(Received for publication, December 23, 1996)
From the Departments of Protein Glycosylation and
¶ Molecular and Instrumental Structural Research, Gesellschaft
für Biotechnologische Forschung, Mascheroder Weg 1, D-38124 Braunschweig, Germany
Stable BHK-21 cell lines were constructed
expressing the Golgi membrane-bound form and two secretory forms of the
human 1,3/4-fucosyltransferase (amino acids 35-361 and 46-361). It
was found that 40% of the enzyme activity synthesized by cells
transfected with the Golgi form of the fucosyltransferase was
constitutively secreted into the medium. The corresponding enzyme
detected by Western blot had an apparent molecular mass similar to
those of the truncated secretory forms.
The secretory variant (amino acids 46-361) was purified by a single
affinity-chromatography step on GDP-Fractogel resin with a 20% final
recovery. The purified enzyme had a unique NH2
terminus and contained N-linked endo H sensitive
carbohydrate chains at its two glycosylation sites. The
fucosyltransferase transferred fucose to the O-4 position of GlcNAc in
small oligosaccharides, glycolipids, glycopeptides, and glycoproteins
containing the type I Gal1-3GlcNAc motif. The acceptor
oligosaccharide in bovine asialofetuin was identified as the Man-3
branched triantennary isomer with one Gal
1-3GlcNAc. The type II
motif Gal
1-4GlcNAc in bi-, tri-, or tetraantennary neutral or
2-3/
2-6 sialylated oligosaccharides with or without
N-acetyllactosamine repeats and in native glycoproteins
were not modified.
The soluble forms of fucosyltransferase III secreted by stably transfected cells may be used for in vitro synthesis of the Lewisa determinant on carbohydrates and glycoproteins, whereas Lewisx and sialyl-Lewisx structures cannot be synthesized.
The Lewis blood group 1,3/4-fucosyltransferase
(FucT-III)1 has been reported to catalyze
the formation of the Lewisa (Lea),
Lewisx (Lex), sialyl Lewisa
(sLea), and sialyl Lewisx (sLex)
determinants (1). The determinant sLex is found on
glycoproteins and glycolipids at the surface of leukocytes and tumor
cells (2), whereas sLea is mostly found at the surface of
cancer cells of the digestive system (3). It has been found that both
determinants are components of ligands for L-, E-, and P-selectins (4,
5). During inflammatory processes, the leukocytes interact with
E-selectin induced at the surface of endothelial cells via the
sLex structure (1) and adhesion between tumor cells and the
endothelium is mediated by the sLe x and the sLe
a structures (3). Molecules containing the sLe
x and sLe a determinants at their surface can
be efficiently used for inhibition of cellular interactions and,
consequently, are potential tools for the therapeutic treatment in
inflammatory diseases and in metastasis. The recombinant FucT-III or
soluble variants have been reported to synthesize the two structures
in vitro (6-9).
The purification of FucT-III from human milk and from the supernatant of the human epidermoid carcinoma cell line A431 has been reported (6, 8). Soluble chimeric forms of FucT-III fused to a sequence of protein A via the amino terminus have been characterized from transiently transfected COS cells (9, 10). Solubilization of the enzyme due to the deletion of its cytoplasmic domain, transmembrane, and part of the stem region has been reported to change its enzymatic properties. The activity with type II glycolipid acceptors is decreased to non-detectable levels, and the activity with glycoprotein acceptors, particularly asialofetuin, is increased (9).
In the present work, we describe the construction of plasmids encoding soluble forms of the recombinant human FucT-III, where the human IL-2 signal sequence is linked to Ala-47 or Val-36 of the FucT-III and their expression from stably transfected BHK-21 cell lines. The purification of a secreted enzyme form by a single-step affinity chromatography procedure using GDP-Fractogel from supernatants of recombinant stable BHK-21 cells, which do not express any Lewis-type fucosyltransferase activity in their wild-type form, is described for the first time. The purified recombinant enzyme was characterized, and its specificity toward the acceptor substrate motif in the N-glycans of bovine asialofetuin is reported.
GDP-fucose, bovine asialofetuin, native fetuin,
and bovine thyroglobulin were purchased from Sigma.
GDP-[14C]fucose (285 mCi/mmol) was from Amersham
(Braunschweig, Germany (FRG)). Recombinant antithrombin III expressed
from CHO cells, -trace protein, and erythropoietin from BHK-21 cells
were purified and characterized as described previously (11, 12, 13). GDP-Fractogel was from Merck, Darmstadt, FRG).The soluble form of
recombinant human IL-4 receptor expressed from CHO cells was a gift
from Dr. G. Zettlmeißl (Behringwerke Marburg, Germany). Lacto-N-tetraose (LNT, Gal
3GlcNAc
3Gal
4Glc),
lacto-N-neotetraose (LNnT, Gal
4GlcNAc
3Gal
4Glc),
lacto-N-fucopentaose I (LNFP-I, Fuca2Gal
3GlcNAc
3Gal
4Glc), LS-tetrasaccharide a (LST-a,
NeuAca2-3Gal
3GlcNAc
3Gal
4Glc), and
3
-sialyl-N-acetyllactosamine (SLN, NeuAca2-3Gal
4GlcNAc) were bought from Oxford Glycosystems, UK. N-Linked
complex-type bi-, tri-, and tetraantennary structures with one to three
N-acetyllactosamine repeats and one to four
2,3-linked
NeuAc residues were isolated from recombinant glycoproteins expressed
from BHK-21 and CHO cells and were structurally characterized by NMR
and mass spectrometric techniques as described previously (11-15).
Purified anti-human Fuc-TIII antibody was a gift from Dr. J. Lowe
(University of Michigan Medical School). The monoclonal antibody A3C5
was a gift from Dr. W. Lindenmaier (Gesellschaft für
Biotechnologische Forschung, Braunschweig, Germany). The
Gal
3GlcNAc-O(CH2)8COOMe and
Gal
4GlcNAc-O(CH2)8COOMe acceptors were a
gift from Dr. O. Hindsgaul (University of Alberta, Canada). The
glycolipid Gal
1,3GlcNAc
1,3Gal
1,4Glc-ceramide was obtained from
Dr. B. Kniep (University of Leipzig, Germany), and the glycopeptide
ASTTTN(
Gal1, 3
GlcNAc
1-)YT was a gift from Dr. Kunz (University
of Mainz, Germany).
Peptide-N4-(N-acetyl-
-glucosaminyl)asparagine
amidase (PNGase F) (recombinant enzyme from Flavobacterium
meningosepticum expressed in Escherichia coli) and
endoglycosidase H was purchased from Boehringer (Mannheim, Germany),
Vibrio cholerae sialidase was from Calbiochem (La Jolla, CA), HPLC-grade trifluoroacetic acid and acetonitrile were from Pierce
and J.T. Baker (Deventer, The Netherlands), respectively, puromycin-dihydrochloride was bought from Sigma (Deisenhofen, Germany).
DMEM was prepared using Dulbecco's modified Eagle's medium
(purchased from Life Technologies, Inc., Eggenstein, Germany), which was supplemented prior to use with 10 mM Hepes, 45 mM NaHCO3, 2 mM glutamine, 0.061 g
liter
1 ampicilline and 0.1 g liter
1
streptomycin sulfate and which was adjusted to pH 7.1 for use in cell
culture.
Mutants of the human 1,3/4-fucosyltransferase were
generated by PCR-based site-directed mutagenesis of a FucT-III cDNA
(a gift from Dr. S. Gonski, Hoechst AG, Frankfurt, FRG) encoding the
full length of the enzyme. PCR was performed using the Expand High
Fidelity DNA-polymerase-mixture (Boehringer) and the supplied buffer
according to the manufacturer's protocol at standard concentrations of
0.3 µM for each primer and 0.2 mM each
deoxynucleotide. PCR conditions, if not otherwise stated, were a 2-min
denaturation step at 94 °C, followed by 30 cycles with 15 s of
denaturation at 94 °C, 20 s of annealing at 50 °C, 2 min of
elongation at 72 °C, and final elongation for 8 min at 72 °C. DNA
fragments were cloned into the eukaryotic expression vector pCR3 using
a TA cloning kit (Invitrogen, Leek, The Netherlands). Positive clones
were identified by using standard techniques and were verified by using the CircumVent Thermal Cycle Dideoxy Sequencing kit (New England Biolabs, Schwalbach, Germany).
The mutant pCRFT3T2 encodes a full-length human FucT-III that is
COOH-terminally elongated with a tripeptide-spacer GAG followed by the
epitope FDKNYVANSGK derived from human cytomegalovirus glycoprotein ICP
36 (that is recognized by the monoclonal antibody A3C5).2 The construct was generated in one
step using FucT-III cDNA template, the primer FT6-N (forward)
5-ACT CTG ACC CAT GGA TCC CCT-3
, and the mutagenesis primer FT-TAG2
(reverse) 5
-TCA CTT GCC GCT GTT TGC GAC GTA ATT TTT GTC GAA TCC AGC
TCC GGT GAA CCA AGC CGC.
Mutant pCRS1FT3 encodes a FucT-III containing the human IL-2 signal
peptide (17 amino acids using the second start codon) and the first two
amino acids, Ala-Pro, of the mature IL-2 protein linked
NH2-terminally to Val-36 of FucT-III. This construct was generated in a one-step PCR using the FucT-III cDNA template, the
mutagenesis primer s1FT3 (forward) 5-AGG ATG CAA CTC CTG TCT TGC ATT
GCA CTA AGT CTT GCA CTT GTC ACA AAC AGT GCA CCT GTG TCC CGA GAC GAT-3
and primer FT6-C (reverse) 5
-CTC TCA GGT GAA CCA AGC CGC TAT
GC-3
.
Mutant pCRS2FT3T2 encodes a FucT-III containing a 20-amino acid human
IL-2 signal peptide (using the first start codon) fused NH2-terminally to Ala-47 of FucT-III and containing GAG
linker and the A3C5 epitope at the COOH terminus as described above for mutant pCRFT3T2. The vector was generated in a two-step PCR procedure essentially as described (16, 26). The first PCR was performed using
human IL-2 cDNA as a template and the mutagenesis primers IL2N
(forward) 5-AAG ATG TAC AGG ATG CAA CTC C-3
and FT3N2IL (reverse)
5
-CGG GAG GAC CCA CTA GGT GCA CTG TTT GTG-3
(PCR conditions: 2 min at
94 °C, 30 cycles with 15 s at 94 °C, 20 s at 50 °C,
20 s at 72 °C, and a final 8 min at 72 °C). One fifth of the
first reaction mixture was used in a second PCR using human FucT-III cDNA as a template, fresh IL2N primer, and the mutagenesis primer FT-TAG2 (shown above) at reduced primer concentrations of 0.04 µM. The fragment of the expected length was recovered by
agarose gel electrophoresis and was extracted from the gel using
JETsorb reagents (Genomed, Bad Oeynhausen, Germany) prior to TA
cloning.
BHK-21 cells were grown as
described previously (12) and transfected by the calcium phosphate
precipitation method. Semi-confluent cells were typically transfected
with 4 µg of plasmid DNA (pCRFT3, pCRS1FT3, or pCRS2FT3T2) and 1 µg
of the plasmid DNA pSV2pac to confer resistance to puromycin.
Puromycin-resistant cells were selected using several medium exchanges
with DMEM containing 10% fetal calf serum (FCS) and 0.8 µg
ml1 puromycin-dihydrochloride during a time period of
2-3 weeks. Confluent cells were grown for 2 days in the absence of FCS
and the corresponding supernatants were tested for fucosyltransferase activity after a 5-10-fold concentration in a Speed-Vac or by ultrafiltration using Centricon 10 cartridges (Millipore, Eschborn, FRG).
Cell extracts were freshly prepared prior to each assay. Cells were washed with 5 ml of DMEM, trypsinized, and spun at 1000 × g. After resuspending in ice-cold extraction buffer (1 ml of 20 mM Mops-KOH buffer, pH 7.5, containing 2% Triton X-100/107 cells) cells were disrupted using a Potter-Elvhjem homogenizer at 0 °C. If not otherwise stated, extracts were used within the same day of homogenization for fucosyltransferase activity determinations.
Enzyme PurificationThe culture supernatant from stably
transfected BHK-21 cells (1600 ml) was 3.5-fold concentrated by
ultrafiltration and was applied on a 25-ml GDP-Fractogel column
equilibrated with 20 mM Mes-KOH, pH 6.8, containing 0.02%
NaN3 and 1 mM dithioerythreitol at a flow rate
of 1mlmin1 at room temperature. The column was washed
with 20 mM Mes-KOH buffer, pH 6.8, containing 50 mM NaCl, 0.02% NaN3, and 30% glycerol (120 ml) and subsequently with 100 ml of the same buffer containing 500 mM NaCl. Elution of the enzyme was performed at a flow rate of 1.7 ml min
1 with the same buffer containing 1.5 M NaCl (90 ml). The enzyme was concentrated by
ultrafiltration using Centricon 10 cartridges to a final protein
concentration of 30 µg ml
1 and was stored in elution
buffer containing 30% glycerol and 0.02% NaN3 at
20 °C without any loss of activity over a period of 6 months.
SDS-PAGE was performed
according to Laemmli (17) using 12.5% and 3% acrylamide in the
resolution and stacking gels, respectively.For Western blot analysis,
proteins were transferred to nitrocellulose (Millipore) in a semidry
instrument (Bio-Rad). The membrane was blocked with Tris-buffered
saline containing 10% horse serum and 3% bovine serum albumin for
1 h and incubated overnight with biotinylated anti-cytomegalovirus
tag monoclonal antibody (A3C5) or anti-FucT-III antiserum in blocking
buffer at 1:500 and 1:1000 dilutions, respectively, overnight. The
second antibody, streptavidin or anti-rabbit immunoglobulin coupled to
horseradish peroxidase, respectively, was used at a 1:1000 dilution.
The blots were developed with Tris-buffered saline containing 0.5 mg
ml1 4-chloro-1-naphthol solubilized in methanol and 0.2%
perhydrol. Endoglycosidase treatment of the enzyme with recombinant
PNGase F and endoglycosidase H as well as mild acid treatment were
performed as described (12).
The fucosyltransferase activity
with oligosaccharides, glycolipids, glycopeptide, and
8-methoxycarbonyloctyl glycoside acceptors type I and II was tested at
37 °C in 60 µl of the following reaction mixture using 20-60
microunits of enzyme: 50 mM Mops/NaOH buffer, pH 7.5, 20 mM MnCl2, 0.1 M NaCl, 4 mM ATP, 0.1 mM GDP-[14C]Fuc
(containing 60,000 cpm of GDP-[14C]fucose/nmol).
Glycolipid and glycopeptide acceptors were used at 1 mM and
8-methoxycarbonyloctyl glycoside acceptors at 0.3 mM
concentrations. SLN, LnNT, LNT, and LST-a were used at a 0.33 mM concentration in the presence of 0.42 mM
GDP-Fuc. Reducing N-glycans of the bi-, tri- and
tetra-antennary complex type, with one to three
N-acetyllactosamine repeats and one to four 2-3 linked
NeuAc, were used at 0.1-0.2 mM concentrations in the
presence of 0.20 mM GDP-Fuc for 4 and 24 h. Aliquots
of reaction mixtures with oligosaccharides and N-glycans as
acceptors were analyzed by HPAE-PAD after desialylation with V. cholerae sialidase and by matrix-assisted laser
desorption/ionization time of flight mass spectrometry (MALDI/TOF-MS)
after desalting. Some of the samples for MALDI/TOF-MS analysis were
reduced and permethylated. The reaction mixtures with glycolipid and
8-methoxycarbonyloctyl glycoside as acceptors were diluted with water
to 1 ml and applied to Sep-Pak C18 cartridges, which were
washed with 5 ml of water. The products were then eluted with 1 ml of
methanol. The incorporation of [14C]fucose was determined
by liquid scintillation counting. The glycopeptide reaction mixture was
separated by reversed-phase chromatography on a C18 Vydac
column as described previously (19). Absorbance was monitored at 202 nm, and fractions corresponding to individual peaks were analyzed by
MALDI/TOF-MS.The fucosyltransferase activity toward glycoprotein
substrates was tested in 20 µl of the following reaction mixture: 20 mM Tris-Mes buffer, pH 6.8, 50 mM NaCl, 1 mM dithioerythritol, 25 mM MnCl2,
10 mM Fuc, 5 mM ATP, 0.05 mM
GDP-[14C]Fuc (1 nmol contained 300,000 cpm), and 2 mg
ml
1 of glycoprotein acceptor (1 nmol of bovine
asialofetuin, 1.7 nmol of
-trace protein, and 1.8 nmol of soluble
form of the IL-4 receptor). After incubation the reaction mixtures were
precipitated for 10 min with 1 ml of 1% phosphotungstic acid in 0.5 M HCl (0 °C) and transferred under vacuum to glass
microfiber filters (Whatman GF/C). Filters were washed with 1%
tungstophosphoric acid in 0.5 M HCl and methanol, dried,
and counted for radioactivity in a Beckmann LS 6000 LC scintillation
counter. There was no incorporation of Fuc in reaction mixtures without
enzyme or without acceptor. For a detailed characterization of the
fucosylated N-linked oligosaccharides from asialofetuin, 10 mg of the protein were incubated with S2FT3T2 in the buffer described
above containing 750 nmol of GDP-[14C]Fuc for 24 h
at 37 °C. The resulting protein was dialyzed against 0.5% acetic
acid, lyophilized, and the corresponding oligosaccharides were released
by automated hydrazinolysis in a GlycoPrep instrument (N+O mode, Oxford
Glycosystems, UK). The oligosaccharides were then separated by
amino-bonded phase high performance liquid chromatography (13), and the
radiolabeled peaks analyzed by HPAE-PAD, MALDI/TOF-MS, and methylation
analysis.
Enzyme kinetics of S2FT3T2 was performed in reaction mixtures as described above where the concentration of the type I 8-methoxycarbonyloctyl glycoside varied between 0.0095 and 0.91 mM. The apparent kinetic parameters were determined from a Michaelis-Menten curve fit to the experimental data using the least square method. One unit of enzyme activity was defined as the amount of enzyme catalyzing the transfer of 1 µmol of fucose/min to the 8-methoxycarbonyloctyl glycoside type I substrate.
Methylation Analysis of CarbohydratesFor methylation analysis, oligosaccharides were permethylated according to Hakomori (18), purified on a Sephadex LH2O column, hydrolyzed, reduced, and peracetylated as described (14). Separation and identification of partially methylated alditol acetates was performed on a Finnigan gas chromatograph (Finnigan MAT Corp., San Jose, CA), equipped with a 30-meter DB5 capillary column, connected to a Finnigan GCQ ion trap mass spectrometer.
Analytical HPAE-PAD of Native and Desialylated OligosaccharidesA Dionex BioLC System (Dionex, Sunnyvale, CA)
equipped with a CarboPac PA1 column (4 mm × 250 mm) was used in
combination with a pulsed amperometric detector (detector potentials
and pulse durations: E1 = +50 mV, T1 = 480 ms; E2 = +500
mV, T2 = 120 ms; E3 = 500 mV, T3 = 60 ms). Prior to
HPAE-PAD analysis, N-glycans were desialylated by
solubilizing in sialidase buffer (10 mM sodium acetate, 1 mM CaCl2, 0.02% sodium azide) and by
incubating with 0.2 units ml
1 V. cholerae
sialidase for 2 h at 37 °C. The NeuAc/oligosaccharides mixtures
were injected onto the column before or after desalting. Elution was
performed by applying a 2-min isocratic run with 100% solvent A
followed by a linear gradient from 0 to 20% solvent B over 38 min and
a linear gradient to 100% solvent B within 10 min. The flow rate was 1 ml min
1; solvent A: 0.2 M NaOH, solvent B:
0.2 M NaOH containing 0.6 M sodium acetate.
Separation of small oligosaccharides and their fucosylated products was
performed by applying a linear gradient from 0-20% 0.1 M
NaOH over 40 min and to 100% 0.1 M NaOH containing 0.6 M sodium acetate within 10 min as described (19).
2,5-Dihydroxybenzoic acid was used as
UV-absorbing matrix. 2,5-Dihydroxybenzoic acid (10 mg
ml1) was dissolved in 10% ethanol in water. For analysis
by MALDI/TOF-MS, the solutions of the native or reduced and
permethylated oligosaccharides were mixed with the same volume of
matrix. 1 µl of the sample was spotted onto a stainless steel tip and
dried at room temperature. The concentrations of the Analyte mixtures
were approximately 10 pmol µl
1.
Measurements were performed on a Bruker ReflexTM MALDI/TOF mass spectrometer using a N2 laser (337 nm) with 3-ns pulse width and 107-108 watts/cm2 irradiance at the surface (0.2 mm2 spot). Spectra were recorded at an acceleration voltage of 20 kV using the reflectron for enhanced resolution.
Electrospray Ionization Tandem Mass Spectrometry (ESI-MS/MS)A Finnigan MAT TSQ 700 triple quadrupole mass spectrometer equipped with a Finnigan electrospray ion source was used for ESI-MS. The reduced and permethylated samples were dissolved in acetonitrile saturated with NaCl (about 10 pmol/µl) and injected at a flow rate of 1 µl/min into the electrospray chamber. A voltage of +5.5 kV was applied to the electrospray needle. For collision-induced dissociation experiments, parent ions were selectively transmitted by the first mass analyzer and directed into the collision cell (argon was used as collision gas) with a kinetic energy set around minus 60 eV.
BHK-21B cells were
transfected with plasmids encoding the membrane-bound form of FucT-III
with a tag at its carboxyl terminus (FT3T2), and two soluble forms of
FucT-III containing the human IL-2 leader peptide sequence, at amino
acid 36 (S1FT3) or amino acid 47 (S2FT3T2, with a tag at its COOH
terminus) (Fig. 1). After transfection, cells were
selected in medium containing 0.8 µg ml1 puromycin
hydrochloride. Resistant cell clones were isolated, and, after reaching
confluence, supernatants and cellular extracts were assayed for
fucosyltransferase activity with
Gal
1-3GlcNAc-O(CH2)8COOMe as an acceptor
substrate (Table I). The highest activity of secreted FucT-III was measured in the supernatant of stable BHK-21 cells transfected with pCRS2FT3T2. Secretion of enzyme activity increased for
up to 40 h in confluent cultures supplemented with fresh medium. This clone (S2FT3T2) was used for the production and purification of
the recombinant enzyme (see below). For the cell clones expressing secretory forms of FucT-III, some fucosyltransferase activity was
detected in the cellular extracts (10-20% of the secreted form, see
Table I) presumably representing the enzyme fraction transported along
the secretory pathway of cells. For cells transfected with the
membrane-bound form of FucT-III, most of the activity was found to be
associated with cellular extracts; however, considerable amounts (about
40% of the total activity measured with
Gal
1-3GlcNAc-O(CH2)8COOMe as a substrate)
were detected in the culture supernatant of confluent monolayers
(viability higher than 95% based on trypan blue exclusion) that were
cultivated for a 24-h period in fresh medium. The Western blotting
analysis of concentrated supernatants from the three cell lines using
the anti-FucT-III antibody showed bands of similar molecular masses
around 40 kDa. This indicates a significant shedding of the
membrane-bound form (FT3T2), presumably after proteolytic cleavage
within the stem region.
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For the purification of S2FT3T2,
confluent recombinant monolayers were grown for 4 weeks in DMEM
containing 2% FCS, the medium being harvested every 2 or 3 days and
being frozen at 20 °C until further use. The enzyme was purified
from 1600 ml of culture supernatant by affinity chromatography on a
GDP-Fractogel column using the one-step procedure as described under
"Experimental Procedures." The eluate containing the S2FT3T2 was
concentrated by ultrafiltration and was stored in the presence of 30%
(v/v) glycerol. The activity of the pooled supernatants after thawing
decreased by approximately 2.5-fold. The purified S2FT3T2 preparation
(4.5 ml) had a protein concentration of 30 µg ml
1 and
an activity of 33 units/liter with the type I 8-methoxycarbonyloctyl glycoside as a substrate). Thus, the final recovery of recombinant S2FT3T2 was approximately 20%.
Upon SDS-PAGE analysis followed by Coomassie staining, the enzyme
preparation was found to consist of a closely spaced doublet of about
40-42 kDa molecular mass, which was also detected with the
anti-FucT-III antibody as well as with the anti-tag monoclonal antibody
A3C5 (Fig. 2). Gas-phase sequencing of the purified
enzyme yielded the expected 20 amino-terminal amino acids
(1APSGSSRQDTTPTRPTLLIL20) that are deduced from
the S2FT3T2 cDNA. This result indicates that the difference in
molecular mass between the two S2FT3T2 forms seen after SDS-PAGE is not
due to proteolytic degradation of the polypeptide.
N-Glycosylation of the Recombinant Human FucT-III
The recombinant S2FT3T2 contains two potential N-glycosylation sites (Asn-154 and Asn-185, according to the numbering of the FucT-III wild-type sequence). Incubation of the purified recombinant enzyme with PNGase F gave rise to a 3-4-kDa decrease in apparent molecular mass, resulting in two clearly distinguishable bands. The mass shift observed suggests that both glycosylation sites of S2FT3T2 are occupied by N-linked oligosaccharides. The lower molecular mass band after PNGase digestion had an apparent molecular mass of approximately 38 kDa (predicted molecular mass: 38,357). After incubation with Endo H, a small part of S2FT3T2 was not sensitive to the enzyme, suggesting the presence of small amounts of complex-type glycans. However, most of the enzyme showed a mobility shift in SDS-PAGE similar to that obtained with PNGase F, indicating that the majority of the oligosaccharides are of the oligomannosidic or hybrid type (Fig. 2). Furthermore, mild acid hydrolysis of the enzyme before and after PNGase F or Endo H treatment did not produce any detectable shift in molecular weight (not shown), suggesting the absence of NeuAc in N- or, if present, O-linked glycans.
Substrate Specificity of the Soluble Form of the Recombinant Human FucT-IIIThe S2FT3T2 catalyzed the fucosylation of the type I but
not of the type II 8-methoxycarbonyloctyl glycoside. The apparent kinetic parameters determined with the type I acceptor using saturating concentrations of the GDP-Fuc were V = 0.83 ± 0.07 pmol min1ml
1, and Km = 0.54 ± 0.08 mM, assuming a Michaelin behavior for the
enzyme.
The specificity of the S2FT3T2 toward the small oligosaccharide
acceptors LNFP, LNT, LNnT, LST-a, and SLN was analyzed after incubations of 2, 4, and 21 h. The formation of the fucosylated products was monitored by HPAE-PAD (Fig. 3), and the
molecular masses of the products were determined by MALDI/TOF-MS (Table II). It was found that the reaction was linear for at
least 4 h, so the activities shown in Table II were calculated
based on 2-h incubation values. The S2FT3T2 activity with the type I
acceptor (LNT) is 1.5-fold higher than with the type II acceptor
(LNnT). Substitution of the terminal monosaccharide residue of the LNT with 2,3-linked sialic acid causes a 1.3-fold increase in activity of the S2FT3T2. Substitution of the LNT with
2-linked Fuc causes a
3.3-fold increase in activity of the S2FT3T2 (Table II). To identify
the linkage position of fucose residues, after reduction and
permethylation, the oligosaccharides were analyzed by collision-induced decomposition mass spectrometry and by methylation analysis (Fig. 4 and Table III). For LNT, it was found
that S2FT3T2 transferred Fuc to the O-4 position of GlcNAc (70%), to
the O-3 position of Glc (20%), or to both positions (10%). In LNnT,
all the Fuc was found to be attached to the O-3 position of Glc. With
LNFP as an acceptor, Fuc was transferred exclusively to the O-4
position of GlcNAc with no fucosylation being detected on the Glc
residue. 80% of the product obtained with LST-a contained fucose at
the O-4 position of GlcNAc, and 20% was modified at the O-3 position of Glc; additionally, based on the molecular ions detected in ESI
spectra, small amounts of bifucosylated and trace amounts of
trifucosylated structures were observed in the product with this
substrate (compare Table II). No fucosylation was detected in the
trisaccharide SLN when incubated under identical conditions.
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S2FT3T2 activity toward glycolipids was tested with
Gal3GlcNAc
3Gal
4Glc
1-1ceramide as a substrate. It was found
that 63 pmol/min/ml of Fuc transferred to 50 nmol of the acceptor.
The chemically synthesized glycopeptide
ASTTTN(Gal1,3
GlcNAc1)YT (mass of 1243) was almost totally
fucosylated by S2FT3T2 during an overnight incubation, yielding a
product with m/z = 1389 [M + Na]+ as
detected by ESI-MS.
Complex-type bi-, tri-, and tetraantennary structures with zero to four
N-acetyllactosamine repeats and with or without
2,3/6-linked NeuAc residues were incubated with the enzyme for 4 and
24 h. Analyses by HPAE-PAD and MALDI/TOF-MS revealed that no
fucosylation had occurred irrespective of the antennarity, number of
lactosamine repeats or sialylation degree after a 24- h incubation
period.
The glycoproteins, bovine asialofetuin, native fetuin and bovine
thyroglobulin, human -trace protein from hemofiltrate (20), recombinant human
-trace protein, recombinant human antithrombin III, and recombinant human IL-4 receptor from CHO cells, were tested as
acceptors for S2FT3T2 (Table IV) by determination of [14C]fucose incorporation. Only very low incorporation of
fucose was achieved. The enzyme showed the highest activity with
asialofetuin as a substrate. The activity with recombinant
-trace
protein from BHK-21 cells was higher than with its natural counterpart isolated from hemofiltrate (15). No fucosylation of bovine
thyroglobulin or recombinant antithrombin III from CHO cells was
detected. Fucosylated glycoproteins were subjected to SDS-PAGE, and,
following subsequent autoradiography, radiolabeled bands were detected
at migration positions corresponding to the molecular masses of the
untreated glycoproteins.
|
For the determination of the linkage position of
fucose in asialofetuin oligosaccharides, 10 mg of the glycoprotein were
incubated with S2FT3T2 in the presence of 750 nmol of
GDP-[14C]Fuc (2 × 105 cpm) for 18 h. The oligosaccharides from the unmodified and modified glycoprotein
were released by automated hydrazinolysis and subjected to HPAEC-PAD
(Fig. 5). Three major oligosaccharide peaks were obtained for the glycan mixture from unmodified asialofetuin: biantennary, triantennary 2,4-branched, and triantennary 2,4-branched with one Gal1-3GlcNAc antenna in a ratio of 10:55:35. The glycan mixture from in vitro fucosylated asialofetuin yielded a new
peak eluting at 15.5 min (C1) with a concomitant decrease of peak C (C1, A, B, C in ratio of 27:11:53:9). The glycan mixture from S2FT3T2-treated asialofetuin was subjected to separation on
NH2-bonded phase. Two major peaks were obtained, which were
not completely separated (data not shown). Two molecular ions
corresponding to reduced and permethylated triantennary
N-glycans (m/z = 2537) and a fucosylated
triantennary structure (m/z = 2710), respectively, were
detected after MALDI/TOF-MS. The native material obtained after
NH2-bonded phase was subjected to preparative HPAEC-PAD yielding peaks C1 and B (Fig. 5, panel 3). Methylation
analysis revealed the presence of only 4-substituted GlcNAc in peak B
and a mixture of 4-substituted and 3,4-disubstituted GlcNAc as well as
terminal fucose in peak C1. Peak C1 eluted at 15.5 min in HPAEC-PAD and
was completely converted to a structure eluting at the position of peak
C upon mild acid treatment (Fig. 5, panel 4), whereas peak B
was not affected. Thus, the results indicate that the
N-linked oligosaccharide of asialofetuin with one type I
antenna is modified by S2FT3T2 with fucose linked to position 4 of
GlcNAc in the type I motif. No indication of the presence of
fucosylated O-linked glycans was obtained in our
experiments.
The construction of a soluble form of FucT-III through the replacement of amino acids 1-35 and 1-46 by the signal sequence of human IL-2 (constructs S1FT3 and S2FT3T2, respectively) produced catalytically active secreted forms of the enzyme when expressed from stably transfected BHK-21 cell lines.
The purification of human 1,3/4-fucosyltransferase from milk and
from the culture supernatant of the A431 carcinoma cell line has been
reported by Johnson et al. (6, 8). Their methodology involved four purification steps with a final recovery of approximately 25%. In the present work the purification of a soluble recombinant form of FucT-III was achieved by a single affinity chromatography step
using GDP-Fractogel. Comparable final yields were obtained with this
procedure, which is more convenient and can also be used for the
purification of other recombinant human
fucosyltransferases.3 It is also
advantageous over other methods, where part of the protein A
polypeptide sequence is linked to the amino terminus of the FucT-III,
resulting in chimeric enzyme forms that can be purified by adsorption
on and elution from IgG-Sepharose. However, in the procedure described
in the present work, no fusion with unrelated bulky protein moieties
that might alter enzyme specificity toward different substrates (9) is
required.
For the purified enzyme described here, two closely spaced bands with a
unique amino-terminal sequence were detected. Apparent molecular masses
of about 42 kDa were calculated from their mobility in SDS-PAGE and
Coomassie staining as well as in Western blotting analysis using an
antibody that recognizes the tag sequence fused to the COOH terminus of
the enzyme. Since the expected NH2 terminus of the S2TF3T2
polypeptide was unequivocally detected upon gas-phase sequencing of the
product, the observed difference in apparent molecular mass is not due
to proteolytic degradation at any part of the recombinant enzyme. The
results obtained after the incubation with Endo H or PNGase F and
neuraminidase/mild acid treatment indicated that the two glycosylation
sites of S2FT3T2 are occupied with oligomannosidic or hybrid-type
glycans that are not decorated with significant amounts of NeuAc. Based
on binding studies with concanavalin A (8), occupancy of
N-glycosylation site(s) has been suggested previously for an
1-3/4-fucosyltransferase purified from the culture medium of the
A431 carcinoma cells.
The BHK-21 cells stably transfected with the membrane-bound wild-type form of the human FucT-III unexpectedly secreted considerable amounts of a soluble form of the enzyme into the culture supernatant (about 40% of the total activity that was measured after a 24-h production period of confluently growing and more than 95% viable BHK-21 cells). The detection of enzyme activity in supernatants of transiently transfected COS cells has been previously reported by Kukowska-Latallo (10) using expression plasmids encoding the human wild-type FucT-III. The detection of significant enzyme activity in the supernatants of stably transfected BHK-21 (>95% viable) cell clones in the present study suggests a common mechanism of endoproteolytic cleavage of human fucosyltransferase III within the stem region inside Golgi/endoplasmic reticulum compartments, since the apparent molecular weight detected for the secreted form of FT3T2 was very similar to that observed for the truncated genetically engineered secretory variants S2FT3T2 and S1FT3. This would also explain the high amounts of this enzyme that are detected in human milk and supernatants of A431 cells. Recently, Kimura et al. (21) have reported the preparation of a monoclonal antibody against human FucT-III that recognized enzyme forms in Western blotting with approximate molecular masses of 42-45 kDa in a variety of human tumor cell lines including A431 cells. Unfortunately, no data were presented on immunodetection of the FucT-III forms in the culture supernatants of these cells.
The truncated enzyme form S2FT3T2 showed virtually no activity with the type II 8-methoxycarbonyloctyl glycoside acceptor. Similar results were obtained by de Vries et al. (9) when using cell extracts of transiently transfected COS cells with constructs encoding the membrane-bound form of the FucT-III, whereas Johnson et al. (7) measured 10% of enzyme activity with this substrate compared with the value obtained with the type I acceptor. The Km value of S2FT3T2 with the type I 8-methoxycarbonyloctyl glycoside acceptor (0.54 mM) is similar to that found for the enzyme purified from human milk (0.5 mM) (7); however, it is approximately half of that determined by de Vries et al. (9) for a truncated form of the FucT-III (amino acids 43-361) linked to protein A. It might be speculated that the protein A moiety affects the specificity of the enzyme toward the substrate. Recent results obtained by Xu et al. (22) indicate that amino acid deletions in this region do not affect enzyme activity.
The S2FT3T2 efficiently fucosylates small oligosaccharides containing the type I and type II structures. Fuc was transferred preponderantly to the O-4 position of GlcNAc in LNT (70%), LNFP (100%), and LST-a (80%), whereas virtually all the Fuc was transferred to the O-3 position of Glc in LNnT. These results are in agreement with those described for enzyme preparations purified from human milk and the medium of A431 cells (7, 8). However, in our studies with S2FT3T2 and LNT or LST-a as acceptors, additional monofucosylation at the reducing Glc occurred, and bifucosylated products that contained fucose at the O-4 and O-3 positions of GlcNAc and Glc were detected. These structures were not detected in previously reported work. SLN was not fucosylated by S2FT3T2, in contrast to what was described for FucT-III from milk or secreted from A431 cells.
All glycoproteins tested, except for asialofetuin, were found to be
very poor substrates for S2FT3T2 based on [14C]fucose
incorporation studies. The asialofetuin used in the present work
contained triantennary glycans with terminal Gal1-3 and Gal
1-4
linked to GlcNAc as described by Rice et al. (23), but no
branched O-linked oligosaccharide described by Edge and
Spiro (24) was detected. This probably is due to the different
commercial origin of the protein preparation used here. The fucosylated
N-linked oligosaccharide of asialofetuin was identified as a
2,4-branched triantennary structure containing one Gal
1-3GlcNAc
antenna. This type of oligosaccharide is present, if any, in only very
small amounts in the other glycoproteins which were used in this work as substrates for S2FT3T2. The observed substrate specificity of the
enzyme enables us to suggest that human FucT-III provides a useful tool
for detection of type I structures in N-glycosylated glycoproteins.
Based on antibody binding studies, COS-1 cells start to express
Lex, sLex, Lea, and
sLea structures at their surface after transfection with
the FucT-III gene (1, 10); some of the fucosylated molecules are
glycoproteins (e.g. PSGL-1) (25). However, the secreted
FucT-III produced in BHK-21 cells only had the capability of in
vitro modifying type I structures, resulting in Lea
and sLea type motives in small oligosaccharides,
glycolipid, glycopeptide, and glycoproteins. In contrast to previously
reported work (10), in our study no structure could be detected,
indicating that the enzyme recognizes the GlcNAc in type II
N-glycans as a substrate; therefore, a biosynthetic
involvement of FucT-III in the formation of Lewisx or
sialyl-Lewisx motifs on glycoconjugates seems questionable.
This difference in specificity might be due to the truncation at the
amino terminus of the enzyme, or it may result from differences in the
intracellular environment/compartmentalization and the in
vitro assay conditions applied. However, it should be emphasized
that solubilization of membrane-bound glycosyltransferases after
disruption of cells in the presence of detergents will frequently
result in a mixture of proteolytically cleaved and intact forms, which
makes it difficult to unequivocally assess the substrate specificity of
the native Golgi enzymes by in vitro assays. Finally, this
problem could be solved by coexpression experiments using
glycosyltransferase genes and, e.g., secretory model
glycoproteins, which must be modified properly by the recipient cell
line and must be structurally characterized thoroughly with respect to
their carbohydrates. In preliminary studies of our laboratory using
coexpression of human erythropoietin and FucT-III in BHK cells, no
peripheral fucosylation of the secreted erythropoietin could be
detected. This supports our view that the enzyme acts as a
1-4-fucosyltransferase also in vivo.
We gratefully acknowledge the excellent technical assistance of Susanne Pohl and Christiane Kamp. We thank Drs. O. Hindsgaul, B. Kniep, and R. Kunz for the gift of acceptors, and W. Lindenmaier and J. Lowe for the gift of antibodies.