(Received for publication, August 21, 1996)
From the Department of Biochemistry and Molecular
Biology, University of Georgia, Athens, Georgia 30602, the
¶ Howard Hughes Medical Institute and Department of Pathology,
University of Michigan Medical School, Ann Arbor, Michigan 48109, and the ** Department of Biochemistry and Molecular Biology, University
of Oklahoma Health Sciences Center,
Oklahoma City, Oklahoma 73190
The human H(O) blood group is specified by the
structure Fuc1-2Gal
1-R, but the factors regulating expression of
this determinant on cell surface glycoconjugates are not well
understood. To learn more about the regulation of H blood group
expression, cDNA encoding the human H-type
GDPFuc:
-D-galactoside
1,2-fucosyltransferase (
1,2FT) was stably transfected into Chinese hamster ovary (CHO) cells. The new cell line, designated CHO(
1,2)FT, expressed surface neoglycans containing the H antigen. The structures of the fucosylated neoglycans in CHO(
1,2)FT cells and the distribution of these glycans
on glycoproteins were characterized. Seventeen percent of the
[3H]Gal-labeled glycopeptides from CHO(
1,2)FT cells
bound to the immobilized H blood group-specific lectin Ulex
europaeus agglutinin-I (UEA-I), whereas none from parental CHO
cells bound to the lectin. The glycopeptides from CHO(
1,2)FT cells
binding to UEA-I contained polylactosamine
[3Gal
1-4GlcNAc
1-]n with the terminal sequence
Fuc
1-2Gal
1- 4GlcNAc-R. Fucosylation of the polylactosamine sequences on complex-type N-glycans in CHO(
1,2)FT cells
caused a decrease in both sialylation and length of polylactosamine. Unexpectedly, only small amounts of terminal fucosylation was found in
diantennary complex-type N-glycans. The
O-glycans and glycolipids were not fucosylated by the
H-type
1,2FT. Two major high molecular weight
glycoproteins, one of which was shown to be the lysosome-associated
membrane glycoprotein LAMP-1, preferentially contained the
H-type structure and were bound by immobilized UEA-I. These
results demonstrate that in CHO cells the expressed H-type
1,2FT does not indiscriminately fucosylate terminal galactosyl residues in complex-type N-glycans, but it favors glycans
containing polylactosamine and dramatically alters their length and
sialylation.
The A and B blood group antigens and the Lewis B and Y antigens
are formed by the action of specific glycosyltransferases, which act on
a precursor oligosaccharide containing the H blood group structure
Fuc1-2Gal
1-3(4)GlcNAc
1-R (1, 2). This precursor H-substance
can be synthesized by either of two
GDPFuc:
-D-galactoside
1,2-fucosyltransferases
(
1,2FT).1 One enzyme, designated the
H-type, is the product of the human H blood group locus (3,
4) and the other enzyme, designated the SE-type, is a
product of the Secretor locus (5, 6).
Although H blood group determinants are expressed on some
glycoproteins, such as band-3 glycoprotein of erythrocytes (7) and
respiratory mucins (8-10), the expression of H antigens on glycoproteins appears to be restricted, since relatively few
glycoproteins contain significant amounts of the determinant. Because
the H-type 1,2FT can add fucose to a wide variety of
simple acceptors in vitro, including
phenyl-
-D-galactose (11) and small glycans terminating
in the lactosamine sequence Gal
1-3(4)GlcNAc-R (12), it would seem
possible for H antigens to occur indiscriminately on glycoproteins,
since lactosamine sequences are commonly found on mammalian
glycoproteins (13). There has not been a systematic study, however,
about the specificity of the H-type
1,2FT toward complex
glycoconjugates and the consequences of the
1,2-fucosylation on
overall glycoconjugate biosynthesis in cells.
To address the issues about the mode of action of the H-type
1,2FT on glycoprotein acceptors, we have expressed the cDNA encoding the enzyme in Chinese hamster ovary cells. These cells have
well defined glycans on glycoproteins and glycolipids and do not
synthesize the Fuc
1-2Gal linkages (14-17). CHO cells have been
successfully used by several groups investigating the effects of newly
introduced glycosyltransferases on glycoconjugate biosynthesis (15,
18-20). We have compared the types of glycans synthesized by CHO cells
expressing the H-type
1,2FT with parental CHO cells lacking the
1,2FT. Our results demonstrate that the
H-type
1,2FT acts preferentially to fucosylate
polylactosamine sequences in these cells, resulting in decreased
2,3-sialylation of polylactosamine and truncation in the
length of the polylactosamine. These studies demonstrate that the human
H-type
1,2FT does not indiscriminately act on all
glycoproteins glycans, but the enzyme favors certain glycoproteins
containing polylactosamine units.
Galactose, lactose, fucose, raffinose,
-methylmannoside,
-methylglucoside, Triton X-100, Nonidet P-40,
dimethyl sulfoxide, iodomethane,
2-acetamido-2-deoxy-D-glucose, anhydrous sodium acetate, phenyl-
-D-galactoside,
-methylgalactoside, and
1-methylimidazole, Sephadex G-25, Arthrobacter urefaciens
neuraminidase, pepstatin, leupeptin, trypsin inhibitor,
phenylmethylsulfonyl fluoride, Tetragonolobus purpureas
agglutinin, Ulex europaeus agglutinin I (UEA-I),
Anguilla anguilla agglutinin, Griffonia
simplicifolia I-B4 isolectin, and Euonymus
europaeus agglutinin were obtained from Sigma.
Concanavalin A-Sepharose was obtained from Pharmacia Biotech Inc. Beef
kidney
-L-fucosidase and Pronase were purchased from
Boehringer Mannheim, Inc. Endo-
-galactosidase was obtained from
V-Labs (Covington, LA); D-[6-3H]galactose
(20-40 mCi/mmol) and D-[6-3H]glucosamine
hydrochloride (20-45 mCi/mmol) were purchased from ICN Biomedicals
Inc. and DuPont NEN, respectively. GDP-[14C]fucose (268 mCi/mmol) was obtained from Amersham. Affi-Gel 10, acrylamide, and
bis-acrylamide were obtained from Bio-Rad. Antiserum to murine LAMP-1,
which cross-reacts with hamster LAMP-1, was generously provided
by Dr. Thomas August (The Johns Hopkins University, Baltimore, MD).
Plasmid pCDM7
(1,2)FT (3) was linearized by digestion at a unique NheI
site within the M13 origin of replication in the vector pCDM7. The
linearized DNA was transfected with the G418 resistant plasmid pSV2Neo,
with a 10:1 molar ratio of linearized pCDM7 (
1,2)FT and pSV2Neo,
using the calcium phosphate transfection procedure of Chen and Okayama
(21). Clonal cell lines were derived from within the G418 resistant
transfectant population using cloning cylinders. Clonal transfectant
cell lines analyzed by flow cytometry for expression of cell surface
blood group H antigen, using a monoclonal anti-H antibody and
procedures described previously (22, 23). A representative clonal line
that stably expressed cell surface blood group H-determinants was
termed CHO(
1,2)FT. Construction of a control CHO cell line (CHO-V
cells) stably transfected with the vector pCDM7 has been described
previously (23). CHO cells lacking expressed
1,2FT were designated
parental CHO cells.
Extracts were prepared from CHO cell
lines by homogenizing washed cell pellets in 1% Triton X-100, as
described previously (24). Reaction mixtures for assaying 1,2FT
activity contained cell extracts and 3 µM
GDP-[14C]fucose, 25 mM
phenyl-
-D-galactoside, and 5 mM ATP, in 25 mM sodium phosphate, pH 6.1. Reactions were conducted at
37 °C for a period of time allowing a linear rate of incorporation
of radiolabeled fucose, which was generally less than 1% of the counts
incorporated into the added artificial acceptor (22).
CHO-V or CHO(1,2)FT cells were subjected
to flow cytometry analysis, using mouse monoclonal IgM antibodies (22,
25). Cells were stained with a monoclonal anti-H antibody
(Chembiomed, 10 µg/ml) or a control anti-Lex
monoclonal antibody (anti-SSEA-1, Citation, 1:25 dilution). The cells
were then washed, and stained with fluorescein conjugated goat
anti-mouse IgM (Sigma, 40 µg/ml), and subjected to
analysis by flow cytometry on FACScan (Becton-Dickinson, Mountain View, CA), as described previously (22, 26). Cell staining was measured in
arbitrary fluorescent intensity units and displayed on a four-decade log scale.
CHO-V cells, CHO clone 3 cells (15), and CHO
(1,2)FT cells were cultured in 10% fetal calf serum in
-modified
Eagle's medium. The CHO cell line Ade
C (27, 28) was
obtained from M. Van Keuren (Howard Hughes Medical Institute,
University of Michigan) and grown in
-modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% fetal calf serum (HyClone
Laboratories, Logan, UT). Transfected CHO cells were grown in media
containing G418 (Life Technologies, Inc.) at 400 µg/ml (active
drug).
Cells were removed from dishes by treatment with 0.25% trypsin solution containing 2 mM EDTA and the cells were washed three times in Tris buffer (50 mM Tris, 1 mM CaCl2, 1 mM MgCl2, pH 8.0). Cell suspensions for analyses were prepared by gently suspending cells to a concentration of 3 × 106 cells/ml. Aliquots (10 µl) of cell suspensions were placed on microscope slides, and an equal volume of different concentrations of lectin solutions (1 mg/ml or 0.1 mg/ml) were added. The microscope slides were gently shaken and agglutination was determined by direct and microscope observation 3 min after addition of the lectin. Immediate agglutination was defined as 3+, an intermediate level of agglutination was 2+, perceptible but weak level of agglutination was 1+, and lack of agglutination was 0 .
Preparation of Metabolically Radiolabeled Glycoconjugates from Cultured CellsPlates of cells (60 mm) at 50% confluency were incubated in 2 ml of the corresponding medium containing 1 mCi of [3H]galactose or [3H]glucosamine for 48 h. Medium was removed from incubation dishes and the cells were gently washed in the plates three times with 1-ml portions of cold PBS. Radiolabeled cells were scraped from the culture plates with a rubber policeman, and cell suspensions in cold PBS were gently aspirated with sterile pipettes and transferred to 1-ml centrifuge tubes. After washing three times in cold PBS, the cell pellets were collected by centrifugation. Metabolically labeled glycopeptides and glycolipids from the same cell pellets were separately obtained essentially according to the method of Finne and Krusius (29), modified as described previously (15).
Glycolipids were applied to aluminum-backed Silica Gel 60 10 × 10-cm plates (Merck); lower phases were developed in CHCl3:CH3OH:H2O (65:25:4) and upper phase glycolipids in CHCl3:CH3OH:0.25% KCl (5:4:1). Radiolabeled glycolipids were detected on thin layer chromatograms by autoradiography (15). The pelleted residues obtained after extraction of radiolabeled glycolipids were washed with ethanol to remove excess organic solvents and resuspended in 0.1 M Tris, 1 mM CaCl2, pH 8.0, containing Pronase (10 mg/ml). After incubation for 18 h at 60 °C in a toluene atmosphere, the sample was boiled 5 min and desalted by passage over a column (1 × 50 cm) of Sephadex G 25-80 in 7% n-propanol. The radiolabeled glycopeptides were recovered from the void factions, pooled, and dried in a shaker bath evaporator under reduced pressure.
Chromatography of GlycoconjugatesRadiolabeled glycopeptides were fractionated on ConA-Sepharose as described (30). Ulex europaeus agglutinin-I was immobilized on Affi-Gel-10 according to the manufacturer's instructions using 4 mg/ml free fucose in the coupling reaction to protect activity of the lectin. The final coupling density was approximately 10 mg/ml. For affinity chromatography of glycopeptides or free oligosaccharides, a column (0.5 × 8 cm) of UEA-I-agarose was prepared and equilibrated in TBS-NaN3 (10 mM Tris, pH 8.0 containing 1 mM CaCl2, 1 mM MgCl2, and 0.02% NaN3). For analysis of total cell extracts, the columns were equilibrated in TBS-NaN3 containing 0.1% Triton X-100 and 10 mg/ml phenylmethylsulfonyl fluoride. Fractions (0.5 ml) were collected and material bound by the lectin were eluted with buffer containing 4 mg/ml fucose. HPLC of oligosaccharides was carried out on a Beckman model 334 system using an Alltech carbohydrate column (5 µm, 4.1 × 30 cm), as described previously (31). Initial mobile phase consisted of a mixture of 70% acetonitrile in water decreasing to a 50% acetonitrile during 40 min. Fractions (500 µl, 0.5 min/fraction) were collected, and aliquots were assayed for radioactivity.
SDS-Polyacrylamide Gel Electrophoresis of Metabolically Radiolabeled GlycoproteinsAfter lectin affinity chromatography of extracts from metabolically radiolabeled cells, the column fractions were collected for further experiments. One-third of each fraction was precipitated in 10% trichloroacetic acid. The resulting protein pellets were centrifuged and washed twice with 500 µl of chilled acetone. The labeled glycoproteins were resuspended in 20-40 µl of sample buffer and 1-5 µl of 0.1 M Tris, pH 8, were added to insure basic conditions. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the methods of Laemmli (32) using 7.5% acrylamide at 20 A for approximately 3.5 h. The resulting gels were immersed in fixing solution (ethanol, acetic acid, water) overnight. Fixing solution was removed and the gels were immersed in EN3HANCE (Dupont) for 30 min with gentle agitation. Excess enhancer was removed, and the gels were dried and exposed to Kodak X-Omat x-ray film (Sigma) for 2-7 days. LAMP-1 was immunoprecipitated from the UEA-I-bound glycoproteins using methods described by Do et al. (33), and the immunoprecipitate was analyzed by the above procedures.
Glycosidase TreatmentsMetabolically radiolabeled
glycopeptides were treated with 500 milliunits of
endo--galactosidase in 100 µl of 0.05 M sodium acetate
buffer, pH 5.5, for 18 h at 37 °C in a toluene atmosphere. The
products were separated by descending paper chromatography in a solvent
system of ethyl acetate:pyridine:glacial acetic acid:water (5:5:1:3).
After the chromatograms were dried, 1-cm segments were transferred to
12 × 75-mm test tubes containing 1 ml of 0.1 M pyridine acetate buffer, pH 5.4, to elute radiolabeled
oligosaccharides, and aliquots were assayed for radioactivity by liquid
scintillation counting. The separated oligosaccharides were pooled.
Aliquots were dried in 1-ml microcentrifuge tubes for
-L-fucosidase digestions. Beef kidney
-L-fucosidase (Boehringer Mannheim) was added (200-400 milliunits) in 100 µl of 0.05 M sodium acetate buffer, pH
5.0. The mixtures were incubated for 18 h under a toluene
atmosphere. The samples were boiled for 5 min, and oligosaccharides
were analyzed by HPLC, as described above.
The [3H]Gal-labeled glycopeptides and oligosaccharides were methylated by the method of Hakomori (34), hydrolyzed, reduced, acetylated, and analyzed by condensational gas chromatography, essentially as described by Cummings and Kornfeld (35). The partially methylated alditol acetates obtained in this way from glycopeptides were separated by gas chromatography using a flame ionization detector with a detection temperature of 250 °C. The radioactive combustion product of tritiated water was collected in chilled scintillation vials for periods of 10 or 20 s/collection between 10 and 20 min after injection. Each condensate was mixed with 2.5 ml of scintillation fluid and 250 µl of water to determine the retention time of the radiolabeled partially methylated derivative. The partially methylated alditol acetate standards of galactose were prepared according to the procedure of Doares et al. (36), and separated using Hewlett Packard 5890 gas chromatograph fitted with a SP2330 column (Supelco Inc.). Each peak was identified by its mass spectrum (electron impact) fragmentation pattern on a Hewlett Packard 5970 mass selective detector operated at an ionization potential of 70 eV. The retention times for each of the partially O-methylated, alditol acetates were consistent with previously reported values (36).
The isolation and
characterization of a cloned cDNA that encodes a human
1,2-fucosyltransferase (
1,2FT) has been described previously (22,
24). The catalytic properties of the enzyme encoded by this cDNA
and its chromosomal localization strongly suggest that this cDNA
corresponds to the human H blood group locus. Recent studies analyzing
the structure and function of this gene in H blood group 8-deficient
individuals (Bombay and paraBombay) are consistent with this assignment
(4). We sought to further define the biochemical nature of
oligosaccharide products constructed by this enzyme by expressing it in
CHO cells. The oligosaccharide precursors synthesized by CHO cells have
been extensively characterized previously (14-17), and represent a
prototypical cell line with which to explore the in vivo
acceptor substrate utilization properties of the human H blood group
1,2FT.
To construct such cells, a human cDNA encoding this enzyme was
cloned into the mammalian expression vector pCDM7 and was stably transfected into a parental CHO cell background, using a calcium phosphate transfection procedure and the G418 selection plasmid pSV2Neo, as described under "Experimental Procedures." As we have noted previously (23), approximately 25% of the resulting
G418-resistant transfectants stably expressed the corresponding cell
surface H blood group oligosaccharide determinant, as assessed by flow cytometry. A clonal cell line that exhibited stable expression of cell
surface blood group H-determinants was isolated from this population
and designated CHO(1,2)FT. Flow cytometry analyses demonstrate that
virtually 100% of the cells derived from this clone exhibit bright
fluorescence when stained with a monoclonal anti-H antibody (Fig.
1). As expected, the cell line also expressed substantial amounts of
1,2FT activity. Cell extracts were prepared from CHO(
1,2)FT cells and assayed for
1,2FT activity using
phenyl-
-galactoside, an artificial acceptor specific for
1,2FT
(11). Extracts prepared from CHO(
1,2)FT cells contain an
1,2FT
activity (specific activity of 99.8 pmol/mg h). By contrast, the
control CHO-V cell line contains no detectable
1,2FT activity, nor
do these cells express any detectable amounts of cell surface H blood
group determinants (Fig. 1).
Cell Agglutination with Fucose-binding Lectins
To aid in
characterizing the carbohydrate structures containing the H blood group
antigen synthesized by CHO(1,2)FT cells, we sought to identify a
fucose-binding lectin that bound with high affinity to the cells. As
controls, we assessed the agglutinability of the parental CHO cells and
a CHO cell clone, designated Clone 3, which stably expresses the murine
UDPGal:Gal
1-4GlcNAc
1,3 galactosyltransferase and synthesizes
surface glycans with the terminal sequence
Gal
1-3Gal
1-4GlcNAc
1-R (15). Agglutination was measured at
both 1.0 and 0.1 mg/ml amounts of the lectins and was visually scored.
The fucose-binding lectins tested with reported reactivity to H blood
group determinants were E. europaeus agglutinin (37),
A. anguilla agglutinin (38-40), Ulex europaeus I
(UEA-I) (41-43), and T. purpureas agglutinin (44, 45). The CHO(
1,2)FT cells were agglutinated by all the fucose-binding lectins
at 1.0 mg/ml, but UEA-I was the best agglutinin of cells at 0.1 mg/ml
(Fig. 2). E. europaeus agglutinin also
agglutinated CHO Clone 3 cells, which is consistent with the ability of
this lectin to also bind terminal galactosyl residues (37). The
parental CHO cells were not agglutinated by any of these lectins. The
CHO Clone 3 cells were also agglutinated by G. simplicifolia
I-B4 isolectin, which is specific for terminal
-galactosyl residues (46). Since the CHO(
1,2)FT cells were highly
agglutinated by UEA-I, this lectin was selected for use in the lectin
affinity chromatographic separations of glycans from these cells.
Glycoproteins from CHO(
To identify whether glycoproteins in the
CHO(1,2)FT cells contain the H-blood group antigen, both
parental CHO cells and CHO(
1,2)FT cells were metabolically
radiolabeled with [3H]galactose or
[3H]glucosamine, as described previously and under
"Experimental Procedures." Extracts of the cells in 0.1% Triton
X-100 were passed over an affinity column of immobilized UEA-I.
Approximately 5% of the total [3H]Gal-labeled material
(this includes radiolabeled glycolipids and glycoproteins) from the
CHO(
1,2)FT cells bound to the column and was eluted with fucose,
whereas very little material from the parental cells was bound (Fig.
3). A similar result was obtained with
[3H]GlcN-labeled material, except that approximately 30%
of the radiolabeled material was bound by UEA-I-agarose (data not
shown). The samples from [3H]GlcN-labeled glycoproteins
in CHO(
1,2)FT cells bound and unbound by UEA-I-agarose were analyzed
by SDS/PAGE and autoradiography (Fig. 4). A number of
glycoproteins were not bound by the lectin, whereas only two
predominant glycoproteins in CHO(
1,2)FT cells of
100 kDa and
120 kDa were bound by immobilized UEA-I. A similar result was
obtained with [3H]Gal-labeled material (data not
shown).
To confirm that the column chromatography on UEA-I-agarose was
efficient for all those glycoproteins containing the H antigen structure, the gel lanes containing the [3H]Gal-labeled
material UEA-I unbound and bound material were sliced into 0.5-cm
sections and treated with Pronase to generate total glycopeptides. The
pooled, radiolabeled glycopeptides from each lane were then re-applied
to a column of UEA-I-agarose. Approximately 40% of the radiolabeled
glycopeptides from the UEA-I-bound glycoproteins was bound by the
immobilized lectin, but the glycopeptides derived from the originally
unbound glycoproteins did not bind to the column (data not shown).
These results demonstrate that UEA-I-agarose is effective in binding
those glycoproteins containing H antigen and that the bound
glycoproteins are enriched for glycans containing this determinant. As
discussed below, the UEA-I-agarose column contains a high density of
lectin and can bind simple oligosaccharides containing a single
terminal 1,2-fucosyl residue. Thus, the lack of binding of the
majority of glycoproteins to the UEA-I-agarose column is due to their
lack of H antigen.
We have previously shown that in CHO cells polylactosamine sequences
are enriched on two major glycoproteins, one identified as
lysosome-associated membrane glycoprotein 1 (LAMP-1), and the other,
which has the properties of LAMP-2 (33). In CHO cells, LAMP-1 has a
size of 120 kDa and LAMP-2 has a size of
100 kDa. A polyclonal
antibody reactive against murine LAMP-1, which cross-reacts with
hamster LAMP-1, was used to assess whether the ~120-kDa glycoprotein was indeed LAMP-1. The UEA-I-bound material in the
[3H]GlcN-labeled material was immunoprecipitated with the
anti-LAMP-1 antibody and analyzed by SDS-PAGE. A single protein was
immunoprecipitated migrating with a mass of ~120 kDa (Fig. 4). These
results demonstrate that fucosylation is preferential to glycoproteins
containing polylactosamine sequences and that LAMP-1 is the ~120-kDa
glycoprotein containing the H antigen.
To further study the glycans on glycoproteins in
CHO(1,2)FT cells containing the H blood group antigen, the
[3H]Gal-labeled total glycoproteins from both parental
CHO and CHO(
1,2)FT cells were delipidated by organic extraction and
treated with Pronase to generate glycopeptides. These total
glycopeptides were first fractionated by lectin affinity chromatography
on ConA-Sepharose. O-Glycans and the tri- and tetraantennary
complex-type N-glycans do not bind ConA-Sepharose, whereas
diantennary complex-type N-glycans bind and are eluted with
10 mM
-methylglucoside (30, 47, 48). The high
mannose-type N-glycans are bound tightly by ConA-Sepharose and eluted with 100 mM
-methylmannoside. The elution
profile on ConA-Sepharose of [3H]Gal-labeled
glycopeptides from both parental CHO cells and CHO(
1,2)FT cells were
identical (data not shown) and approximately 80% of the radioactivity
was not bound. This unbound material from both cell types was then
applied to a column of UEA-I-agarose. Approximately 17% of the total
[3H]Gal-labeled glycopeptides from CHO(
1,2)FT cells
bound to the column and were eluted with fucose, but no material from
parental CHO cells was bound (Fig. 5). When the
diantennary complex-type N-glycans from CHO(
1,2)FT cells
bound by ConA-Sepharose were analyzed for binding to UEA-I-agarose,
very little material (<5%) was bound and eluted with fucose. This
material was not analyzed further.
The glycopeptides from CHO(1,2)FT not bound by ConA-Sepharose and
bound by UEA-I-agarose were isolated and reapplied to the column either
before or after treatment with
-L-fucosidase. As expected, untreated glycopeptides quantitatively rebound to the column,
whereas the glycopeptides treated with
-L-fucosidase did
not bind (data not shown). These results demonstrate that a specific
subset of glycopeptides derived from CHO(
1,2)FT contain terminal
fucosyl residues recognizable by UEA-I-agarose.
The complex-type
N-glycans of CHO cells are well defined and are mixtures of
di-, tri-, and tetraantennary structures, some of which contain
polylactosamine [-3Gal1-4GlcNAc
1-]n (14-17). The
O-glycans of CHO cells have no polylactosamine sequences and
are simple core 1 structures (Gal
1-3GalNAc
1-Ser/Thr-R) that are
either mono- or disialylated (19). The glycopeptides bound by
UEA-I-agarose (Fig. 5) were all large sized on gel filtration on
Sephadex G-25, in contrast to O-glycans, which have an
intermediate elution on this column (data not shown) (49). This
indicates that O-glycans are not present in the material
bound by UEA-I-agarose.
To determine whether fucosylation occurred on polylactosamine, the
[3H]Gal-labeled glycopeptides from both parental CHO and
CHO(1,2)FT cells not bound by ConA-Sepharose were treated with
endo-
-galactosidase, an enzyme capable of cleaving polylactosamine
at internal unsubstituted galactosyl residues (50). The resulting
fragments were separated by descending paper chromatography (Fig.
6). It has been demonstrated previously that
endo-
-galactosidase treatment of [3H]Gal-labeled
glycopeptides from parental CHO cells produces a sialylated
tetrasaccharide (NeuAc
2-3Gal
1-4GlcNAc
1-3Gal), a trisaccharide (Gal
1-4GlcNAc
1-3Gal), and a disaccharide
(Gal
1-4GlcNAc) (14, 16). Endo-
-galactosidase released 67% of
the radioactivity from parental CHO cells, and the released material
migrated as the three expected products (Fig. 6A). In
contrast, endo-
-galactosidase treatment of total
[3H]Gal-labeled glycopeptides from CHO(
1,2)FT cells
released only 48% of the radioactivity and the pattern of the
fragments produced was different from that obtained from parental cells
(Fig. 6B). There was a marked reduction in the sialylated
tetrasaccharide and a large reduction in the trisaccharide and
disaccharide material. A new fragment migrating slightly slower than
the trisaccharide was present and was designated peak a.
This peak a fragment was enriched in the glycopeptides from
CHO(
1,2)FT cells bound by UEA-I-agarose compared to those unbound
(Fig. 6C and D). We predicted that peak a is the
tetrasaccharide Fuc
1-2Gal
1-4GlcNAc
1-3Gal. To confirm this,
peak a was isolated following preparative descending paper
chromatography and first analyzed for size on amine adsorption HPLC.
The fragment elutes in the position of a tetrasaccharide and treatment
of the fragment with
-L-fucosidase converts it to a
trisaccharide (Fig. 7). In addition, the majority of
radioactivity in this tetrasaccharide is bound by UEA-I-agarose (data
not shown) and eluted with 4 mg/ml fucose. The results indicate that
the tetrasaccharide component identified in glycans released from CHO(
1,2)FT cells by endo-
-galactosidase is the tetrasaccharide Fuc
1-2Gal
1-4GlcNAc
1-3Gal. Methylation analysis, described
below, confirms that galactose residues in glycopeptides from
CHO(
1,2)FT cells bound by UEA-1-agarose are substituted at
the C-2 position by fucose.
The results of endo--galactosidase treatment and HPLC analysis for
the [3H]Gal-labeled material are compiled in Table
I. From this table it is evident that the lengths of
polylactosamine in glycopeptides from CHO(
1,2)FT cells are shorter
than those from the parental cells, based on the recovery of
disaccharide Gal
1-4GlcNAc and the total amount of released
material. The disaccharide provides a measure of polylactosamine chain
length, since long polylactosamines release more releasable
disaccharide than shorter ones (16). In addition, the presence of
terminal fucosylation of glycopeptides in CHO(
1,2)FT cells occurs at
the expense of sialylation, indicating a competition reaction between
1,2-fucosylation and
2,3-sialylation.
|
To determine the
substitution pattern of galactosyl residues, the
[3H]Gal-labeled glycopeptides form parental CHO and
CHO(1,2)FT cells were methylated before and after treatment with
neuraminidase and the methylated species analyzed by gas
chromatography. The results are compiled in Table II.
Only two species of methylated galactosyl residues are obtained from
the methylation of [3H]Gal-labeled glycopeptides from
parental CHO cells. The two species are
2,3,4,6-tetra-O-methylgalactose, which arises from terminal galactosyl residues, and 2,4,6-tri-O-methylgalactose, which
arises from 3-substituted galactosyl residues within both sialylated sequences NeuAc
2-3Gal-R and polylactosamine sequences
[-3Gal
1-4GlcNAc
1-]n. No galactose residues in parental
CHO cells are substituted at the C-2 position, as evidenced by the lack
of 3,4,5-tri-O-methylgalactose. In contrast, 28% of the
galactose residues in glycopeptides from CHO(
1,2)FT cells are
substituted at C-2 and there is a decrease in the other two methylated
species. The 2-substituted galactose residues is expected to
arise by fucosylation at C-2 by the
1,2FT in the CHO(
1,2)FT
cells. Methylation analysis of the glycopeptides from CHO(
1,2)FT
cells after
-L-fucosidase treatment shows a decrease in
the 3,4,6-tri-O-methylgalactose and an increase in the
2,3,4,6-tetra-O-methylgalactose (data not shown). The
decrease in 3-substituted galactosyl residues in the CHO(
1,2)FT
cells is consistent with a decreased sialylation and reduction in
polylactosamine chain length. As expected, the amount of 2-substituted
galactose residues is enriched in glycopeptides bound by UEA-I-agarose
(Table II). These results demonstrate that a high percentage of the
total galactosyl residues in glycopeptides derived from CHO(
1,2)FT cells are substituted at the C-2 position by fucose and that there is
an overall decrease in sialylation and polylactosamine chain length in
these cells compared to parental CHO cells. These results demonstrate
that there is preferential fucosylation of glycoproteins in CHO cells
by the
1,2FT and that this preferential fucosylation is occurring on
polylactosamine.
|
Metabolically radiolabeled glycolipids from both
CHO(1,2)FT and parental CHO cells were compared by thin layer
chromatography analysis, as described previously (15). The detectable
[3H]Gal-labeled glycolipids in both lower and upper
phases fractions from both parental CHO and CHO(
1,2)FT cells were
identical (Fig. 8, A and B). These
results indicate that the
1,2FT does not efficiently use endogenous
lactosylceramide as an acceptor and does not compete for sialylation of
this glycosphingolipid.
The results of our study demonstrate that the human
H-type 1,2FT is effective in fucosylating terminal
galactosyl residues within polylactosamine resulting in both a
shortening of the chain length and a decrease of
2,3-sialylation of
terminal galactosyl residues. Because of its preference for
polylactosamines, the
1,2-fucosylation is preferentially restricted
to two major glycoproteins. It has been shown previously that in CHO
cells polylactosamine is enriched on only two major glycoproteins,
LAMP-1 and another glycoprotein tentatively identified as LAMP-2 (33).
The preferential presence of polylactosamine on these glycoproteins in
CHO cells correlates well with studies showing that polylactosamine
also occurs preferentially on LAMPs in human granulocytes (51, 52). In
CHO cells the H-type
1,2FT does not efficiently act on
terminal galactosyl residues of diantennary complex-type
N-glycans, nor is it efficient in fucosylating either
O-glycans or lactosylceramide.
Our findings are interesting in light of the evidence that the
H-type 1,2FT can utilize a wide variety of acceptors
in vitro, including lactose,
phenyl-
-D-galactoside, and a wide variety of small
oligosaccharides terminating in Gal
1-3/4GlcNAc-R (11, 12).
Interestingly, neither the purified H-type
1,2FT nor
SE-type
1,2FT can use simple natural glycosphingolipids
such as paragloboside (Gal
1-4GlcNAc
1-3Gal
1-4Glc-Cer) as
acceptors in vitro (53), although H antigen has been
observed on Fuc
1-2Gal
1-4GlcNAc
1-3Gal
1-4Glc-Cer and Fuc
1-2Gal
1-3GlcNAc
1-3Gal
1-4Glc-Cer (54, 55).
Lactosylceramide is poorly modified if at all by the H-type
1,2FT, although Fuc
1-2Gal
1-4Glc-Cer has been described in
rat intestine (56). The broad acceptor specificity of the
H-type
1,2FT supports a prediction that terminal fucosylation by this enzyme could be indiscriminate and could occur on
many different types of N- and O-glycans, and thus be present on many different glycoproteins. The results of our study indicate otherwise. Within CHO cells during glycoprotein biosynthesis, the
1,2FT is highly selective in fucosylating glycoproteins and demonstrates a marked preference for polylactosamine.
There is very little information about the expression of H blood group
on glycoproteins resulting from the action of the human H-type 1,2FT, and often, it is unclear whether expression
is due to the SE-type or H-type
1,2FT. In
general, however, the evidence indicates the H antigen expression is
highly limited to certain glycoproteins. For example, band 3 glycoprotein (the anion transporter) is the major ABO(H)-containing
glycoprotein on human erythrocytes and it carries H antigens on its
polylactosamines (7, 57, 58). In hamster pancreas ductal
adenocarcinomas, it was found that blood group A antigen (and by
inference the H blood group precursor) occur on only three major
glycoproteins of 120, 135, and 150 kDa, and the antigen is present on
highly branched N-glycans (62). A small number of surface
glycoproteins of 140, 120, and 80 kDa from human endothelial cells bind
to UEA-I-agarose (60). In a high tumorigenic clone of rat colon
carcinoma cells, H antigen expression was essentially limited to splice
variants of the CD44 molecule (61). Although most plasma glycoproteins lack either polylactosamine sequences or blood group antigens, there is
recent evidence that at least three glycoproteins contain minor levels
of H blood group antigen. Factor VIII (62) and von Willebrand factor
(63) contain a small amount of H antigen on di- and triantennary
N-glycans lacking polylactosamine. Human plasma
2-macroglobulin contains H- antigen on
N-glycans, but only about 10% of the glycoprotein is
precipitable anti-H antibody (64). Transgenic mice expressing the human
H-type
1,2FT under the control of a mammary
gland-specific promoter produce milk containing very high levels of
2
-fucosyllactose, but only a single glycoprotein contains the H
antigen (80). Although a systematic survey of glycoproteins expressing
H antigen in human cells and tissues under the control of the
H-type
1,2FT has not been conducted, the evidence
strongly suggests that H antigen expression by this enzyme is limited
to certain glycoproteins, and even when present on glycoproteins
lacking polylactosamine sequences, the quantitative amounts of the
antigen are low.
The human epidermoid carcinoma cell line A431, from which the cDNA
encoding the human H-type 1,2FT was originally isolated, synthesizes the epidermal growth factor receptor, which contains polylactosamine sequences (66) and is a major carrier of blood group A
and fucose determinants (and by inference H antigen) (66-68). These
blood group determinants are on N-glycans and not
O-glycans, based on two observations. First, the epidermal
growth factor receptor lacks O-glycans (68) and the well
characterized O-glycans on the human low density lipoprotein
receptor, synthesized by the same cells, lack any fucosyl residues
(69). These observations, and the fact that in the CHO
(1,2)FT cells
O-glycans are not modified by the H-type
1,2FT, raise the question whether this enzyme can utilize
O-glycans on glycoproteins as acceptors. In some cases, such
as that observed for the respiratory mucins, O-glycans
contain the H antigen, but the occurrence of this structure is due to the SE-type
1,2FT and not the H-type
1,2FT
(9, 70).
There are several possible explanations for the observation that the
H-type 1,2FT exhibits preferential fucosylation of
glycoproteins. One possibility is that the enzyme has better access to
polylactosamine sequences on glycoproteins than to other carbohydrate
acceptors. Such a mechanism has been proposed to explain the branching
of some diantennary N-glycans in specific glycoproteins by
N-acetylglucosaminyltransferase V (GlcNAcT-V) (71, 72). In
this case GlcNAcT-V acts on diantennary complex-type
N-glycans but only if they are accessible on the intact
glycoprotein. Thus, glycoprotein conformation has an important role in
allowing accessibility of glycans to enzymes. Surprisingly, most
diantennary N-glycans are inaccessible to this GlcNAcT-V, and this was demonstrated to be the case for glycoproteins in Chinese
hamster ovary cells (72). The important role of accessibility and
protein conformation has also been demonstrated for the
UDPGlc:glycoprotein glucosyltransferase, which transfers glucose
residues to high mannose-type N-glycans of glycoproteins in
a partially folded state and does not act efficiently on high mannose
glycans in native glycoproteins (73). It is thus possible, that the
human H-type
1,2FT acts on terminal
-galactosyl
residues in glycoproteins to which it has access, and
polylactosamine-containing glycans may be the most accessible.
It is also possible that the human H-type 1,2FT may
recognize some peptide determinant in specific glycoproteins as a
prelude to the fucosylation reaction. Precedent for specific
recognition mechanisms of glycosylation have been described for
lysosomal enzymes, which acquire GlcNAc
-1-phosphate on mannosyl
residues by the lysosomal phosphotransferase (74), and pituitary
glycoprotein hormones, which acquire terminal
1-4-linked GalNAc
residues through the action of a specific
N-acetylgalactosaminyltransferases that recognizes peptide
sequences within the acceptors (75). At present we do not know the
basis for this preferential fucosylation by the H-type
1,2FT. Future experiments will also explore the
1,2-fucosylation of glycoproteins when the enzyme is expressed in other cell types and
the specificity of the recombinant enzyme for specific glycoprotein acceptors in vitro.
The reduction in polylactosamine chain length and decrease in
sialylation we observed in CHO(1,2)FT cells is consistent with proposals about importance of competition reactions between
glycosyltransferases in regulating glycoconjugate biosynthesis (76,
77). Related studies on glycoconjugates synthesized in the presence and
absence of expression of the SE
1,2FT suggest that
fucosylation by this enzyme alters surface expression of Lewis
antigens, probably by competing with sialyltransferases acting on
terminal Gal residues of the lactosamine unit and
1,3-fucosyltransferases acting on GlcNAc residues of the lactosamine
unit (78, 79). Recent studies on respiratory mucins demonstrate that
expression of the SE
1,2FT has profound effects on the
structures of O-glycans, presumably through competition
reactions with other glycosyltransferases (9, 10, 70). Thus,
fucosylation of lactosamine units by the human H-type
1,2FT "commits" an oligosaccharide to a defined pathway
preventing sialylation of terminal lactosamine units and precluding
further elongation of polylactosamine
[-3Gal
1-4GlcNAc
1-]n.
In previous studies we demonstrated that CHO cells transfected with the
murine UDPGal:Gal1-4GlcNAc (Gal to Gal)
1,3-galactosyltransferase (
1,3GT), contain polylactosamine
modified by the addition of terminal
1-3 linked galactosyl residues
(15).
-Galactosylation by this enzyme decreases sialylation of
polylactosamine sequences, but has much less effect on length of
polylactosamine than is observed in the present study with the human
H-type
1,2FT. The biochemical basis for this difference
is unclear. Perhaps the H-type
1,2FT localizes in CHO
cells to a more proximal Golgi compartment than the
1,3GT, and can
more effectively compete for polylactosamine elongation compared to the
1,3GT. Such differential localization has recently been
proposed as a possible explanation for differential effects on
polylactosamine synthesis in CHO cells by two
1,3FTs (FTIII and
FTIV) (20). Alternatively, the H-type
1,2FT and the
1,3GT may differ in their acceptor preference for polylactosamine
length on acceptor glycoproteins.
The results of our study raise many questions about the specificity of
the both the H-type and the SE-type 1,2FTs and
suggest new directions for research on these enzymes. It would now be important to conduct a parallel study using CHO cells expressing the
SE-type
1,2FT to determine whether that enzyme causes a
different type of glycosylation than we have observed for the
H-type
1,2FT. The specificity of the H-type
and the SE-type
1,2FT for glycoprotein acceptors should
now be studied more thoroughly in vitro using either
purified or recombinant enzymes and glycoprotein acceptors containing
complex-type N-glycans with and without polylactosamine sequences. It should be noted, however, that in some cases recombinant, soluble enzymes have altered specificity compared to native enzymes (65). In addition, the specificity of the
1,2FTs for
O-glycans should be studied. The predilection of the
H-type
1,2FT for polylactosamine during glycoprotein
biosynthesis suggest that the level of expression of the enzyme could
also have a major effect on limiting polylactosamine length and
modification. Future studies should also address the effects of
differential levels of
1,2FT expression in cells on overall
sialylation and polylactosamine structure. It is also possible that in
cells expressing very high levels of
1,2FT that the enzyme might
begin to compete favorably for sialylation of those
N-glycans lacking polylactosamine, and thus cause more
pronounced effects on the terminal glycosylation (e.g.
2,3/6-sialylation or
1,3-galactosylation) of glycans in
cells.