text]
|
21.
|
Ikeda, Y.,
Fujii, J.,
and Taniguchi, N.
(1993)
J. Biol. Chem.
268,
3980-3985[Abstract/Free Full Text]
|
22.
|
Chu, G.,
Hayakawa, H.,
and Berg, P.
(1987)
Nucleic Acids Res.
15,
1311-1325[Abstract]
|
23.
|
Laemmli, U. K.
(1970)
Nature
227,
680-685[Medline]
[Order article via Infotrieve]
|
24.
|
Taniguchi, N.,
Nishikawa, A.,
Fujii, S.,
and Gu, J. G.
(1989)
Methods Enzymol.
179,
397-408[Medline]
[Order article via Infotrieve]
|
25.
|
Bradford, M. M.
(1976)
Anal. Biochem.
72,
248-54[CrossRef][Medline]
[Order article via Infotrieve]
|
26.
|
Nagai, K.,
Ihara, Y.,
Wada, Y.,
and Taniguchi, N.
(1997)
Glycobiology
7,
769-776[Abstract]
|
27.
|
Chen, L.,
Zhang, N.,
Adler, B.,
Browne, J,
Freigen, N.,
and Pierce, M.
(1995)
Glycoconj. J.
12,
813-823[Medline]
[Order article via Infotrieve]
|
28.
|
Gardell, S. J.,
and Tate, S. S.
(1979)
J. Biol. Chem.
254,
4942-4945[Abstract]
|
29.
|
Nash, B.,
and Tate, S. S.
(1982)
J. Biol. Chem.
257,
585-588[Abstract/Free Full Text]
|
30.
|
Taniguchi, N.,
Iizuka, S.,
Zhe, Z. N.,
House, S.,
Yokosawa, N.,
Ono, M.,
Kinoshita, K.,
Makita, A.,
and Sekiya, C.
(1985)
Cancer Res.
45,
5835-5839[Abstract]
|
31.
|
Opat, A. S.,
Houghton, F.,
and Gleeson, P. A.
(2000)
J. Biol. Chem.
275,
11836-11845[Abstract/Free Full Text]
|
32.
|
Chen, C.,
Ma, J.,
Lazic, A.,
Backovic, M.,
and Colley, K. J.
(2000)
J. Biol. Chem.
275,
13819-13826[Abstract/Free Full Text]
|
33.
|
Levin, J. M.,
Robson, B.,
and Garnier, J.
(1986)
FEBS Lett.
205,
303-308[CrossRef][Medline]
[Order article via Infotrieve]
|
34.
|
Luthy, R.,
McLachlan, A. D.,
and Eisenberg, D.
(1991)
Proteins
10,
229-239[Medline]
[Order article via Infotrieve]
|
35.
|
Chou, P. Y.,
and Fasman, G. D.
(1978)
Adv. Enzymol. Relat. Areas. Mol. Biol.
47,
45-148[Medline]
[Order article via Infotrieve]
|
36.
|
Earl, P. L.,
Moss, B.,
and Doms, R. W.
(1991)
J. Virol.
65,
2047-2055[Medline]
[Order article via Infotrieve]
|
37.
|
Willey, R. L.,
Bonifacino, J. S.,
Potts, B. J.,
Martin, M. A.,
and Klausner, R. D.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
9580-9584[Abstract]
|
38.
|
Poumbourios, P.,
Wilson, K. A.,
Center, R. J.,
El, A. W.,
and Kemp, B. E.
(1997)
J. Virol.
71,
2041-2049[Abstract]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.