THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 273, No. 47, Issue of November 20, pp. 30985–30994, 1998
Printed in U.S.A.
In Vivo Specificity of Human a1,3/4-Fucosyltransferases III-VII
in the Biosynthesis of LewisX and Sialyl LewisX Motifs on
Complex-type N-Glycans
COEXPRESSION STUDIES FROM BHK-21 CELLS TOGETHER WITH HUMAN b-TRACE PROTEIN*
(Received for publication, July 21, 1998, and in revised form, September 3, 1998)
Eckart Grabenhorst‡, Manfred Nimtz‡, Júlia Costa§, and Harald S. Conradt‡¶
From ‡Protein Glycosylation, Gesellschaft für Biotechnologische Forschung mbH, D-38124 Braunschweig, Germany and
§Instituto de Tecnologia Quı́mica e Biológica, P-2780 Oeiras, Portugal
Each of the five human a1,3/4-fucosyltransferases
(FT3 to FT7) has been stably expressed in BHK-21 cells
together with human b-trace protein (b-TP) as a secretory reporter glycoprotein. In order to study their in
vivo properties for the transfer of peripheral Fuc onto
N-linked complex-type glycans, detailed structural analysis was performed on the purified glycoprotein. All
fucosyltransferases were found to peripherally fucosylate 19 –52% of the diantennary b-TP N-glycans, and all
enzymes were capable of synthesizing the sialyl LewisX
(sLex) motif. However, each enzyme produced its own
characteristic ratio of sLex/Lex antennae as follows: FT7
(only sLex), FT3 (14:1), FT5 (3:1), FT6 (1.1:1), and FT4
(1:7). Fucose transfer onto b-TP N-glycans was low in
FT3 cells (11% of total antennae), whereas the values for
FT7, FT5, FT4, and FT6 cells were 21, 25, 35, and 47%,
respectively. FT3, FT4, FT5, and FT7 transfer preponderantly one Fuc per diantennary N-glycan. FT4 preferentially synthesizes di-Lex on asialo diantennary N-glycans and mono-Lex with monosialo chains. In contrast,
FT6 forms mostly a1,3-difucosylated chains with no, one,
or two NeuAc residues. FT3, FT4, and FT6 were proteolytically cleaved and released into the culture medium
in significant amounts, whereas FT7 and FT5 were
found to be largely resistant toward proteolysis. Studies
on engineered soluble variants of FT6 indicate that
these forms do not significantly contribute to the in vivo
fucose transfer activity of the enzyme when expressed
at activity levels comparable to those obtained for the
wild-type Golgi form of FT6 in the recombinant host
cells.
The involvement of fucosyltransferases in the biosynthesis of
Lewis-type and sialylated Lewis-type carbohydrate structures
as ligands for selectins present on endothelial cells, platelets
and lymphocytes has been reviewed in several excellent recent
reviews (1– 4). The interaction of selectins with fucosylated
glycoprotein or glycolipid ligands is ascribed a central role in
biological phenomena like tumorigenesis, tissue differentiation, and leukocyte adhesion during inflammatory processes;
* This work was supported in part by European Union Grant BIO2CT94-3069 (to H. S. C.) and by a Deutscher Akademischer Austauschdienst grant (to J. C. and H. S. C.). The costs of publication of this
article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Protein Glycosylation, Gesellschaft für Biotechnologische Forschung mbH, Mascheroder
Weg 1, D-38124 Braunschweig, Germany. Tel.: 49-531-6181-287; Fax:
49-531-6181-202; E-mail: [email protected].
This paper is available on line at http://www.jbc.org
however, the specific role of each of the enzymes within the
assembly of these structures is not fully understood.
The in vivo enzyme specificity of the five human a1,3/4-FTs1
(FT3–FT7) that have been cloned so far (5–11) is difficult to
assess. Although in vitro activity assays using small oligosaccharide acceptor substrates might be valuable for monitoring
the purification of glycosyltransferases from natural or recombinant sources, the substrate specificities determined in vitro
might not allow for final conclusions on their in vivo functional
role. The structural analysis of a large number of glycoproteins
from human tissues indicates a biosynthetic pathway where a
sequential and ordered action of glycosyltransferases might be
operating (12–14). However, the mechanisms by which this is
accomplished in vivo, e.g. how the different subcompartmental
localization and temporal action of glycosyltransferases that
could compete for the same acceptor substrate in the Golgi is
regulated, has not been studied in detail.
Results obtained from in vitro assays of enzymes isolated
from tissues or body fluids are often difficult to interpret due to
the fact that several enzymes with different or overlapping
acceptor substrate specificities might be present. Furthermore,
heterogeneity in the polypeptide primary structure of the glycosyltransferase preparations due to different proteolytic cleavage that might occur during tissue/cell disruption procedures
must be considered as well when data from the literature are
compared. This must also be considered when activity measurements are carried out on recombinantly expressed enzymes
from insect or mammalian host cells, which in some cases have
been expressed as fusion proteins to facilitate their purification
(10, 15–18).
According to the data published so far, FT7 has been re1
The abbreviations used are: FT, fucosyltransferase; b-TP, b-trace
protein; CHO, Chinese hamster ovary; CTS, cytoplasmic, transmembrane, and stem; dHex, deoxyhexose; DHFR, dihydrofolate reductase;
DMEM, Dulbecco’s modified Eagle’s medium; Endo H, endo-b-N-acetylD-glucosaminidase H; ESI, electrospray ionization; ESI-MS/MS, nanospray tandem mass spectrometry; FBS, fetal bovine serum;
Gal(b33)GlcNAc-Oct, Gal(b33)GlcNAc-O(CH2)8COOCH3; Gal(b34)GlcNAc-Oct, Gal(b34)GlcNAc-O(CH2)8COOCH3; Hex, hexose, HexNAc,
N-acetylhexosamine; HexNAc-ol, N-acetylhexosaminitol; HPAE-PAD,
high-pH anion-exchange chromatography with pulsed amperometric
detection; HPLC, high performance liquid chromatography; IL, interleukin; Lea, LewisA (Gal(b33)[Fuc(a134)]GlcNAc-R); Lex, LewisX;
(Gal(b34)[Fuc(a133)]GlcNAc-R); MALDI/TOF-MS, matrix-assisted
laser desorption/ionization time of flight mass spectrometry; Mes,
2-morpholinoethanesulfonic acid; Mops, 3-morpholinopropanesulfonic
acid; NeuAc, N-acetylneuraminic acid; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PNGase F, peptide-N 4(N-acetyl-b-D-glucosaminyl)asparagine amidase F; sLea, sialyl LewisA
(NeuAc(a233)Gal(b33)[Fuc(a134)]GlcNAc-R); sLex, sialyl LewisX
(NeuAc(a233)Gal(b34)[Fuc(a133)]GlcNAc-R); ST, sialyltransferase;
rhu, recombinant human.
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In Vivo Specificity of Human a1,3/4-Fucosyltransferases
ported to fucosylate only a2,3-sialylated, small N-acetyllactosamine-type structures and is inactive with neutral acceptors
(10, 11, 18). FT4 acts almost exclusively on unsialylated
Gal(b134)GlcNAc-R (type II) structures (15, 19), whereas FT5
and FT6 have been reported to act on both a2,3-sialylated and
unsialylated type II acceptors (15, 16, 20, 21). FT3 has been
reported to mainly transfer Fuc in a1,4-linkage onto GlcNAc in
type I chains (15, 21, 22). Activity with type I acceptors has also
been reported for human FT5 (15), while FT4, FT6 and FT7 are
not active with Gal(b133)GlcNAc-R substrates (10, 15, 21).
In view of their important biological role in the biosynthesis
of selectin ligands by a1,3/4-FTs, there is a need to define and
compare their in vivo specificities toward a suitable acceptor or
reporter glycoconjugate that is constitutively expressed and
supplied in stable concentrations within the biosynthetic glycosylation pathway. Inflammation-induced expression of sLex
and Lex on N-acetyllactosamine-type N-glycans of several
acute phase proteins in human serum has been reported (23,
24). Walz et al. (25) have shown that in vitro fucosylated human
a1-acid glycoprotein leads to interaction with E-selectin expressed on inflamed endothelial cells. These reports have discussed a possible biological role of peripherally fucosylated
(acute phase) serum glycoproteins in context with a competitive inhibition of the primary interaction of leukocytes with the
selectins (23).
To our knowledge, no reports have been published comparing
the in vivo specificity of human a1,3/4-FTs toward N-glycosylated protein substrates. A recent report by Kimura et al. (26)
describes the activity of recombinantly introduced human a1,3/
4-FTs on glycolipids of HeLa and Namalwa cells. In the present
paper, we have used stable coexpression of human b-TP together with the full-length forms of the five known human
a1,3/4-FTs from BHK-21 cells in order to assess the in vivo
substrate specificity of the different transferases by carbohydrate structural characterization of the secreted b-TP using
complementary chromatographic and mass spectrometric techniques. Human b-TP is a 168-amino acid glycoprotein with two
N-glycosylation sites, which has been shown previously to contain peripheral Fuc when the native polypeptide is isolated
from human cerebrospinal fluid (27, 28) or from human serum
(28). By contrast, no peripheral Fuc was found attached to
human b-TP expressed from BHK-21 cells in a previous study
(29).
EXPERIMENTAL PROCEDURES
Materials—Peptide-N 4-(N-acetyl-b-D-glucosaminyl)asparagine amidase F (from Flavobacterium meningosepticum) and endo-b-N-acetyl-Dglucosaminidase H (from Streptomyces plicatus), both recombinant
from Escherichia coli, were bought from Boehringer Mannheim, Vibrio
cholerae sialidase was purchased from Calbiochem (La Jolla, CA), DNazol and Tri-Reagent were from Molecular Research Center, Inc. (Cincinnati, OH), Jetsorb was obtained from Genomed (Bad Oeynhausen,
Germany), fetal bovine serum was purchased from ITM (Munich, Germany), GDP-[14C]Fuc (285 Ci/mol) was from Amersham Pharmacia
Biotech (Braunschweig, Germany), and bovine fetuin, GDP-Fuc, and
G418 sulfate were bought from Sigma. DMEM was prepared from
Dulbecco’s modified Eagle medium (Life Technologies, Inc.) by supplementation prior to use with 10 mM Hepes, pH 7.2, 45 mM NaHCO3, 2 mM
glutamine, 0.061 g/liter ampicillin, 0.1 g/liter streptomycin sulfate, and
0, 2, or 10% FBS. N-Linked complex-type diantennary and tetraantennary structures with zero to four a2,3-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 (29 –32). The
Gal(b133)GlcNAc-O(CH2)8COOCH3 and Gal(b134)GlcNAc-O(CH2)8COOCH3 acceptors were a gift from Dr. O. Hindsgaul (University of
Alberta, Canada).
Nomenclature—The nomenclature of the five known human a1,3/4FTs has been described (10). In the present paper, these a1,3/4-FTs as
well as their corresponding cDNAs are abbreviated FT3 to FT7. The
BHK-21 cell lines that were constructed in the present study were
termed according to the coexpressed FT, e.g. “FT5 cells” are BHK-21
cells coexpressing human FT5 and human b-TP. The nomenclature for
sialyltransferases is according to Tsuji (33).
Molecular Cloning of Human a1,3/4-Fucosyltransferases—The
cDNAs encoding human a1,3/4-FTs were obtained by PCR according to
sequences published in the literature. If not otherwise stated, PCR was
performed in 50 ml with 15 pmol of each of the primer using the Expand
High Fidelity DNA polymerase system (Boehringer Mannheim). cDNAs
encoding human FT5 (7) and FT6 (8, 9) were cloned from HL-60
genomic DNA using the sense primer 59-ACT CTG ACC CAT GGA TCC
CCT and the antisense primer 59-CTC TCA GGT GAA CCA AGC CGC
TAT GC for both constructs. The DNA was prepared using DNazol
according to the manufacturer’s protocol, and 0.9 mg of HL-60 genomic
DNA was used in a PCR with the following conditions: a 3-min denaturation step at 94 °C, 35 cycles with 15 s of denaturation at 94 °C, 20 s
of annealing at 55 °C, 150 s of elongation at 72 °C, increased by 5 s per
cyle during the last 20 cycles, and a final elongation step for 10 min at
72 °C.
The cDNAs for human FT4 and FT7 were cloned from reversetranscribed HL-60 mRNA. For this, total RNA from HL-60 cells was
isolated with Tri-Reagent according to the protocol provided by the
supplier and poly(A)1 RNA was separated by using the Oligotex mRNA
purification kit (Qiagen). The cDNA was prepared from poly(A)1 RNA
by using the first strand synthesis protocol of a cDNA synthesis kit (Life
Technologies, Inc.), except that random hexamers (Amersham Pharmacia Biotech) were used instead of the oligo(dT) primer. The reverse
transcription product was purified by phenol extraction, and 1 ml corresponding to 1 mg of total RNA was used in the PCR. The long form of
human FT4 (6) was cloned using the sense primer 59-CAA GAG TAG
CGG ATG AGG CGC TTG and the antisense primer 59-CGC TTC CAG
GGG AGC GCG GCT TCA in the presence of 1.3 M betaine as has been
recommended for GC-rich sequences (34), using the PCR conditions: 3
min at 94 °C, 35 cycles with 15 s at 93 °C, 20 s at 49 °C, 150 s at 68 °C,
increased by 4 s/cycle during the last 20 cycles, and 10 min at 68 °C. For
cloning of FT7 (10, 11), the sense primer 59-TCT CTT GGC TGA CTG
ATC CTG GG and the antisense primer 59-TCA GGC CTG AAA CCA
ACC CT were applied using the conditions: 3 min at 94 °C, 35 cycles
with 15 s at 94 °C, 20 s at 45 °C, 120 s at 72 °C, increased by 10 s/cycle
during the last 20 cycles, and 10 min at 72 °C. The construct for FT3
was the same as used previously (22); this cDNA was originally cloned
by Dr. B. Seed (Boston, MA).
All DNA fragments generated by PCR were cloned into the eukaryotic expression vectors pCR3 or pCR3.1 (Invitrogen) according to the
manufacturer’s instructions. Positive clones were identified by restriction enzyme analysis, and the correct sequences were verified by automated sequencing of both DNA strands.
Construction of Soluble FT6 Mutants—The FT6 mutant s-FT6 encodes a human FT6 where the first 51 amino acids comprising the
cytoplasmic, transmembrane, and stem region (CTS) region of the enzyme are replaced by the human IL-2 signal peptide. A first DNA
fragment encoding the IL-2 signal peptide was generated by PCR using
the sense primer 59-CGG AAT TCG AGC TCG CCC GGG GAT CC, the
antisense mutagenesis primer 59-CTT TGT AGA ACT GTC GAC AGG
TGC ACT GTT TGT G (SalI site underlined), and a human IL-2 cDNA
(35) as a template (conditions: 3 min at 94 °C, 25 cycles with 15 s at
94 °C, 20 s at 50 °C, 30 s at 72 °C, and 8 min at 72 °C). A second
fragment was obtained by PCR of genomic DNA from human A172 cells,
which was prepared as described (36), using the sense mutagenesis
primer 59-GGG TCC CGC TTC GTC GAC AGC ACA GGG ACC (SalI
site underlined) and the antisense primer 59-GAA GCT TCA GAT CTA
CGA GTC CTT AGG at identical PCR conditions except that a 2-min
elongation time was used. Both fragments were cloned as described
above into pCR3 that contains a NotI site at the 39-end of the multiple
cloning site, yielding the vectors pCRIL43.1 and pCRFucN2C. The
insert of pCRFucN2C was cut with SalI and NotI, separated by agarose
gel electrophoresis, and isolated from the gel by using the Jetsorb kit.
The purified fragment was cloned into the unique SalI and NotI sites of
pCRIL43.1 using standard molecular biology techniques (36), yielding
the vector pCRs-FT6.
The construct BT-FT6 was generated to encode a fusion protein
where the full-length human b-TP is linked N-terminally to amino acids
55–359 of FT6. In a first PCR, using the sense primer 59-GAT CGA ATT
CGC ACA CCT GCT CGG CTG CAG, the antisense mutagenesis primer
59-GGG CGG GGG TCC CTT GTT CCG TCA TGC A and b-TP cDNA
(29) as a template, a fragment was generated that contained the whole
b-TP coding sequence and 13 base pairs from human FT6 at the 39-end
(PCR conditions: 3 min at 94 °C and 25 cycles with 15 s at 94 °C, 20 s at
50 °C, 30 s at 72 °C). One fifth of the reaction product was used as a
In Vivo Specificity of Human a1,3/4-Fucosyltransferases
59-megaprimer in a second PCR together with 5 pmol of the antisense
primer 59-CTC TCA GGT GAA CCA AGC CGC TAT GC and FT6 cDNA
as a template under the following conditions: 3 min at 94 °C, 35 cycles
with 15 s at 94 °C, 20 s at 55 °C, 150 s at 72 °C, increased by 5 s/cycle
during the last 20 cycles, and 10 min at 72 °C. The new PCR fragment
was isolated by agarose gel electrophoresis, purified with Jetsorb reagents, and cloned using the pCR3.1 TA-cloning system as described
above, generating plasmid pCRBT-FT6.
Expression of Human a1,3/4-Fucosyltransferases in BHK-21 Cells—
BHK-21 cells stably expressing human b-TP (29) were transfected with
the different FT constructs by using the calcium phosphate precipitation method as described (36). In each case, 4 mg of plasmid-DNA was
added to 4 3 105 cells grown to a density of 4 3 104 3 cm22. All vectors
used contained a neomycin phosphotransferase gene driven by the
SV40 promoter conferring G418 resistance to cells harboring these
plasmids. Three days after transfection, the cells were subcultivated 1:5
and selected in DMEM containing 10% FBS and 1.5 g/liter G418 sulfate.
Using this selection procedure, usually 100 –500 clones survived and
where propagated in selection medium for a further time period of 2–3
weeks. Cell culture supernatants of confluently growing monolayers
were then analyzed for similar b-TP expression levels by Western blot
analysis and for FT activity as described below.
SDS-PAGE and Western Blot Analysis—SDS-PAGE was performed
according to Laemmli (37) 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 rabbit anti-b-TP antiserum (38) in
blocking buffer at 1:1000 dilution. The second antibody, goat anti-rabbit
immunoglobulin coupled to horseradish peroxidase, was used at a 1:500
dilution. The blots were developed with Tris-buffered saline containing
0.5 g/liter 4-chloro-1-naphthol solubilized in methanol and 0.2%
perhydrol.
Immunodetection of rhu-FT6 was performed essentially as described
for b-TP; in this case, a rabbit antiserum (prepared by Eurogentec,
Seraing, Belgium) raised against the human FT6 peptide 125RRQGQRWIWFSMESPSHCWQLK(C) was used following immunoaffinity purification on the peptide antigen coupled to Affi-Gel-15 (Bio-Rad).
Production and Purification of Recombinant Human b-TP—BHK
cells expressing human b-TP were grown to confluence in 175-cm2
culture flask in DMEM containing 10% FBS and where then used for
the production of b-TP using medium exchanges every 2–3 days with
DMEM alternatingly containing 0 or 2% FBS for a period of up to 3
weeks. The production of rhu-b-TP was monitored by Western blot
analysis of cell culture supernatants at different time points during the
cultivation periods and the yield for all cell lines was found to be about
1.0 mg of b-TP 3 1026 cells 3 48 h21. The purification of rhu-b-TP was
performed by immunoaffinity chromatography using the monoclonal
anti-b-TP antibody Y248D9 (39) coupled to Affi-Gel-15 matrix (BioRad). In the case of BHK-21 cells coexpressing b-TP and BT-FT6, the
latter protein was removed from supernatants of BT-FT6 cells by GDPFractogel affinity chromatography before immunoaffinity purification
of rhu-b-TP. Purity of the final preparations was analyzed by SDSPAGE and Western blotting as well as N-terminal amino acid
sequencing.
Fucosyltransferase Assays—Cell extracts were freshly prepared prior
to FT assays. The cells from confluently grown monolayers were washed
with PBS and suspended in PBS by scraping them from the plastic
surface. Following centrifugation at 800 3 g, the cells were resuspended
in lysis buffer (10 mM Mops, pH 7.5, 0.1% Triton X-100, 10 mg/liter
aprotinin, 10 mg/liter leupeptin, 1 mM phenylmethylsulfonyl fluoride)
at densities of 1–5 3 107 3 ml21. The FT activity of extracts and culture
supernatants of cells transfected with different FTs was tested at 37 °C
with up to 8 ml of the sample in 20 ml of a reaction mixture containing
100 mM NaCl, 50 mM Mes, pH 6.5, 20 mM MnCl2, 0.055 mM GDP-Fuc
(1.1 nmol), 60,000 cpm of GDP-[14C]Fuc, and 0.8 mM Gal(b134)GlcNAcOct acceptor (Gal(b133)GlcNAc-Oct in the case of FT3). Following
incubation for up to 3 h, the reaction mixtures were diluted with water
to 0.5 ml and applied to Sep-Pak (Waters) C18 cartridges. The columns
were washed two times with 2.5 ml of water and eluted with 1.5 ml of
methanol, and incorporation of [14C]Fuc was determined by liquid scintillation counting of the eluate. The FT activity of FT7 was tested under
identical conditions except that 23 g/liter bovine fetuin was used instead of the Gal(b134)GlcNAc-Oct acceptor. In this case, the reaction
mixture was precipitated with 1 ml of 0.5 M HCl containing 1% phosphotungstic acid at 0 °C and transferred under vacuum to glass microfiber filters (Whatman GF/C). The filters were washed once with 0.5 M
30987
HCl containing 1% phosphotungstic acid and twice with methanol,
dried, and analyzed for radioactivity by scintillation counting. Under
the conditions used, incubation with FT6 yielded similar incorporation
of Fuc into Gal(b134)GlcNAc-Oct and bovine fetuin used as acceptors.
In all cases, 1 unit of enzyme activity corresponds to transfer of 1 mmol
of Fuc/min.
Fucosyltransferase Purification—Culture supernatants from stably
transfected BHK-21 cells were applied onto a 5-ml GDP-Fractogel column (21) equilibrated with 20 mM Mes, pH 6.8, containing 0.02% NaN3
and 1 mM dithioerythritol, at a flow rate of 1 ml/min at room temperature. The column was washed with buffer A (20 mM Mes, pH 6.8,
containing 0.02% NaN3 and 30% glycerol), first in the presence of 50 mM
NaCl and subsequently in the presence of 500 mM NaCl. Elution of the
enzyme was performed with buffer A containing 1.5 M NaCl, and the
eluate was used for in vitro fucosylation studies.
For SDS-PAGE analysis, the eluate was concentrated 7-fold using
Diaflo YM10 ultrafiltration membranes (Amicon, Witten, Germany),
diluted with six volumes of 10 mM Mes, pH 6.8, and again concentrated
7-fold by ultrafiltration. The concentrate was used directly for enzymatic characterization of the eluted FTs by incubation with 0.1 unit/ml
sialidase at pH 5.5, with 0.1 unit/ml Endo H at pH 6.0 (pH values were
adjusted with acetic acid), and with 10 units/ml PNGase F for 20 h at
37 °C.
Preparative in Vitro Fucosylation of Glycoconjugates—For in vitro
fucosylation of purified wild-type b-TP from BHK-21 cells, 0.5 mg of the
protein was incubated for 24 h at 37 °C with 0.05 milliunit of purified
s-FT6 in a buffer containing 100 mM NaCl, 50 mM Mes, pH 6.8, 20 mM
MnCl2, 4 mM GDP-Fuc, 0.5% Triton X-100, and 0.02% NaN3. The
protein was then precipitated with 220 °C ethanol, dried, and analyzed
as described below.
Reducing N-glycans of the di- and tetraantennary complex type were
incubated at 0.1– 0.2 mM concentrations with 0.2 unit/liter s-FT6 in the
presence of 0.2 mM GDP-Fuc, 100 mM NaCl, 50 mM Mops, pH 7.5, 20 mM
MnCl2, and 0.02% NaN3 for 6 and 24 h. Aliquots of the reaction mixtures were analyzed by HPAE-PAD before and after desialylation and
by MALDI/TOF-MS after desalting as detailed below.
Enzymatic Release of N-Glycans Bound to Recombinant Human
b-TP—Purified rhu-b-TP was reduced, carboxamidomethylated, and
digested with trypsin as described previously for natural b-TP isolated
from human cerebrospinal fluid (27). The tryptic glycopeptides were
isolated by reversed-phase HPLC and were incubated with PNGase F
as described (27). The oligosaccharides were separated from peptides by
passing the incubation mixture through a reversed-phase C18-column
and were recovered in the flow-through.
High pH Anion-exchange Chromatography (HPAE-PAD) of Oligosaccharides—A Dionex BioLC System (Dionex, Sunnyvale, CA) equipped
with a CarboPac PA1 column (4 mm 3 250 mm) was used in combination with a pulsed amperometric detector (detector potentials and pulse
durations: E1 5 150 mV, T1 5 480 ms; E2 5 1500 mV, T2 5 120 ms; E3
5 -500 mV, T3 5 60 ms). The oligosaccharide material was desalted
before HPAE-PAD analysis by injecting onto a Fast Desalting column
(Amersham Pharmacia Biotech) and eluting with doubly distilled H2O
by using the FPLC system (Amersham Pharmacia Biotech). In some
cases, N-glycans were also desialylated prior to HPAE-PAD analysis by
incubation with 0.2 unit/ml V. cholerae sialidase for 2 h at 37 °C in a
buffer containing 10 mM NaOAc, pH 5.5, 1 mM CaCl2, and 0.02% NaN3.
The oligosaccharides were then injected onto the CarboPac PA1 column
that was equilibrated with 100% solvent A. Elution was performed by
applying a linear gradient from 0 –20% solvent B over a period of 40 min
followed by a linear increase from 20 –100% solvent B over 5 min.
Solvent A 5 0.1 M NaOH in doubly distilled H20, solvent B 5 0.6 M
NaOAc in solvent A, flow rate 5 1 ml/min.
Mass Spectrometric Characterization of N-Glycans—The unseparated glycan pools obtained after enzymatic liberation from rhu-b-TP
purified from the cell culture medium of the different FT cell lines were
characterized after reduction and permethylation by MALDI/TOF-MS
(molecular masses of all components), by ESI-MS/MS (monosaccharide
sequence, presence of isomeric structures, some linkage information),
and methylation analysis (substitution position). Additionally, all major
carbohydrate structures detected by HPAE-PAD mapping of the native
and desialylated glycans were isolated from preparative runs and were
characterized individually by using the same techniques.
Matrix-assisted Laser Desorption Time of Flight Mass Spectrometry
(MALDI/TOF-MS)—For analysis by MALDI/TOF-MS, the solutions of
the reduced and permethylated oligosaccharides were mixed with the
same volume of matrix (10 g/liter 2,5-dihydroxybenzoic acid in 10%
ethanol in water). 1 ml of the sample was then spotted onto a stainless
steel tip and dried at room temperature. The concentrations of the
30988
In Vivo Specificity of Human a1,3/4-Fucosyltransferases
analyte mixtures were approximately 10 mM. 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 watt/cm2
irradiance at the surface (0.2 mm2 spot). Spectra were recorded at an
acceleration voltage of 20 kV using the reflectron and the delayed
extraction facility for enhanced resolution. The following series of diantennary complex type structures bearing the proximal Fuc only or
1–2 a1,3-linked Fuc and 0 –2 NeuAc residues were typically identified
by their characteristic masses. Their approximate ratios were determined by assessment of the relative intensities of the respective molecular ion signals (sodium adducts, compare Fig. 1): asialo, [Hex5 HexNAc3 dHex(1–3) HexNAc-ol 1 Na]1 (m/z 2262, 2436, 2610); monosialo,
[NeuAc Hex5 HexNAc3 dHex(1–3) HexNAc-ol 1 Na]1 (m/z 2623, 2797,
2972); disialo, [NeuAc2 Hex5 HexNAc3 dHex(1–3) HexNAc-ol 1 Na]1
(m/z 2985, 3158, 3332).
MALDI/TOF-MS of total b-TP N-glycans did not separate structures
bearing three Fuc residues from the singly undermethylated species
with one Fuc and an additional NeuAc, e.g. diantennary asialo complex
type with proximal and two peripheral Fuc at m/z 2610 [Hex5 HexNAc3
dHex3 HexNAc-ol 1 Na]1 from diantennary monosialo complex type
structure with proximal Fuc lacking one methyl group at m/z 2609
[NeuAc Hex5 HexNAc3 dHex HexNAc-ol - CH2 1 Na]1. The abundance
of the trifucosylated structures were corrected by subtracting the expected intensity of the undermethylated species obtained from the
average degree of undermethylation observed in the spectrum, which
could be easily determined from nonsuperimposed clusters of molecular
ion signals. In most cases, the ratio of [M 2 CH2]1 to [M]1 was approximately 1:3 for structures in the molecular mass range of 2200 –3000
Da.
Nanospray Tandem Mass Spectrometry (ESI-MS/MS)—A Finnigan
MAT TSQ 700 triple quadrupole mass spectrometer (Finnigan MAT
Corp., San Jose, CA) equipped with a nanospray ion source (Protana,
Odense, Denmark) was used for ESI-MS. The reduced and permethylated samples were dissolved in MeOH (concentrations approximately
10 mM) saturated with NaCl, and approximately 3 ml of this solution was
filled into gold-coated nanospray glas capillaries (Protana, Odense,
Denmark). The tip of the capillary was placed directly in front of the
entrance hole of the heated transfer line of the mass spectrometer, and
a voltage of 800 V was applied. 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. In contrast to
MALDI/TOF-MS, the doubly charged molecular ion species [M 1
2Na]21 of the reduced and permethylated oligosaccharide compounds
described above were detected by ESI-MS. Individual molecular ion
species (parent ions) were then selected using the first mass analyzer,
and fragments were generated by collision induced dissociation with
argon atoms. These daughter ions were then separated by the second
mass analyzer. The following characteristic low molecular weight fragment ions at m/z 398 [NeuAc 1 Na], 486 [Hex HexNAc 1 Na], 490
[dHex HexNAc-ol 1 Na], 660 [Hex dHex HexNAc 1 Na], 847 [NeuAc
Hex HexNAc] accompanied by the secondary fragment 472 [HOHex
HexNAc 1 Na], and 1021 [NeuAc Hex dHex HexNAc] accompanied by
646 [HOHex dHex HexNAc] were used for the detailed structural characterization of individual glycans (compare Fig. 5). In this way, the
terminal substitution pattern of N-acetyllactosamine antennae and the
discrimination between possible linkage positions of Fuc (proximal or
peripheral, to sialo- or asialo-antennae) was achieved. The well known
preferred elimination of the substituent linked to O-3 of GlcNAc residues (40) was used to differentiate between Lex (3-linked Fuc, 4-linked
Gal) and Lea (3-linked Gal, 4-linked Fuc) motifs. Only relatively weak
signals due to the elimination of Fuc could be detected, indicating the
presence of Lex structures. The exact linkage position of individual
N-acetyllactosamine antennae to the branched common core mannose
(O-3 or O-6), however, could not be determined.
Methylation Analysis—Oligosaccharides were permethylated according to Hakomori (41), purified, hydrolyzed, reduced, and peracetylated
as described (31). Separation and identification of partially methylated
alditol acetates were 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. The linkage types present in each oligosaccharide pool were determined in this way, and particularly the degree of sialylation and
peripheral fucosylation obtained by MALDI mapping and by HPAEPAD were corroborated by the detected ratio of the partially methylated
alditol acetates characteristic for terminal or 3-substituted Gal, 4-substituted or 4,6-disubstituted reduced GlcNAc, and non-reduced 4-substituted or 3,4-disubstituted GlcNAc. For isolated N-glycans, the sub-
stitution pattern of all monosaccharide constituents was determined,
and in combination with the ESI-MS/MS data, a detailed structural
characterization of all major components was achieved. An unequivocal
discrimination between the Lea and Lex structural motifs, which both
yield the 3,4-disubstituted GlcNAc derivative, was performed by methylation analysis after elimination of the Fuc residues by mild acid
treatment of the respective oligosaccharides. No 3-substituted GlcNAc
was detected by this procedure excluding the presence of type I motifs/
Lea structures.
RESULTS
Coexpression of Human a1,3/4-Fucosyltransferases Together
with Human b-TP in BHK-21 Cells—BHK-21 cells stably expressing human b-TP (29) were cotransfected with plasmids
encoding human a1,3/4-FTs (FT3 to FT7) or recombinant soluble forms of FT6 (s-FT6, BT-FT6) where the catalytic domain
of FT6 was fused to the C terminus of the IL-2 signal peptide or
the full-length b-TP. Stably transfected cells were selected in
the presence of 1.5 g/liter G418 sulfate and were used for
subsequent production and characterization of the secreted
b-TP. The a1,3/4-FT activity was analyzed using Gal(b134)GlcNAc-Oct or the type I derivative Gal(b133)GlcNAc-Oct in
the case of FT3 as acceptors. b-TP expression levels as well as
that of the BT-FT6 fusion protein were calculated from Western blot analyses of the supernatants using anti-b-TP antibodies. All stable cell lines secreted about 1 mg of recombinant
b-TP/106 cells/48 h in serum-free medium. For characterization
of N-linked oligosaccharides of b-TP, 1–2 liters of supernatant
were produced from confluently growing cells and b-TP was
purified by immunoaffinity chromatography (27, 29) with a final
yield of .90% as described under “Experimental Procedures.”
In Vitro and in Vivo Synthesis of Lex and sLex Motifs in b-TP
N-Glycans by Recombinant Human FT6 —Human b-TP expressed from BHK-21 cells in the absence of recombinant FTs
contains almost exclusively diantennary complex-type II oligosaccharides at its two N-glycosylation sites. A complete structural characterization of b-TP oligosaccharides has been published elsewhere (29). About 90% of the oligosaccharides are
mono- or disialylated with exclusively a2,3-linked NeuAc, and
all glycans are completely proximally fucosylated in a1,6-linkage (compare molecular ion signals detected by MALDI/
TOF-MS after reduction and permethylation; Fig. 1A).
When recombinant b-TP is incubated with purified s-FT6
expressed from BHK-21 cells in the presence of GDP-Fuc, three
novel molecular ion peaks are detected in the MALDI-spectrum
of the liberated oligosaccharides (Fig. 1B), indicating efficient
peripheral fucosylation of the b-TP glycoprotein with one or
two a1,3-linked Fuc resulting in Lex and sLex motifs as determined by methylation analysis (detection of the 3,4-disubstituted GlcNAc-derivative, which was not detected in oligosaccharides from wild-type b-TP, data not shown). As calculated
from the peak areas of the native oligosaccharides obtained by
HPAE-PAD mapping (see below), 46% of the a2,3-monosialylated oligosaccharides acquired one additional a1,3-linked Fuc,
whereas 33 and 6% of the a2,3-disialylated oligosaccharides
were modified with one or two a1,3-linked Fuc residues, respectively. This result is in agreement with published reports for
the in vitro specificity of rhu-FT6, which indicate that the
enzyme can form Lex as well as sLex motifs with small type II
oligosaccharides (16, 21). For b-TP N-glycans isolated from the
supernatant of cells stably transfected with human FT6, a
MALDI spectrum was obtained that differed from that of the in
vitro modified b-TP N-glycans (Fig. 1C). The di-sLex diantennary structure (m/z 3332) is formed in only small amounts by in
vitro incubation of b-TP with s-FT6, whereas this structure is
significantly increased in the oligosaccharide mixture from
b-TP isolated from the medium of FT6 cells. Since MALDI/
TOF-MS does not allow to distinguish between, e.g., reduced
In Vivo Specificity of Human a1,3/4-Fucosyltransferases
FIG. 1. In vitro and in vivo substrate specificity of rhu-FT6:
MALDI/TOF-MS analysis of permethylated total b-TP oligosaccharides. Total N-glycans of b-TP expressed from BHK-21 cells were
obtained from the purified product by enzymatic liberation and, after
reduction and permethylation, were finally analyzed by MALDI/
TOF-MS as detailed under “Experimental Procedures.” The symbols
indicate N-acetyllactosamine-type diantennary chains with proximal,
a1,6-linked Fuc (m/z 2262) containing NeuAc (indicated by S) and/or
peripheral Fuc (Lex or sLex) as explained in the mass spectrometric
section under “Experimental Procedures.” Panel A, N-glycans from
purified human b-TP expressed from BHK-21 cells without coexpressed
FT; panel B, N-glycans from purified rhu-b-TP subjected to in vitro
fucosylation with s-FT6; panel C, N-glycans from b-TP stably coexpressed together with wild-type FT6 in BHK-21 cells.
and permethylated diantennary chains containing three Fuc or
containing one Fuc, one NeuAc, and lacking one CH2 group due
to undermethylation, and furthermore MALDI/TOF-MS does
not allow absolute quantification of signal intensities of different molecular ions, native oligosaccharide mixtures were also
subjected to HPAE-PAD mapping, as exemplified in Fig. 2.
This HPAE-PAD mapping procedure allows the separation of
all possible peripherally fucosylated asialo, mono and disialo
diantennary oligosaccharides that could evolve from in vivo or
in vitro Fuc transfer onto the type II b-TP N-glycans except for
the structure at m/z 2797, which comprises the monosialylated
isomers with a2,3-linked NeuAc attached to a Lex or a nonfucosylated N-acetyllactosamine branch. These two structures,
however, can be distinguished by the application of an ESIMS/MS technique as described under “Experimental Procedures.” The almost identical PAD responses for all structures
therefore allows accurate quantification by peak integration.
About 50% of the b-TP oligosaccharides contain a1,3-linked
Fuc when the protein is expressed from FT6 cells and the
sialylation degree is lower when compared with structures of
b-TP from cells without FT6. Most of the oligosaccharides are
found to be modified with two peripheral Fuc. The ratio of
sLex:Lex antennae in the total N-glycan mixture is 1.1:1. The
lower degree of sialylation of b-TP from FT6 cells could result
from in vivo competition of the recombinant FT6 with the
endogenous a2,3-STs for the common asialo oligosaccharide
substrate, but obviously, as is the case for in vitro incubation
conditions, the enzyme can act also in vivo on both a2,3-sialylated as well as unsialylated N-linked oligosaccharides.
When purified s-FT6 was analyzed for in vitro substrate
specificity toward free complex-type N-glycans at 0.1– 0.2 mM
acceptor concentration (compare Table I), for both the asialo
diantennary and tetraantennary type II N-glycans, only 20% of
the substrate was a1,3-monofucosylated, whereas with the
a2,3-disialylated diantennary glycan, 90% conversion to prod-
30989
ucts containing one or two sLex motifs in an almost identical
ratio was observed. The a2,6-sialylated diantennary N-glycan
was not recognized as a substrate. About 60% of an a2,3tetrasialylated tetraantennary structure was a1,3-fucosylated,
with 20% of the product containing two sLex antennae.
Soluble Forms of FT6 Do Not Contribute to the in Vivo
Specificity of the Enzyme—As we have previously reported for
the wild-type Golgi form of human FT3 expressed from BHK-21
cells (22), the full-length Golgi form of human FT6 is also
subjected to considerable intracellular proteolysis, and secreted forms of the enzyme (termed sec-FT6) can be isolated
from the medium of stably transfected cells. As shown in Fig. 3,
sec-FT6 produces a diffuse band in SDS-PAGE with an apparent mass of about 50 kDa, whereas incubation with PNGase F
reduces the apparent mass by generating two closely spaced
sec-FT6 bands of about 40 kDa. Further analysis of sec-FT6
reveals only slight sensitivity to sialidase treatment, but partial resistance to Endo H treatment, indicating that in sec-FT6
most likely all four N-glycosylation sites of FT6 are present and
occupied by a mixture of oligomannosidic and complex-/hybridtype N-glycans.
About 75% of the total FT6 activity was found in the supernatant of confluent FT6 cells after 48 h. In order to rule out the
possibility that this proteolytic activity is a property of our host
cell line, we compared a second BHK cell line (BHK-21A, see
Ref. 29) and CHO DHFR2 cells after transfection with human
wild-type FT6. As shown in Table II, all FT6 cells showed
similar expression values and similar ratios of cell-associated
and cell supernatant FT activities. Therefore, the susceptibility
of Golgi forms of glycosyltransferases toward intracellular proteolysis seems to be a general phenomenon, since similar observations have been reported for other recombinant glycosyltransferases by several groups (22, 42– 46). However, since it
was not clear to what extent intracellularly cleaved sec-FT6
contributes to the overall in vivo modification of b-TP N-glycans, we generated b-TP-secreting cell lines, which coexpress
the recombinant soluble FT6 variants s-FT6 and BT-FT6 (see
above). A comparison of intracellular FT6 activity and FT6
activity measured after 48 h in supernatants of confluent s-FT6
cells and BT-FT6 cells is also included in Table II. We isolated
the secreted b-TP from the supernatants of all cell lines and
compared the fucosylation of the diantennary oligosaccharides
liberated from the purified glycoprotein. The HPAE-PAD elution profiles of the desialylated oligosaccharides are shown in
Fig. 4. FT6 cells synthesize b-TP oligosaccharides with no, one
or two peripheral Fuc residues in a ratio of 5:1:4 (Fig. 4A). The
s-FT6(I) cell line expresses a total FT6 activity comparable to
FT6 cells (cf. Table II), but no Lex-containing oligosaccharides
were detected in the b-TP secreted thereof (Fig. 4B), whereas in
the case of s-FT6(II) cells that express a 20-fold higher FT6
activity, small amounts of the a1,3-monofucosylated (10%) and
a1,3-difucosylated structure (7%) were observed (Fig. 4C). The
cell line expressing the chimeric BT-FT6 construct had a 80fold higher total FT6 activity, and in this case, total b-TP
oligosaccharides contained about 12% of the a1,3-monofucosylated and 12% of the a1,3-difucosylated diantennary structure
(Fig. 4D). This result let us conclude that soluble forms of
human FT6 including those generated by intracellular proteolysis of the full-length Golgi enzyme do not contribute significantly to the in vivo fucosylation properties of stably transfected FT6 cells toward secreted glycoproteins. A similar
situation should also be expected in natural cells/tissues, which
usually express about 50-fold lower total FT activity when
compared with the recombinant BHK-21 cells (as is the case for
HL-60 cells).
Stable Coexpression of FT3, FT4, FT5, or FT7 from BHK-21
In Vivo Specificity of Human a1,3/4-Fucosyltransferases
30990
FIG. 2. In vivo substrate specificity of rhu-FT6: HPAE-PAD analysis of human b-TP after coexpression in BHK-21 cells. Total b-TP
N-glycans were enzymatically released from purified glycopeptides and were analyzed by HPAE-PAD mapping as detailed under “Experimental
Procedures.” The structural symbols depicted are the same as used in Fig. 1. Indicated peaks were calculated to represent .90% of the total
oligosaccharides of b-TP (masses were confirmed by MALDI/TOF-MS and methylation analysis as described under “Experimental Procedures”).
Unidentified peaks represent either noncarbohydrate material or small amounts of triantennary structures (29) and were not considered for
calculation of the results (compare also asialo oligosaccharide profiles in Fig. 4 and MALDI/TOF-MS in Fig. 1).
TABLE I
In vitro fucosylation by s-FT6 of sialylated and asialo complex-type
oligosaccharide acceptors
Complex-type oligosaccharides containing a1,6-linked Fuc (except for
structure III) were characterized as reported previously (29, 32) and
were used at 0.1– 0.2 mM concentrations for in vitro fucosylation with
s-FT6. A dash indicates that no product was detected by MALDI/
TOF-MS and HPAE-PAD analysis.
Acceptor oligosaccharide
% of oligosaccharide structures containing
a1,3-linked Fuc
0 Fuc
I. Asialo diantennary
II. a2,3-Disialylated
diantennary
III. a2,6-Disialylated
diantennary
IV. Asialo
tetraantennary
V. a2,3-Tetrasialylated
tetraantennary
1 Fuc
2 Fuc
3 Fuc
4 Fuc
80
10
20
45
—
45
—
—
—
—
100
—
—
—
—
80
20
—
—
—
41
47
12
Trace
—
FIG. 3. Characterization of secreted forms of wild-type rhuFT6 (sec-FT6) generated by intracellular proteolysis. Sec-FT6
was partially purified from the culture supernatant of FT6 cells by
GDP-Fractogel affinity chromatography and was not treated (lane 1), or
was treated with sialidase (lane 2), Endo H (lane 3), or PNGase F (lane
4) prior to SDS-PAGE and Western blot analysis by using a rabbit
anti-FT6-peptide antiserum. In each case, sec-FT6 purified from 15 ml
of supernatant was applied.
Cells Together with b-TP—Since we have corroborated differences in in vivo and in vitro specificity of rhu-FT6, it is conceivable that such differences also exist for other human a1,3/
4-FTs. We therefore have constructed and selected stable cell
lines expressing human FT3, FT4, FT5, or FT7 together with
human b-TP as a secretory reporter glycoprotein. After purification of b-TP from each culture supernatant as described for
FT6 cells, oligosaccharides were liberated and characterized by
MALDI/TOF-MS and HPAE-PAD mapping. Apart from FT3
and FT6 cells, also FT4 cells were found to secrete substantial
amounts of FT activity into the culture medium (Table III).
About 30 –50% of the N-linked oligosaccharides of b-TP secreted from the stable cells where a1,3-fucosylated except for
the FT3 cell line, where about 13% of the total disialylated
b-TP glycans were modified with one peripheral Fuc. A comparison of all b-TP N-glycan structures formed by the a1,3/4FT-transfected cells is detailed in Table IV.
TABLE II
Fucosyltransferase activities in cellular extracts and in supernatants
of different cell lines and stably transfected FT6 cells
Enzyme assays were performed on samples taken 48 h after confluent
BHK-21 cells were supplied with fresh medium. Activity values were
determined with Gal(b1 3 4)GlcNAc-Oct as acceptor. Values for HL-60
cells were determined for cells grown in suspension culture. A dash
indicates incorporation of [14C]Fuc at background levels (,200 cpm
corresponding to ,0.08 microunits 3 1026 cells and ,4.0 microunits 3
ml21 3 48 h21, respectively).
Fucosyltransferase activity
Cell line
BHK-21B
HL-60
FT6 (BHK-21B)
FT6 (BHK-21A)
FT6 (CHO-DHFR2)
s-FT6(I)
s-FT6(II)
BT-FT6
Cellular extract
Culture medium
microunits 3 1026 cells
microunits 3 ml21 3 48 h21
—
4
45
23
70
13
265
380
—
—
125
185
300
160
2700
9000
FT7 cells were found to synthesize exclusively sLex structures. This was corroborated also for the monosialylated oligosaccharide fraction that contained no a1,3-difucosylated structure and only a single Fuc, which was exclusively present as
the sLex and not as the Lex motif (determined by ESI-MS/MS
as described under “Experimental Procedures”). In the small
amount of asialo oligosaccharide fraction, the Lex epitope could
not be detected. This in vivo specificity is in agreement with
recently published work on the in vitro activity of the enzyme
(10, 11, 18).
Also in agreement with the literature is the detection of Lex
in of the vast majority of fucosylated b-TP glycans from FT4
cells. This is also the case for the monosialylated a1,3-monofucosylated N-glycan, as demonstrated by ESI-MS/MS (Fig. 5).
However, a significant amount of mono-sLex was observed in
the disialo oligosaccharide fraction, which contradicts published data on the in vivo specificity of the enzyme as measured
by E-selectin binding studies (10, 19, 47, 48). Interestingly, no
a1,3-difucosylated disialo structures are observed, supporting
the view of the preferential action of FT4 on nonsialylated
structures.
FT5 cells secrete b-TP with preponderantly sLex motifs, but
Lex-containing structures are also formed by the cells and are
detected as a1,3-difucosylated asialo and a1,3-difucosylated
monosialo structures. The a1,3-monofucosylated monosialo oligosaccharide was found to consist of a mixture of Lex and sLex
chains.
Most surprisingly, we were able to detect sLex motif in the
disialo fraction of b-TP oligosaccharides from FT3 cells. In a
previous study (22), we have reported that human FT3 from
BHK-21 cells failed to fucosylate type II N-acetyllactosamine
structures in several glycoproteins when incubated in vitro,
and furthermore we clearly demonstrated that in bovine fetuin
In Vivo Specificity of Human a1,3/4-Fucosyltransferases
30991
TABLE IV
In vivo synthesis of Lex and sLex on b-TP N-glycans by full-length
human a1,3/4-fucosyltransferases expressed in BHK cells
For each cell line, the secreted reporter glycoprotein b-TP was isolated and the total oligosaccharides were characterized by MALDI/
TOF-MS and were quantified by HPAE-PAD mapping as detailed for
FT6 in Fig. 1 and Fig. 2. The a1,3-monofucosylated monosialo diantennary chains were found to contain exclusively Lex in the case of FT4
cells, exclusively sLex in the case of FT7 cells, and a mixture of both
isomers in the case of FT3, FT5, and FT6 cells.
% of total diantennary b-TP
oligosaccharides
Diantennary structure
Asialo
1 3 Lex
2 3 Lex
Monosialo
1 3 Lex* or 1 3 sLex**
1 3 Lex and 1 3 sLex
Disialo
1 3 sLex
2 3 sLex
FIG. 4. In vivo functional activity of recombinant soluble forms
of human FT6 coexpressed together with b-TP in BHK-21 cells.
The diagram shows the HPAE-PAD elution profiles of b-TP N-glycans
after enzymatic desialylation. Arrows indicate the elution position of
(proximally fucosylated) di-Lex diantennary and mono-Lex diantennary
chains, respectively, as indicated by the symbols. Panel A, coexpression
of wild-type FT6 (same preparation as in Fig. 3 after desialylation);
panel B, coexpression with s-FT6; panel C, coexpression with highly
overexpressed s-FT6; panel D, coexpression with highly overexpressed
BT-FT6 fusion protein (cf. expression levels in Table II). The BT-FT6
fusion protein was removed from the medium prior to purification of
rhu-b-TP by quantitative adsorption onto a GDP-Fractogel column. A
minor peak marked by an asterisk represents an asialo-triantennary
structure, which was not considered for the calculation of the results in
this work.
TABLE III
Comparison of fucosyltransferase activities and in vivo
specificities of a1,3/4-FT-transfected BHK cells
The percentage of peripheral fucosylation of b-TP oligosaccharides
was calculated from peak areas upon HPAE-PAD mapping. FT activity
was tested with Gal(b1 3 4)GlcNAc-Oct and, in the case of FT3, with
Gal(b1 3 3)GlcNAc-Oct acceptors. Since no low molecular weight substrate was available for assessment of FT7 activity, this enzyme was
measured using bovine fetuin as a substrate. A dash indicates incorporation of [14C]Fuc at background levels (,200 cpm corresponding to
,4.0 microunits 3 ml21 3 48 h21).
Cell line
FT3
FT4
FT5
FT6
FT7
Peripherally
fucosylated b-TP
oligosaccharides
Fucosyltransferase activity
Cellular extract
Culture medium
%
microunits 3
1026 cells
microunits 3
ml21 3 48 h21
19
46
34
52
31
22
54
1
55
3
80
20
—
125
—
only a triantennary oligosaccharide containing one type I
branch is modified with a1,4-linked Fuc, although an 8 times
higher type II acceptor concentration was present. Similarly,
we were unable to show sLex or Lex forming in vitro activity in
extracts of FT3 cells with low molecular weight type II oligosaccharide acceptors. This finding was confirmed in a recent
publication (21) describing the failure to in vitro fucosylate
diantennary type II oligosaccharides with large amounts of a
purified enzyme preparation. Almost no fucosylation of asialo
branches was observed in b-TP oligosaccharides from FT3 cells,
and using ESI-MS/MS technique, it was confirmed that only
the sLex and no sLea was present in the oligosaccharide preparations (data not shown).
As summarized in Fig. 6, the results of our in vivo specificity
FT3
FT4
FT5
FT6
FT7
2
—
—
20
3
—
59
13
3
Trace
3
17
9
21*
4
40
5
—
2
—
2
18
7
5
46
12
8
3
Trace
13
16
5
15
29
5
13
4
—
—
22
9**
—
43
11
11
study of rhu-a1,3/4-FTs indicate that each enzyme exhibits its
characteristic fucosylation pattern with the type II chains on
coexpressed human b-TP resulting in different sLex/Lex ratios:
FT7 (only sLex) . FT3 (14:1) . FT5 (3:1) . FT6 (1.1:1) . FT4
(1:7). From the results obtained, rhu-FT6 turns out to have a
high in vivo preference to form a1,3-difucosylated structures
with asialo, mono, and disialo diantennary acceptor oligosaccharides. A similar high preference for the synthesis of a1,3difucosylated diantennary glycans is also detectable for FT4,
however, only for the asialo structure (cf. Table IV). Apart from
its strict specificity toward a2,3-sialylated antennae, FT7 appears to have very similar preference for a1,3-mono- and a1,3di-Fuc transfer onto N-linked oligosaccharides, whereas FT3
and FT5 predominantly attach a single peripheral Fuc residue
to diantennary N-glycans. This is reflected in the decreased
amount of modified antennae compared with modified oligosaccharide chains (11% versus 19% for FT3 and 25% versus 34%
for FT5).
DISCUSSION
It is difficult to confidently assess the contribution of individual FTs in the biosynthesis of specific Lewis-type carbohydrate structures as ligands for selectins in natural cells or
tissues, since, based on immunochemical methods, in vitro
enzyme activity assays as well as on RNA analyses (10, 49 –52),
most natural tissues or cells express more than a single FT
species. This is even more difficult when considering the complex in vivo situation with different potential acceptor substrates that are presumably recognized in a different physicochemical environment inside the cell, e.g. glycolipids, N-, O-, or
N1O-glycosylated, membrane-anchored, or secreted glycoproteins, which furthermore might be present in variable amounts.
Although most of the studies published so far on the recombinant expression of human a1,3/4-FTs have focused on the
modification of acceptors on the surface of host cells (6 –10, 19,
47, 53–59) that have been discussed in context with selectin
binding studies, little is known about the specificity of human
a1,3/4-FTs in the recognition of polypeptide-linked N-glycans.
In the present report, we have been concentrating on the in
vivo functional activity and the in vivo substrate specificity of
the five different human a1,3/4-FTs toward the N-glycans of
coexpressed and secreted human b-TP. Human b-TP is almost
exclusively decorated with diantennary complex-type II N-glycans with proximal a1,6-linked Fuc when expressed from
BHK-21 cells (29), the ratio of a2,3-di-, monosialylated and
asialo structures being roughly 70:25:5. The protein from nat-
30992
In Vivo Specificity of Human a1,3/4-Fucosyltransferases
FIG. 5. ESI daughter ion mass spectrum of a reduced and permethylated
monosialylated difucosylated diantennary oligosaccharide isolated
from b-TP coexpressed in FT4 cells.
The monosaccharide composition of the
oligosaccharide was deduced from its molecular mass, and the fragment ions generated by collision induced dissociation
allowed its characterization as is explained in the fragmentation scheme. It
should be noted that any fucosylation of
the sialylated antennae of the diantennary structure can be excluded since the
expected fragment ions at m/z 1021
[NeuAc Hex dHex HexNAc 1 Na]1, 646
[HO-Hex dHex HexNAc 1 Na]1, and 486
[Hex HexNAc 1 Na]1 and the corresponding doubly charged ions generated by the
elimination of these fragments were not
detected. The linkage of the peripheral
Fuc to O-3 of a GlcNAc residue is indicated by the weak, but reproducible fragment ion due to the elimination of this
residue which would not be observed in
the case of a1,4-linked Fuc (see also under
“Experimental Procedures”).
FIG. 6. Biosynthesis of sLex and Lex on b-TP oligosaccharide
antennae by a1,3/4-FT-cotransfected BHK cells. The amount of
b-TP N-glycan antennae containing the sLex or the Lex motif was
calculated from the data shown in Table IV. Black bars, sLex; shaded
bars, Lex.
ural sources contains exclusively diantennary chains with
some peripheral Fuc (27, 28, 39), indicating that the glycoprotein is recognized by FT(s) in vivo; in contrast, rhu-b-TP from
BHK-21 cells lacks any Lex or sLex structures (29). This is in
agreement with our failure to detect any transfer of [14C]Fuc
onto the Gal(b134/3)GlcNAc-Oct acceptors from cellular extracts of wild-type BHK-21 cells.
The results shown here unequivocally demonstrate for the
first time that all five human a1,3/4-FTs have the capability to
form the sLex motif with complex-type II N-glycans. The in vivo
a1,3-fucosylation of b-TP that is secreted from cells stably
transfected with FT4-FT7 turned out to be highly efficient,
with roughly 30 –50% of b-TP N-glycans bearing at least one
peripheral Fuc. FT3 cells showed a significant lower fucosylation efficiency (see below). However, careful examination of the
oligosaccharide mixture obtained from b-TP that was secreted
by the individual cell line indicated that each FT exhibits its
own characteristic fucosylation pattern with type II chains of
rhu-b-TP resulting in different sLex/Lex ratios: FT7 . FT3 .
FT5 . FT6 . FT4. By HPLC/ESI-MS of tryptic b-TP glycopep-
tides from FT5-FT7 cells, we have detected an almost identical
pattern of the oligosaccharides attached at each of the two
N-glycosylation sites (data not shown). It is conceivable that
this is also the case for b-TP from FT4 and FT3 cells.
For FT7 cells, we detected peripheral Fuc exclusively in sLex
antennae, a result that is in perfect agreement with the published data on the in vitro specificity of FT7 toward a2,3sialylated type II low molecular weight acceptors (10, 11, 18).
Furthermore, the detection of exclusively sLex and no Lex motifs derived from FT7 cells indicates that the Lex structures
detected in other glycan preparations do not result from partial
desialylation of b-TP in the culture medium of cells or during
the one-step purification procedure employed, which enabled
us to quantitatively recover the recombinant protein from cell
supernatants.
According to published data, FT4 exhibits substantial enzyme activity only with asialo low molecular weight type II
acceptors (15, 19, 55). Our own results employing diantennary
asialo or a2,3-disialylated type II N-glycans along with sec-FT4
confirmed these findings.2 We found that 46% of the b-TP
glycans from FT4 cells are modified with peripheral Fuc predominantly in the Lex motif. However, small amounts of a1,3difucosylated monosialo as well as a1,3-monofucosylated disialo N-glycans were detected. To our knowledge, this is the
first report ascribing rhu-FT4 a role in formation of the sLex
epitope in vivo by carbohydrate structural analysis. However,
supporting the hypothesis that FT4 is preponderantly a Lexforming enzyme is our almost exclusive detection by ESIMS/MS of the Lex determinant in the a1,3-monofucosylated
monosialo structure.
Interestingly, the b-TP oligosaccharides from FT4 cells were
found to be undersialylated (52% sialylated versus 48% asialo
antennae), whereas in b-TP from wild-type BHK-21 cells, and
also from FT7 cells, about 80% of sialylated and 20% of asialo
antennae were detected. This observation is consistent with
previously published data (56), where the authors reported a
reduced sialylation of cell surface oligosaccharides of a single
CHO clone expressing FT4. However, these authors in contrast
to our findings did not detect any sLex but reported a small
amount of VIM-2 structures to be present.
FT5 cells secrete b-TP with N-glycans containing preponderantly the sLex motif. De Vries et al. (15) have reported that a
2
E. Grabenhorst, M. Nimtz, and H. S. Conradt, unpublished results.
In Vivo Specificity of Human a1,3/4-Fucosyltransferases
soluble form of FT5 from COS-7 cells had a high preference for
desialylated glycoprotein substrates as well as asialo low molecular weight acceptors in vitro. Weston et al. (7) reported a
similar substrate specificity of rhu-FT5 for N-acetyllactosamine and the a2,3-sialylated derivative in cellular extracts
and showed similar binding of Lex- and sLex-specific antibodies
to FT5-transfected COS-1 cells indicating similar recognition of
asialo and sialo structures as substrates for this enzyme. In our
expression studies, however, we found a high preference for the
forming of sLex structures (sLex:Lex about 3:1).
Although the in vivo functional activity of FT4, FT5, and FT7
can be described by their preference for either sialylated or
unsialylated acceptors, FT6 acts with no such a preference.
52% of oligosaccharides of secreted b-TP from FT6 cells contained one or two peripheral Fuc residues with an almost
identical ratio of sLex to Lex antennae (1.1:1). It should be
emphasized that the majority of oligosaccharides contained two
peripheral Fuc, which is not the case for the N-glycans synthesized by the other FT-transfected cells except for the a1,3difucosylated asialo structure formed by FT4. Similar to FT4
cells, the sialylation state of b-TP from FT6 cells is decreased
(65% sialylated and 35% asialo antennae). This could be interpreted by a competition between rhu-FT6 and the endogenous
a2,3-STs for the common Gal(b134)GlcNAc-R acceptor, and
the FT6 in vivo activity detected here should result from at
least some functional co-localization of the enzymes.
The detection of the sLex motif in b-TP glycans after coexpression of FT3 was unexpected since all our in vitro data
indicated the enzyme to act only on type I chains (22). Accordingly, no Fuc transfer onto the Gal(b134)GlcNAc-Oct could be
observed by cellular extracts of FT3 cells while the
Gal(b133)GlcNAc-Oct was an efficient substrate. Since the
BHK-21 cells do not synthesize any significant Gal(b133)GlcNAc-R chains (see Refs. 29 and 32), we cannot exclude that
FT3 would recognize this structure much more efficiently than
the type II chains. It is noteworthy that only 11% of the total
N-acetyllactosamine antennae of b-TP N-glycans from FT3
cells were substituted with peripheral Fuc, whereas with the
other FTs, 22–50% of the antennae were decorated with peripheral Fuc. This again would point to a functional role of
human FT3 to act as a Lea-forming enzyme in vivo and would
support our previous hypothesis.
We have reported large quantities of intracellularly proteolytically cleaved forms of human FT3 expressed from BHK-21
cells that are released into the cell supernatant (22). Similar
observations have been published, e.g. for b1,4-GalNAc-T (44),
a1,3-GalT (45), and FT6 (46). The enzymes responsible for this
proteolytical cleavage have been proposed to be cathepsin-like
proteases or serine proteases, respectively. Here we confirm
that rhu-FT6 is secreted by two different BHK-21 cell lines and
from CHO DHFR2 cells. Whereas FT7 and FT5 were found to
be resistant to proteolysis, we also detected secreted forms of
FT4 in supernatants of transfected cells.
Our results obtained with the coexpression of genetically
engineered soluble variants of FT6 (s-FT6, BT-FT6) together
with b-TP indicate that the secreted enzyme fraction does not
contribute to the in vivo activity, since only after about 20-fold
overexpression of s-FT6, we were able to detect small amounts
of fucosylated b-TP. In a recent paper (45), Cho and Cummings
found by lectin binding studies that a recombinant, soluble
a1,3-GalT (lacking the transmembrane and cytoplasmic domain) is functionally active in vivo when expressed at slightly
higher levels than the full-length form. The reason for this
discrepancy is unknown; however, that truncated forms of glycosyltransferases in general do not contribute significantly to
their in vivo specificity toward secreted glycoproteins is sup-
30993
ported by our finding that a recombinant soluble form of human ST6Gal does not modify co-secreted rhu-b-TP in BHK-21
cells.2
Interestingly, in addition to the very low fucosylation efficiency of the high enzyme activity expressing s-FT6(II) cell line,
the fucosylation pattern of b-TP glycans was also different with
a higher proportion of a1,3-monofucosylated structures observed over the a1,3-difucosylated glycans, which are the major
N-glycans expressed from cells transfected with full-length
FT6. This then supports the view of the importance of the CTS
region not only for the in vivo function of glycosyltransferases
but also for their in vivo specificity. In this context, it seems
attractive to speculate that the CTS region is also involved in
targeting of a1,3/4-FTs into different subcompartments of the
biosynthetic glycosylation pathway of cells. The CTS region
could be responsible for the localization of FT6 and FT4 to
subcompartments where they can compete with endogenous
ST3Gal III/IV for the same acceptor as is evident from the
lower sialylation state of the b-TP N-glycans secreted from the
transfected cells. This is based on the assumption that ST3Gal
III/IV do not act efficiently on Lex motifs (see discussion in
Refs. 10, 17, and 56). The targeting properties of the FT6 CTS
region should result in an intracellular broader distribution of
FT6 and its overlapping with ST3Gal III/IV. Likewise, the CTS
region should direct FT5 and FT7 into a later functional compartment than the BHK cell endogenous a2,3-STs, which must
provide the properly sialylated oligosaccharide precursor substrates. Since the FTs and STs should be localized in transGolgi/trans-Golgi network subcompartments (46, 60), we believe that it will be difficult to dissect such a spatial separation
by, e.g., immunolocalization techniques.
In our opinion, the approach used here for comparison of the
in vivo activities of the five human a1,3/4-FTs provides new
information about the involvement of the enzymes in the sequential biosynthesis of potential ligands for selectins. The
stable recombinant cell lines will also provide a convenient
source for the isolation of well characterized glycoprotein preparations to be used in in vitro binding or competition assays
with cells/tissues expressing selectins or specific Lewis-type
ligands.
Acknowledgments—We thank Gudrun Arnold, Christiane Kamp,
and Susanne Pohl for excellent technical assistance.
REFERENCES
1. Varki, A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7390 –7397
2. Lasky, L. A. (1995) Annu. Rev. Biochem. 64, 113–139
3. McEver, R. P., Moore, K. L., and Cummings, R. D. (1995) J. Biol. Chem. 270,
11025–11028
4. Kansas, G. S. (1996) Blood 88, 3259 –3287
5. Kukowska-Latallo, J. F., Larsen, R. D., Nair, R. P., and Lowe, J. B. (1990)
Genes Dev. 4, 1288 –1303
6. Goelz, S. E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B.,
Chi-Rosso, G., and Lobb, R. (1990) Cell 63, 1349 –1356
7. Weston, B. W., Nair, R. P., Larsen, R. D., and Lowe, J. B. (1992) J. Biol. Chem.
267, 4152– 4160
8. Koszdin, K. L., and Bowen, B. R. (1992) Biochem. Biophys. Res. Commun. 187,
152–157
9. Weston, B. W., Smith, P. L., Kelly, R. J., and Lowe, J. B. (1992) J. Biol. Chem.
267, 24575–24584
10. Sasaki, K., Kurata, K., Funayama, K., Nagata, M., Watanabe, E., Ohta, S.,
Hanai, N., and Nishi, T. (1994) J. Biol. Chem. 269, 14730 –14737
11. Natsuka, S., Gersten, K. M., Zenita, K., Kannagi, R., and Lowe, J. B. (1994)
J. Biol. Chem. 269, 16789 –16794
12. Sadler, J. E. (1984) in Biology of Carbohydrates (Ginsburg, V., Robbins, P. W.,
eds) Vol. 2, pp. 199 –288, John Wiley & Sons, New York
13. Snider, M. D. (1984) in Biology of Carbohydrates (Ginsburg, V., Robbins, P. W.,
eds), Vol. 2, pp. 163–198, John Wiley & Sons, New York
14. Kornfeld, R., and Kornfeld, S. (1985) Annu. Rev. Biochem. 54, 631– 664
15. de Vries, T., Srnka, C. A., Palcic, M. P., Swiedler, S. J., van den Eijnden, D. H.,
and Macher, B. A. (1995) J. Biol. Chem. 270, 8712– 8722
16. de Vries, T., Palcic, M. P., Schoenmakers, P. S., van den Eijnden, D. H., and
Joziasse, D. H. (1997) Glycobiology 7, 921–927
17. Shinoda, K., Morishita, Y., Sasaki, K., Matsuda, Y., Takahashi, I., and Nishi,
T. (1997) J. Biol. Chem. 272, 31992–31997
18. Britten, C. J., van den Eijnden, D. H., McDowell, W., Kelly, V. A., Witham,
S. J., Edbrooke, M. R., Bird, M. I., de Vries, T., and Smithers, N. (1998)
30994
In Vivo Specificity of Human a1,3/4-Fucosyltransferases
Glycobiology 8, 321–327
19. Kumar, R., Potvin, B., Muller, W. A., and Stanley, P. (1991) J. Biol. Chem. 266,
21777–21783
20. Scudder, P. R., Shailubhai, K., Duffin, K. L., Streeter, P. R., and Jacob, G. S.
(1994) Glycobiology 4, 929 –933
21. Nimtz, M., Grabenhorst, E., Gambert, U., Costa, J., Wray, V., Morr, M., Thiem,
J., and Conradt, H. S. (1998) Glycoconj. J., in press
22. Costa, J., Grabenhorst, E., Nimtz, M., and Conradt, H. S. (1997) J. Biol. Chem.
272, 11613–11621
23. de Graaf, T. W., van der Stelt, M. E., Anbergen, M. G., and van Dijk, W. (1993)
J. Exp. Med. 177, 657– 666
24. Brinkman-van der Linden, E. C., de Haan, P. F., Havenaar, E. C., and van
Dijk, W. (1998) Glycoconj. J. 15, 177–182
25. Walz, G., Aruffo, A., Kolanus, W., Bevilacqua, M., and Seed, B. (1990) Science
250, 1132–1135
26. Kimura, H., Shinya, N., Nishihara, S., Kaneko, M., Irimura, T., and
Narimatsu, H. (1997) Biochem. Biophys. Res. Commun. 237, 131–137
27. Hoffmann, A., Nimtz, M., Wurster, U., and Conradt, H. S. (1994)
J. Neurochem. 63, 2185–2196
28. Pohl, S., Hoffmann, A., Rüdiger, A., Nimtz, M., Jaeken, J., and Conradt, H. S.
(1997) Glycobiology 7, 1077- 1084
29. Grabenhorst, E., Hoffmann, A., Nimtz, M., Zettlmeissl, G., and Conradt, H. S.
(1995) Eur. J. Biochem. 232, 718 –725
30. Zettlmeissl, G., Conradt, H. S., Nimtz, M., and Karges, H. E. (1989) J. Biol.
Chem. 264, 21153–21159
31. Nimtz, M., Noll, G., Pâques, E.-P., and Conradt, H. S. (1990) FEBS Lett. 271,
14 –18
32. Nimtz, M., Martin, W., Wray, V., Klöppel, K.-D., Augustin, J., and Conradt,
H. S. (1993) Eur. J. Biochem. 213, 39 –56
33. Tsuji, S. (1996) J. Biochem. (Tokyo) 120, 1–13
34. Henke, W., Herdel, K., Jung, K., Schnorr, D., and Loening, S. A. (1997) Nucleic
Acids Res. 25, 3957–3958
35. Grabenhorst, E., Hofer, B., Nimtz, M., Jäger, V., and Conradt, H. S. (1993)
Eur. J. Biochem. 215, 189 –197
36. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY
37. Laemmli, U. K. (1970) Nature 227, 680 – 685
38. Hoffmann, A., Conradt, H. S., Gross, G., Nimtz, M., Lottspeich, F., and
Wurster, U. (1993) J. Neurochem. 61, 451– 456
39. Hoffmann, A., Nimtz, M., and Conradt, H. S. (1997) Glycobiology 7, 499 –506
40. Egge, H., and Katalinic, J. (1987) Mass Spectrom. Rev. 1987, 331–393
41. Hakomori, S. (1964) J. Biochem. (Tokyo) 55, 205–207
42. Masri, K. A., Appert, H. E., and Fukuda, M. N. (1988) Biochem. Biophys. Res.
Commun. 157, 657– 663
43. Homa, F. L., Hollander, T., Lehman D. J., Thomsen, D. R., and Elhammer,
A. P. (1993) J. Biol. Chem. 268, 12609 –12616
44. Jaskiewicz, E., Zhu, G., Bassi, R., Darling, D. S., and Young, W. W. Jr. (1996)
J. Biol. Chem. 271, 26395- 26403
45. Cho, S. K, and Cummings, R. D. (1997) J. Biol. Chem. 272, 13622–13628
46. Borsig, L., Katopodis, A. G., Bowen, B. R., and Berger, E. G. (1998)
Glycobiology 8, 259 –268
47. Lowe, J. B., Kukowska-Latallo, J. F., Nair, R. P., Larsen, R. D., Marks, R. M.,
Macher, B. A., Kelly, R. J., and Ernst, L. K. (1991) J. Biol. Chem. 266,
17467–17477
48. Gersten, K. M., Natsuka, S., Trinchera, M., Petryniak, B., Kelly, R. J.,
Hiraiwa, N., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., and Lowe, J. B.
(1995) J. Biol. Chem. 270, 25047–25056
49. Mollicone, R., Gibaud, A., François, A., Ratcliffe, M., and Oriol, R. (1990) Eur.
J. Biochem. 191, 169 –176
50. Cameron, H. S., Szczepaniak, D., and Weston, B. W. (1995) J. Biol. Chem. 270,
20112–20122
51. Robinson, N. E., de Vries, T., Davis, R. E., Stults, C. L. M., Watson, S. R., van
den Eijnden, D. H., and Macher, B. A. (1995) Glycobiology 4, 317–326
52. Clarke, J. L., and Watkins, W. M. (1996) J. Biol. Chem. 271, 10317–10328
53. Lowe, J. B., Stoolman, L. M., Nair, R. P., Larsen, R. D., Berhend, T. L., and
Marks, R. M. (1990) Cell 63, 475- 484
54. Meier, W., Leone, D. R., Miatkowski, K., Lobb, R., and Goelz, S. E. (1993)
Biochem. J. 294, 25–30
55. Goelz, S., Kumar, R., Potvin, B., Sundaram, S., Brickelmaier, M., and Stanley,
P. (1994) J. Biol. Chem. 269, 1033–1040
56. Sueyoshi, S., Tsuboi, S., Sawada-Hirai, R., Dang, U. N., Lowe, J. B., and
Fukuda, M. (1994) J. Biol. Chem. 269, 32342–32350
57. Knibbs, R. N., Craig, R. A., Natsuka, S., Chang, A., Cameron, M., Lowe, J. B.,
and Stoolman, L. M. (1996) J. Cell Biol. 133, 911–920
58. Li, F., Wilkins, P. P., Crawley, S., Weinstein, J., Cummings, R. D., and
McEver, R. P. (1996) J. Biol. Chem. 271, 3255–3264
59. Snapp, K. R., Wagers, A. J., Craig, R., Stoolman, L. M., and Kansas, G. S.
(1997) Blood 89, 896 –901
60. Burger, P. C., Lötscher, M., Streiff, M., Kleene, R., Kaissling, B., and Berger,
E. G. (1998) Glycobiology 8, 245–257