(Received for publication, January 6, 1997, and in revised form, March 4, 1997)
From the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma Center for Molecular Medicine, Oklahoma City, Oklahoma 73190
It has been assumed that membrane-bound
glycosyltransferases function within the Golgi apparatus to glycosylate
glycoproteins. We now report, however, that a truncated, soluble
recombinant form of the murine 1,3-galactosyltransferase expressed
in human 293 cells is highly efficient and comparable to the
full-length enzyme in
-galactosylating both newly synthesized
membrane-associated and secreted glycoproteins. Although the soluble
enzyme was secreted by cells as expected, we also found that the
full-length, membrane-associated form was secreted. Unexpectedly, both
secreted forms are cleaved identically at two primary sites within the
stem region by endogenous protease(s) at the indicated positions in the
sequence 73KDWW
FPS
WFKNG. These results
demonstrate that the soluble
1,3-galactosyltransferase is functional
within the cell and that specific proteolysis occurs in the stem
region. The widespread occurrence of different soluble glycosyltransferases secreted by cells suggests that normal
glycoconjugate biosynthesis may involve cooperation between
membrane-bound and soluble enzymes.
The synthesis of the carbohydrate groups in secreted and membrane-bound glycoconjugates occurs in the endoplasmic reticulum and Golgi apparatus and involves glycosyltransferases, which utilize sugar nucleotides imported into the lumen of these organelles by specific import proteins (1-4). Most of the cloned mammalian glycosyltransferases share many features in common, although their primary structures are often unrelated. The deduced amino acid sequences of the cDNA clones encoding many glycosyltransferases reveal that they are type 2 transmembrane proteins containing a short N-terminal cytoplasmic tail consisting of 4-24 amino acids, a membrane-spanning domain, and an extended stem region in the lumen that precedes the large C-terminal catalytic domain (4, 5). The transmembrane domain and flanking sequences of many glycosyltransferases may be important in retaining/targeting the enzymes in the Golgi apparatus (6-17). However, the exact mechanisms and the precise purpose of the targeting have not been elucidated. Because of their transmembrane nature, it has been presumed that Golgi glycosyltransferases function in the membrane-anchored form.
Numerous glycosyltransferases have been detected, however, as soluble
forms in serum, milk, colostrum, and/or growth media from normal and
transformed cell lines (18-23). The mechanism(s) of formation and
secretion of soluble and active glycosyltransferases are not
understood. N-terminal sequence analyses of soluble forms of the
2,6-sialyltransferase and
1,4-galactosyltransferase demonstrate that they arise through proteolysis within the stem region, thereby releasing the catalytic domain from the membrane (24, 25). The
proteases responsible for cleaving the enzymes are not defined, although evidence suggests that a cathepsin D-like protease may be
responsible for the cleavage of
2,6-sialyltransferase (26). The
functions of the soluble glycosyltransferases are not known.
One enzyme that has recently been shown to be secreted from cultured
cells is the murine 1,3-galactosyltransferase
(
1,3GT)1 (27, 28), which is responsible
for the synthesis of terminal
-Gal sequences in Gal
1
3Gal
1
4GlcNAc-R. The murine (29), bovine (30), porcine (31, 32), and New
World monkey (33) cDNA encoding for
1,3GT have been identified.
The
1,3GT is expressed in a variety of mammalian species, but it is
not expressed in Old World monkeys, apes, and humans (34, 35). The
expression of the
1,3GT in murine F9 teratocarcinoma cells is
transcriptionally up-regulated by retinoic acid induction, but most of
the newly synthesized enzyme is eventually secreted into the culture
media of the cells (27). The
1,3GT is secreted by many cell types, since we have detected soluble forms of the
1,3GT in the culture media of all cell lines expressing the enzyme, and we have found enzyme
activity in the sera of different animals expressing the functional
1,3GT gene (28).
The common occurrence of the 1,3GT in a soluble form led us to
question whether it may have a function. One possibility is that the
soluble enzyme may have a biosynthetic role within the Golgi apparatus.
In this report we describe experiments showing that a truncated,
soluble recombinant form of
1,3GT expressed in human 293 cells is
highly efficient in
-galactosylating both newly synthesized
membrane-associated and secreted glycoproteins and that it is
comparable to the wild-type enzyme in its efficiency. Interestingly,
both the truncated and full-length enzymes are secreted from 293 cells
and both are cleaved by endogenous protease(s) at two primary sites in
the stem region. These results demonstrate that a soluble
glycosyltransferase can function within cells and suggest that normal
proteolysis of membrane-bound glycosyltransferases may be important in
generating enzymes that participate in glycoconjugate biosynthesis.
Bovine serum albumin, UDP-Gal, raffinose, EDTA, N-acetyllactosamine, ATP, D-galactono-1,4-lactone, Tween 20, phenylmethylsulfonyl fluoride, pepstatin, aprotinin, leupeptin, UDP-hexanolamine-Sepharose (4 µmol/ml resin), horseradish peroxidase-conjugated Griffonia simplicifolia isolectin I-B4 (GS-I-B4), and fluorescein isothiocyanate-conjugated GS-I-B4 (FITC-GS-I-B4) were obtained from Sigma. Mouse Engelbreth-Holm-Swarm laminin was obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Restriction enzymes were purchased from Boehringer Mannheim. UDP-[6-3H]galactose (15 Ci/mmol) was obtained from American Radiolabeled Chemicals Inc. (St. Louis, MO). The BCA protein assay kit was purchased from Pierce. Triton X-100 was obtained from Bio-Rad. The ECL Western blotting kit was purchased from Amersham Corp. Tissue culture reagents were obtained from Life Technologies, Inc. All other chemicals used were of the highest grade available.
Cell CultureMouse teratocarcinoma F9 cells were cultured
in Dulbecco's modified Eagle's medium containing 15% fetal calf
serum on gelatin-coated tissue culture plates as described (36). For
induction of differentiation, cells were grown under identical
conditions in media supplemented with 107 M
all-trans-retinoic acid for 3 days. Human 293 kidney cells (ATCC CRL 1573) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. CHO-Tag cells, expressing the large T
antigen, were cultured in
-minimal essential media containing 10%
fetal calf serum, ribonucleosides, and G418 (400 µg/ml). CHO-Tag cells were a generous gift of Dr. John B. Lowe (University of Michigan,
Ann Arbor, MI).
A C-terminal fusion of
the murine 1,3GT to a 9-amino acid peptide tag from influenza
hemagglutinin (HA) was generated by PCR using the 5
primer
5
-GGGGAGCACATCCTGGCCCACATCCAG-3
and the 3
primer
5
-ATGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAGACATTATTTCTAACCAAATTATACT-3
as described.2 Briefly, the plasmid
pCDM7-
GT, which contains the cDNA of the murine
1,3GT (29),
was used as the template for the PCR reaction. The resulting PCR
fragment (490 base pairs), containing a SalI restriction
site at the 5
-end and a XbaI restriction site at the
3
-end, was subcloned into the vector pCR II and sequenced. Wild-type
1,3GT cDNA in pcDNAI/Amp (Invitrogen, San Diego, CA) was
excised with SalI and XbaI, and the excised
fragment was replaced with the 490-base pair
SalI/XbaI PCR fragment encoding the epitope tag.
The 9-amino acid HA epitope tag is specifically recognized by the mouse
monoclonal antibody 12CA5 (37).
This full-length construct, designated 1,3GT-HA, was co-transfected
into 293 cells, along with the G418 selection plasmid PBKEF (a gift
from Dr. Kinji Fukudome, Oklahoma Medical Research Foundation, Oklahoma
City, OK), in a 4:1 molar ratio, using lipofectamine reagent (Life
Technologies, Inc.) according to the procedure recommended by the
supplier. Clonal cell lines were derived from the G418 (400 µg/ml)-resistant transfectant population using cloning cylinders. Flow cytometric analyses were performed (27), and a representative clonal line that stably expresses cell surface
-galactosylated glycoconjugates was selected and designated fl-
1,3GT-1.
A mutated form
of the full-length 1,3GT was generated in which the stem sequence
was mutated to contain Asp residues at three potentially critical sites
for cleavage (see Fig. 1C). This construct was generated by
PCR using the 5
primer (HindIII primer)
5
-GGAAGCTTATGATCACTATGCTTCAAGAT-3
and the 3
primer (mutagenic
primer) 5
-ACTGTGGGTCCCATTTTTAAACCAGCTTGGGAACCACCAGTC-3
with a
DraII site. Specifically, the 3
primer was designed to convert amino acid residues 76, 80, and 81 to Asp (see Fig.
1C). The plasmid pcDNAI/Amp, which contains the
full-length construct
1,3GT-HA, was used as the template for the PCR
reaction. The resulting PCR fragment (260 base pairs) was digested with
HindIII and DraII and then ligated into the
DraII/XbaI excised fragment of
1,3GT-HA,
followed by ligation into the HindIII/XbaI
linearized pcDNAI/Amp vector. This mutant construct, designated
st-1-
1,3GT-HA, was sequenced to confirm the mutations. The plasmid
containing the mutated gene was co-transfected into 293 cells along
with the G418 selection plasmid PBKEF. Clonal selection was carried out
in the presence of G418 (400 µg/ml) as described above. Western blot
analysis was performed using microsomes and culture media of
transfectants as described below, and a representative clonal line that
stably expressed the mutated
1,3GT was selected and designated
st-1-
1,3GT-HA-1.
Construction and Transfection of Truncated, Soluble
To produce a soluble form of murine
1,3-galactosyltransferase, a fusion protein containing a 12-residue
Ca2+-dependent peptide epitope for the
monoclonal antibody, HPC4, and the putative lumenal catalytic domain of
the enzyme was constructed in the mammalian expression vector RSV-PL4
(38). Specifically, the lumenal domain of the
1,3GT was amplified by
PCR using the 5
primer
5
-CCAACCCGGGATTCCAGAGGTTGGTGAGAACAGATGGCAG-3
with a SmaI
site and the 3
primer 5
-CGGCTCTAGAGCCTTCAGACATTATTTCTAACCAAATT-3
with a XbaI site. The plasmid pCDM7-
GT, which
contains the cDNA of the murine
1,3GT (28), was used as the
template for the PCR reaction. The PCR product was digested with
SmaI and XbaI and then ligated into the
StuI/XbaI site of RSV-PL4 vector, resulting in a
fusion protein of the soluble
1,3GT in-frame to the transferrin signal sequence present in the vector. This construct was transfected into human 293 cells using lipofectamine reagent (Life Technologies, Inc.), and clonal selection was carried out in the presence of G418
(400 µg/ml) as described above. Enzyme assays were performed (27),
and representative clonal lines that stably secreted enzyme into media
were selected and designated s-
1,3GT-1, s-
1,3GT-2, and
s-
1,3GT-3.
The HPC4-NKR-P1 was constructed in the mammalian expression vector
RSV-PL4 (38). Specifically, both the N-terminal cytoplasmic and the
transmembrane domain of NKR-P1 were removed, and the extracellular domain of the NKR-P1 was amplified by PCR using the 5 primer 5
-CAAAAACCATCAAGAGAAAA-3
and the 3
primer
5
-GGATTCTAGATCAGGAGTCATTACTCGGGG-3
. The cDNA encoding the murine
NKR-P1 used as the template for the PCR reaction was a kind gift of
Drs. Massimo Trucco and Roberto Giorda (Children's Hospital of
Pittsburgh, Pittsburgh, PA). The PCR product was phosphorylated and
digested with XbaI and then ligated into the
StuI/XbaI site of RSV-PL4 vector, resulting in a
fusion protein of the soluble NKR-P1 in-frame to the transferrin signal
sequence present in the vector. This construct was transfected into
human 293 cells using lipofectamine (Life Technologies, Inc.), and
clonal selection was carried out in the presence of G418 (400 µg/ml)
as described above. Western blot analysis was performed using culture
media of transfectants, and representative clonal lines that stably
secreted NKR-P1 into media were selected.
Cell extracts were prepared in the presence of the protease inhibitors phenylmethylsulfonyl fluoride (1 mM), pepstatin (1 µg/ml), aprotinin (10 µg/ml), and leupeptin (10 µg/ml), and microsomes were prepared as described previously (27, 39). To prepare cell culture media for enzyme assays and immunoblot or lectin blot analyses, cells were grown in complete media until they reached ~80% confluency and were then shifted to serum-free media. The media were harvested after 24 h of culture and subjected to low speed centrifugation (300 × g for 10 min), and the supernatant was centrifuged at high speed (100,000 × g for 1 h). Media were concentrated in a Centriprep-10 concentrator (Amicon Inc., Beverly, MA).
AntibodiesMonoclonal anti-HA (clone 16B12) ascites fluid
was purchased from Berkeley Antibody Co. (Berkeley, CA) and was used at
a dilution of 1:500. Monoclonal anti-HPC4 antibody was a gift from Dr.
Charles Esmon (Oklahoma Medical Research Foundation) and was used at a concentration of 10 µg/ml. To prepare rabbit 1,3GT antiserum against the murine
1,3GT, a fusion protein was constructed that contains the putative lumenal domain of the murine
1,3GT and a
6-histidine tag at the N terminus. Specifically, the lumenal domain of
the
1,3GT, comprising amino acid residues 61-326, was amplified by
PCR using the 5
primer 5
-ACCGGATCCCAACAGAATTCCAGAGGT-3
with a
BamHI site and the 3
primer
5
-AGCCTGCAGTTAAAGCACTCCCTGGTG-3
with a PstI site. The
plasmid pCDM7-
GT, which contains the cDNA of the murine
1,3GT
(28), was used as the template for the PCR reaction. The PCR product
was digested with BamHI and PstI and then ligated
into the BamHI/PstI site of PQE-10 vector (QIA express, Qiagen), resulting in a 6-His-tagged fusion protein of truncated
1,3GT. The resulting PCR fragment containing a
BamHI restriction site at the 5
-end and a PstI
restriction site at the 3
-end was subcloned into the vector pBS II
SK
and sequenced. This construct was used to transform
into Escherichia coli strain M15 (pREP4), and the expressed
6-His-
1,3GT was purified using a nitrilotriacetic acid affinity
column according to the procedure recommended by the supplier. The
rabbit
1,3GT antiserum was raised by injecting an animal three times
(in 10-day intervals) with the nickel affinity-purified, bacterial
6-His-tagged fusion protein of the truncated
1,3GT. Sera were
collected and used at a dilution of 1:500. The secondary antibody
conjugates, horseradish peroxidase-conjugated goat anti-rabbit and
horseradish peroxidase-conjugated sheep anti-mouse IgG antibody, were
used at dilutions of 1:500.
The catalytically active, secreted form of 1,3GT was
purified from culture media of human 293 transfectants by affinity
chromatography on a column of UDP-hexanolamine-Sepharose with some
modification (40). Culture media from fl-
1,3GT-1 and s-
1,3GT-2
cells were harvested when the cells were ~90% confluent and then
dialyzed overnight at 4 °C against 20 mM HEPES, 5 mM MnCl2, pH 7.0 (buffer A) with several
changes of buffer. The dialyzed material was applied to a column
containing UDP-hexanolamine-Sepharose (4 µmol/ml resin) and washed
with buffer A. This was followed by additional washing with buffer A
containing 0.75 M NaCl until the absorbance of each fraction at 280 nm reached background levels. The bound enzyme was
eluted with 5 mM UDP in buffer A, and enzyme was
concentrated in Centricon-10. The purified enzyme was resolved on 10%
SDS-PAGE (41) and electroblotted onto polyvinylidine difluoride
membrane (Immobilon-P, Millipore). The spot corresponding to the
1,3GT was located on the paper by Ponceau staining and then excised and subjected to N-terminal amino sequence analysis by Edman
degradation, using the Applied Biosystems 470A protein sequencer (The
University of Oklahoma Health Sciences Molecular Biology Facility,
Oklahoma City, OK).
Cells were harvested and washed once with Hanks' balanced salt solution. Approximately 5 × 105 cells were incubated with FITC-GS-I-B4 at 10 µg/ml in 100 µl of staining buffer (Hanks' balanced salt solution, 1% heat-inactivated goat serum) on ice for 30 min and then analyzed by fluorescence-activated cell sorting as described previously (27).
ImmunoblottingThe 1,3GT was purified by
UDP-hexanolamine affinity chromatography from s-
1,3GT-2 culture
media as described above. NKR-P1 was purified by immunoaffinity
chromatography on immobilized anti-HPC4. The purified proteins were
resolved in 10% SDS-PAGE. The separated proteins were
electrophoretically transferred (42) onto nitrocellulose membranes and
then stained by standard immunoblot analysis. The standard ECL
immunoblotting procedures are as follows. Blots were blocked with TBS
(20 mM Tris, 150 mM NaCl, pH 7.5) containing 5% nonfat dry milk at room temperature for 1 h, and they were incubated with primary antibodies in dilution buffer (TBS containing 0.5% bovine serum albumin, 0.05% Tween 20) for 1 h. Following three 15-min washes in TTBS (TBS, 0.05% Tween 20), the immunoblots were incubated at room temperature for 1 h with horseradish
peroxidase-conjugated secondary antibodies in dilution buffer. Blots
were washed three times (15 min each) in TTBS followed by one wash in
TBS and developed using the ECL Western blotting kit according to the
manufacturer's instructions. For the detection of
1,3GT derived
from fl-
1,3GT-1 cells, the microsomes and soluble protein in the
culture media were resolved in 10% SDS-PAGE and analyzed by standard
ECL immunoblotting using monoclonal anti-HA antibody.
For the detection of -galactosylated
glycoproteins, 30 µg of the microsomal fraction and 100 µg of
soluble proteins in culture media were prepared as described previously
(27) and subjected to electrophoresis on 7.5% SDS-PAGE. The separated
proteins were electrophoretically transferred onto nitrocellulose
membranes (Hybond-ECL Western, Amersham Corp.) and followed by lectin
blot analysis. The ECL lectin blotting was performed as described
previously (27) except that the
-galactosylated glycoproteins were
detected using 10 µg/ml horseradish peroxidase-conjugated
GS-I-B4.
To test the functionality of the soluble form of murine
1,3GT and to compare it to the full-length enzyme, fusion proteins were constructed as described under "Experimental Procedures" and
illustrated in Fig. 1. A full-length chimeric
1,3GT
was constructed in which the 9-amino acid influenza virus hemagglutinin
(HA) epitope tag (37) was fused to the C-terminal end of full-length
1,3GT. This chimera is shown in Fig. 1A and is designated
the
1,3GT-HA construct. A soluble form of the
1,3GT was
constructed in which the fusion protein consists of the transferrin
signal peptide preceding an HPC4 epitope (38) and followed by amino
acids 63-394 of the murine
1,3GT encoding the C-terminal catalytic
domain. This chimera is shown in Fig. 1B and is designated
the HPC4-
GT construct.
Human kidney 293 cells were transfected with either the 1,3GT-HA or
HPC4-
GT, and stably expressing 293 cell lines were isolated as
described under "Experimental Procedures." One clone stably expressing
1,3GT-HA was designated fl-
1,3GT-1. Three clones stably expressing the HPC4-
GT were identified, and these were designated s-
1,3GT-1, s-
1,3GT-2, and s-
1,3GT-3.
Cell surface expression of
terminal Gal1
3Gal determinants requires expression of the
cognate
1,3GT (29). The stably expressing cell lines were tested for
their ability to display cell surface-localized oligosaccharide product
of enzyme using flow cytometry and FITC-GS-I-B4.
GS-I-B4 is a plant lectin that specifically binds terminal
Gal
1
3Gal linkages (43). As expected, the parental 293 cell line
does not display surface Gal
1
3Gal determinants (Fig.
2). In contrast, 293 cells transfected with either
1,3GT-HA or HPC4-
GT stained brightly with
FITC-GS-I-B4 (Fig. 2). The levels of expression of surface
Gal
1
3Gal determinants in the stably expressing clones were
comparable to the normal level observed in retinoic acid-differentiated
F9 cells cells (Fig. 2). These observations demonstrate that the
soluble
1,3GT can participate in biosynthesis of surface-localized
glycoconjugates containing the Gal
1
3Gal determinant. Based on
these flow cytometric analyses and the level of staining with
GS-I-B4, the soluble
1,3GT appears to be comparable to
the full-length enzyme in generating surface Gal
1
3Gal
determinants.
Lectin Blot Analysis of Glycoproteins Containing Gal
To assess whether there are quantitative and/or
qualitative differences between the full-length or soluble forms of the
enzyme in the way they glycosylate proteins, the microsome and
serum-free media prepared from the clones were analyzed on 7.5%
SDS-PAGE, and glycoproteins were probed with peroxidase-conjugated
GS-I-B4. As expected, the glycoproteins from parental 293 cell microsomes and serum-free media were not reactive with
GS-I-B4 (Fig. 3, A and
B). In contrast, significant amounts of
GS-I-B4-reactive membrane-associated glycoprotein was
present in microsomes of fl-1,3GT-1, s-
1,3GT-1, and s-
1,3GT-2
cells, although the levels of expression appear somewhat higher in
fl-
1,3GT-1 microsomes. However, no appreciable differences were
observed in the overall patterns of
-galactosylated membrane-associated glycoproteins in microsomes between the clones expressing either full-length or truncated enzyme (Fig. 3A).
In addition, both the levels and the pattern of
GS-I-B4-reactive material in the serum-free media of all
the clones were similar (Fig. 3B). In all cases the binding
of GS-I-B4 to glycoproteins was specific, since inclusion
of hapten sugar raffinose (200 mM) blocked binding (Fig. 3,
A and B). In control experiments,
GS-I-B4 bound well, as expected, to a commercial
preparation of Engelbreth-Holm-Swarm laminin, which is known to contain
terminal
1,3-galactosyl residues (44, 45). These results demonstrate
that both the full-length and soluble
1,3GT cause the biosynthesis
of high levels of
-galactosylated glycoproteins.
Enzyme Activities in Cell Extracts and Media from Cells Expressing Soluble and Full-length
To determine whether there are
significant differences in the level of expression of the soluble
versus the full-length 1,3GT and whether there are
differences in secretion of the two forms, we measured the enzyme
activity in cell extracts and culture media using
N-acetyllactosamine as the acceptor and
UDP-[3H]Gal as the donor. The results are shown in Table
I. No activity was detected in extracts of the parental
293 cells. Significant
1,3GT activity was present in extracts of
fl-
1,3GT-1 cells (49.0 nmol/h), but approximately two-thirds of the
total activity, which represents activity in the cell extracts plus
culture media, was recovered in the culture media (Table I). The
activities of the
1,3GT in extracts of s-
1,3GT-1, s-
1,3GT-2,
and s-
1,3GT-3 cells were slightly lower than that of fl-
1,3GT-1
cell extracts (Table I). As might be expected, the levels of activity
of
1,3GT in media of s-
1,3GT-1, s-
1,3GT-2, and s-
1,3GT-3
cells were slightly higher than that in the media from fl-
1,3GT-1
cells.
|
We considered the possibility that the
secreted 1,3GT might transfer
-galactosyl residues to surface
glycoproteins after it was secreted from the cells, rather than from
within, possibly using secreted UDP-Gal as the donor. Parental 293 cells were grown to ~80% confluency, and the media were replaced by
freshly harvested culture media of s-
1,3GT-2 cells. The enzyme
activity in the transferred media was 0.3 nmol/12.5 µl/h. Control
parental 293 cells received media from parental 293 cells not
expressing the
1,3GT. After 5 h of incubation with media either
containing or lacking
1,3GT activity, the cells were harvested and
tested for surface Gal
1
3Gal determinants using flow cytometry
and FITC-GS-I-B4. There was no expression of surface
Gal
1
3Gal determinants in cells exposed to the
1,3GT-containing media, and there were no detectable differences
between control parental 293 cells, which had never been exposed to
1,3GT-containing media, and the treated cells (data not shown).
These observations demonstrate that surface Gal
1
3Gal
determinants in cells expressing the
1,3GT arise from enzyme
activity within the cells rather than from addition of the determinants
by the secreted enzyme outside the cells.
To confirm that the
full-length enzyme was released in a soluble form into the media, the
1,3GT in fl-
1,3GT-1 cells was analyzed by Western blot analysis
using the monoclonal anti-HA antibody. The
1,3GT in microsomal
preparations migrates as a diffuse band centered at ~53 kDa in
reducing SDS-PAGE, whereas the apparent size of the
1,3GT from media
is ~50.5 kDa (Fig. 4A). These data
demonstrate that the full-length
1,3GT is expressed by the cells and
secreted as a lower molecular mass soluble form and that both forms
retain the HA epitope at their C termini.
We also attempted to identify the soluble form of the 1,3GT
expressed from s-
1,3GT-2 cells using Western blot analysis and anti-HPC4 monoclonal antibody. Unexpectedly, the soluble, secreted form
of the enzyme from s-
1,3GT-2 cells did not react with the anti-HPC4
monoclonal antibody (Fig. 4B). The recombinant protein was
present, as shown by its reactivity in a Western blot with rabbit
polyclonal antibody prepared against bacterially derived murine
1,3GT. The secreted enzyme had an apparent Mr
~42,000. These results indicate that the recombinant
1,3GT had
lost the HPC4 epitope during secretion of the enzyme.
We considered the possibility that the HPC4 determinant was cleaved
from the protein by a protease(s) that might recognize in some way the
foreign HPC4 determinant on the recombinant protein. To address this
possibility, another HPC4-tagged recombinant glycoprotein was prepared
and expressed in 293 cells. In this experiment 293 cells were stably
transfected with cDNA encoding a chimeric form of the mouse NKR-P1
in which the HPC4 epitope was appended to the N terminus in a construct
designated HPC4-NKR-P1, prepared as described under "Experimental
Procedures." NKR-P1 is a dimeric surface protein with ~30-kDa
subunits expressed on murine natural killer cells (46). It is a type 2 transmembrane protein, and in this regard it resembles
glycosyltransferases in its proposed topological orientation within the
membrane. Media from cells expressing HPC4-NKR-P1 were reactive with
anti-HPC4 monoclonal antibody (Fig. 4B). These results
demonstrate that the HPC4 epitope is retained on this recombinant
protein. Consistent with this finding that the HPC4 determinant is
cleaved from the 1,3GT constructs but not from the HPC4-NKR-P1, we
also observed that the recombinant
1,3GT in the culture media of
s-
1,3GT-2 cells did not bind to immobilized anti-HPC4 in affinity
chromatography, whereas the HPC4-NKR-P1 was recovered upon
immunoaffinity chromatography (data not shown). These results
demonstrate that the HPC4 epitope is selectively cleaved from the N
terminus of the recombinant
1,3GT derived from HPC4-
GT. Direct
peptide sequencing results described below support this conclusion.
To determine the generality of secretion of the 1,3GT from cells, we
also transiently transfected CHO-Tag cells, which express the large T
antigen, with the full-length
1,3GT-HA construct. The media
harvested 48 h after transfection were assayed for
1,3GT enzyme
activity, and protein in the media was used for a Western blot with the
monoclonal anti-HA antibody. The media contained 45 nmol/h of total
activity and the apparent molecular mass of secreted
1,3GT from
CHO-Tag cells was ~50.5 kDa in reducing SDS-PAGE (data not shown),
which is similar to that of secreted
1,3GT from fl-
1,3GT-1 cells.
These observations confirm that secretion of
1,3GT from cells is a
common phenomenon, which is consistent with recent studies showing that
the enzyme is secreted from a large number of cells types (28).
To identify the N
terminus of the 1,3GT secreted by fl-
1,3GT-1 cells, the enzyme
was purified from cell culture media by affinity chromatography on
UDP-hexanolamine. The purified enzyme was resolved on 10% SDS-PAGE and
subjected to N-terminal sequencing as described under "Experimental
Procedures." Two soluble species were identified, with the
predominant species being 3 amino acid residues longer than the minor
species. The N termini of the two species reveal that soluble forms of
1,3GT are derived from the full-length form by proteolytic cleavage
C-terminal to Trp76 and C-terminal to Ser79 in
the putative stem region of the enzyme, as shown in Fig.
1A.
We then determined the N terminus of the secreted 1,3GT derived from
s-
1,3GT-2 cells, which had lost the N-terminal HPC4 epitope as
described above. The enzyme was purified from the culture media of the
cells by affinity chromatography on UDP-hexanolamine and subjected to
SDS-PAGE, on which it had an apparent size of ~42 kDa (Fig.
4B). The gel band containing the enzyme was subjected to
N-terminal sequencing as described under "Experimental Procedures." Again, two N-terminal sequences were identified, and these were identical to those observed in the soluble forms recovered in the
culture media from fl-
1,3GT-1 cells (Fig. 1A). These
results indicate that for both the soluble and full-length forms of the
1,3GT specific proteolysis occurs within the dipeptide sequence Trp76Phe77 and
Ser79Phe80 (Fig. 1A).
These data indicate that proteolysis occurs within a discrete region of
the stem of the 1,3GT. However, it is possible that other cleavage
sites also exist. For example, other cleavage sites could be present
N-terminal to the defined sites, with multiple proteolytic cleavages
generating the two major final forms identified above. To address this
possibility, we prepared a mutated form of the full-length
1,3GT in
which the stem sequence was mutated to contain Asp residues at three
potentially critical sites for cleavage, as shown in Fig.
1C. These charged residues would presumably prohibit
recognition by proteases that potentially require hydrophobic residues
for cleavage. The plasmid encoding this mutant, termed st-1-
1,3GT-HA, was stably transfected into 293 cells. Media and microsomes from these cells were analyzed by Western blot using the
monoclonal anti-HA antibody. Unexpectedly, the st-1-
1,3GT-HA was
cleaved as efficiently as the wild-type protein, and the soluble, mutated enzyme was recovered in the culture media (data not shown). The
results were comparable to those shown in Fig. 4A. The N
terminus of the soluble, mutated enzyme is not yet defined. These data indicate either that proteolysis of the stem region can occur in other
sites in addition to those identified above or that the mutated stem
region in st-1-
1,3GT-HA may represent a new recognition site for a
different protease(s).
These studies demonstrate that a soluble form of the 1,3GT,
which is similar to that formed naturally through proteolysis of the
membrane-associated full-length enzyme, is functional within the Golgi
apparatus and is capable of generating quantitative levels of Gal
1
3Gal determinants in cell surface and secreted glycoproteins. These
observations suggest the hypothesis that soluble glycosyltransferases,
generated by specific proteolysis of the membrane-associated enzymes
within the secretory apparatus, can participate in the biosynthesis of
complex glycoconjugates.
Numerous studies have documented the existence of soluble
glycosyltransferases in body fluids (18, 19, 23, 47-53) and in the
growth media from normal and transformed cell lines (21, 22), although
the functional significance of such soluble enzymes has not been
explored. In addition, the mechanisms regulating formation of soluble
glycosyltransferases have not been clearly defined. It has been shown
that soluble forms of the rat liver 2,6-sialyltransferase and the
bovine
1,4-galactosyltransferase are derived by proteolysis within
the stem domain (24, 25). Cleavage of the membrane-associated
2,6-sialyltransferase may involve a cathepsin D-like protease (26)
that recognizes peptides containing hydrophobic residues (54). Such an
enzyme could participate in cleavage of the
1,3GT, since cleavages
occur adjacent to aromatic residues. However, cleavage also occurred in
the mutant st-1-
1,3GT-HA, which contained mutations in three
residues potentially recognized by cathepsin D-like proteases (Fig.
1C). These data indicate either that other cleavage sites
exist in the stem region, in addition to those we have defined, or that
the mutations in st-1-
1,3GT-HA created recognition sites for other
proteases. Many more experiments along these lines will be required to
determine the mechanisms of cleavage of the stem region of the
1,3GT
and the proteases responsible.
It has been reported that the 2,6-sialyltransferase is not
efficiently secreted from transfected CHO cells (55), implying that CHO
cells lack a protease capable of recognizing the stem region of the
2,6-sialyltransferase. As we found, CHO cells are capable of
efficiently cleaving the stem domain of the murine
1,3GT and
secreting the enzyme. Thus, proteolytic cleavages of glycosyltransferases in heterologous cells may vary. It has also been
found that the human
GDP-L-fucose:
-D-galactoside
2-
-L-fucosyltransferase is not efficiently secreted from
COS-1 cells transfected with the cloned full-length
GDP-L-fucose:
-D-galactoside
2-
-L-fucosyltransferase cDNA (56), although
GDP-L-fucose:
-D-galactoside
2-
-L-fucosyltransferase is found in abundant levels in
human serum (57-59). It is likely that secretion of a
glycosyltransferase from cells is determined by a combination of
factors including the nature of the stem region and cognate endogenous
proteases in cells expressing the enzyme.
Numerous studies have documented the importance of the transmembrane
domains and flanking sequences of glycosyltransferases for enzyme
retention in the Golgi apparatus (6-17). However, in some cases the
transmembrane domain may not be sufficient or even necessary for Golgi
targeting/retention of enzymes, and other domains of the enzymes may
also be important. A soluble form of mannosidase II can interact with
the membrane-associated form of N-acetylglucosaminyltransferase II in a
transmembrane-independent retention mechanism termed "kin
recognition" (60). In this case, a membrane-associated enzyme might
act as a docking protein for a soluble enzyme. In addition, a soluble
form of the 2,6-sialyltransferase containing only the stem region
and the catalytic domain is slowly secreted from COS-1 cells, further
implying that the stem regions and/or the catalytic domains of that
enzyme may contribute to Golgi retention (13). We observed that a
significant amount of the soluble
1,3GT was retained in the cells
and that it appeared to be slowly secreted. Whether a kin
recognition-type mechanism operates for the
1,3GT and whether the
enzyme contains lumenal domains important for Golgi retention are not
known, but experiments are in progress to test these interesting
possibilities.
Since these results indicate that a soluble glycosyltransferase can be
functional within the Golgi apparatus, what is the importance of the
full-length form? It may be that the the soluble and full-length forms
of the enzymes have somewhat different but overlapping functions. The
full-length forms of glycosyltransferases may be important in
glycosylating membrane-associated acceptors such as glycosphingolipids
and membrane-bound glycoproteins. We observed that the soluble 1,3GT
was as efficient as the full-length derived enzyme in glycosylating
secreted proteins from 293 cells. However, the soluble enzyme appeared
to be less efficient than the full-length derived enzyme in its action
on endogenous membrane-associated glycoproteins (Fig. 3A).
Thus, a cooperative mechanism may exist in the Golgi whereby both the
membrane-bound and soluble enzymes function to fully glycosylate the
repertoire of available acceptors.
The apparent molecular mass of secreted 1,3GT from s-
1,3GT-2
cells was ~42 kDa, which is consistent with the expected size of the
cleaved polypeptide (37.5 kDa) plus two N-glycans of ~2.0 kDa each (Fig. 4B). However, the secreted
1,3GT from
fl-
1,3GT-1 had an apparent size of ~51 kDa (Fig. 4A).
(The predicted size of the soluble enzyme, including the HA epitope,
generated by proteolysis of the full-length enzyme in fl-
1,3GT-1
cells is 38.6 kDa.) Assuming that the protein contains two
N-glycans with predicted sizes of ~2.0 kDa each, the size
would be expected to be 42.6 kDa, which is ~8 kDa lower than the
observed size. The larger size of the secreted enzyme derived from the
full-length form may be due to some type of unusual post-translational
modification that preferentially occurs on the full-length,
membrane-associated enzyme. Such a molecular difference was also
observed in
1,4-galactosyltransferase purified from human milk (61).
It has been reported that a truncated soluble form of
1,4-galactosyltransferase carried additional post-translational
modifications compared with the Golgi-localized membrane-bound
full-length
1,4-galactosyltransferase and that these modifications
were added just prior to enzyme secretion (6). Therefore, additional
post-translational modifications may occur in the full-length construct
of
1,3GT as it goes through the biosynthetic secretory pathway, and
these may be different from those that occur in the recombinant,
soluble form of the enzyme. We are currently addressing this issue to
determine the exact molecular mass of the secreted
1,3GT from
fl-
1,3GT-1 and the post-translational modifications of the
enzyme.
In summary, these studies have demonstrated the ability of a soluble
enzyme to efficiently glycosylate glycoproteins during their
biosynthesis within cells. Whether other glycosyltransferases also
function as soluble forms within cells will need to be tested in the
future. In addition, it will be interesting to determine whether the
prevention of cleavage of the 1,3GT, either by further mutagenesis
of the requisite sequence in the stem region or by inhibition of the
protease(s) responsible for cleavage, has an effect on the efficiency
or specificity of
-galactosylation by the enzyme. Finally, it will
also be important to define whether the membrane-bound and soluble
enzymes differ in their recognition of acceptor glycans, recognition of
glycolipids versus glycoproteins, and recognition of
specific N-glycosylation sites in proteins.
We thank Drs. Carlos A. Rivera-Marrero and
Kelley W. Moremen, who provided the 1,3GT-HA construct, and Dr. Qun
Zhou, who provided the HPC4-NKR-P1 construct.