Identification of a Major Carbohydrate Capping Group of the
L-selectin Ligand on High Endothelial Venules in Human Lymph Nodes
as 6-Sulfo Sialyl Lewis X*
Chikako
Mitsuoka
,
Mikiko
Sawada-Kasugai
,
Keiko
Ando-Furui
,
Mineko
Izawa
,
Hayao
Nakanishi§,
Shigeo
Nakamura¶,
Hideharu
Ishida
,
Makoto
Kiso
, and
Reiji
Kannagi
**
From the
Program of Experimental Pathology,
§ Laboratory of Pathology, and ¶ Laboratory for
Clinical Pathology, Aichi Cancer Center, Nagoya 464 and the
Department of Applied Bio-organic Chemistry, Faculty of
Agriculture, Gifu University, Gifu 501-11, Japan
 |
ABSTRACT |
We investigated the molecular species of sulfated
sialyl Lewis X determinants, the putative L-selectin ligand, expressed
on high endothelial venules (HEV) in human lymph nodes. Comparison of
the reactivity pattern of HEV with the reactivity of the pure 6-sulfo,
6'-sulfo, or 6,6'-bissulfo sialyl Lewis X determinant with hitherto
known anti-sialyl Lewis X antibodies strongly suggested 6-sulfo sialyl
Lewis X to be the best candidate for the major sulfated sialyl Lewis X
determinant on HEV, followed by 6,6'-bissulfo sialyl Lewis X, whereas
6'-sulfo sialyl Lewis X was unlikely. We newly generated monoclonal
antibodies (mAbs) G152 and G72 directed against 6-sulfo sialyl Lewis X,
which intensely labeled HEV in immunohistochemical examination and
inhibited binding of recombinant L-selectin-IgG to HEV, suggesting that
the determinant serves as a ligand for L-selectin. To test the
concomitant expression of 6,6'-bissulfo sialyl Lewis X, specific mAbs
(G2706, G27011, G27037, and G27039) were generated, but all antibodies
failed to react to HEV. Next, we established mAbs (AG97 and AG273)
directed against 6-sulfo Lewis X, the asialo form of 6-sulfo sialyl
Lewis X. The antibodies were not reactive to untreated HEV, but
strongly reacted to sialidase-treated HEV. This indicated the
predominance of the sialylated form of 6-sulfo sialyl Lewis X and
minimal expression of its asialo form, corroborating that it was
synthesized by fucosyltransferase VII, the isoenzyme that
preferentially produces the sialylated form of the determinant.
 |
INTRODUCTION |
L-selectin (leukocyte endothelial cell adhesion molecule-1,
leukocyte adhesion molecule-1, Mel-14 antigen) is expressed on human
peripheral lymphocytes and is proposed to be involved in the homing of
lymphocytes into peripheral lymph nodes (1-3). The high endothelial
venules (HEV)1 in peripheral
lymph nodes are thought to express a carbohydrate ligand that binds to
L-selectin. However, the nature of the L-selectin ligand expressed at
the surface of HEV endothelial cells is not yet clear. Some sulfated
carbohydrate determinants are known to bind to L-selectin or inhibit
L-selectin-mediated cell adhesion. On the other hand, it was suggested
that sialyl Lewis X and sialyl Lewis A have a significant capacity to
bind L-selectin, despite the lack of a sulfate residue in their
carbohydrate structures (4, 5), right after they had first been shown
to serve as ligands for E-selectin (6-10). However, most of the known
anti-sialyl Lewis X antibodies as well as anti-sialyl Lewis A
antibodies did not react efficiently to HEV endothelial cells. In an
earlier report, we showed that one of the anti-sialyl Lewis X
antibodies (2H5) specifically detected a sialyl Lewis X-like
determinant on human HEV, and the antigenic determinant served as a
ligand for L-selectin since the antibody inhibited the binding of
lymphoid cells to human HEV endothelial cells in Stamper-Woodruff
assays (11). We suggested that the antigenic determinant detected by the antibody was closely related, but not exactly identical, to genuine
sialyl Lewis X and called it a complex-type sialyl Lewis X determinant,
which was defined by the 2H5 antibody at that time (11).
In studying the specificity of the known anti-sialyl Lewis X
antibodies, we noticed that they can be classified into two groups according to their cross-reactivity to sialyl Lewis X-related structures. One group consists of antibodies that recognize mainly sialic acid and fucose residues, but do not recognize the type 2 chain
core Gal
1
4GlcNAc
, whereas the other group contains antibodies
that unerringly recognize sialic acid residues and type 2 chain core
Gal
1
4GlcNAc
structure, but only weakly recognize fucose
residues (12). The anti-sialyl Lewis X antibodies that reacted to HEV
were all found to belong to the former group, and the antibodies in the
latter group lacked reactivity to HEV endothelial cells. These findings
led us to assume that the complex sialyl Lewis X on HEV could be a
sialyl Lewis X determinant that was somehow modified at the type 2 chain core structure. This modification would not affect the reactivity
of the antibodies in the former group, but would seriously affect the
reactivity of the antibodies in the latter group. The sulfated sialyl
Lewis X determinants, proposed by Hemmerich et al. (13, 14)
to be expressed on HEV in murine lymph nodes, were the sialyl Lewis X
determinants modified by sulfate residues at the type 2 chain core
structure. We thought one of these might be the complex sialyl Lewis X
determinant that had been detected by our antibody on human HEV
endothelial cells.
In a previous study, we investigated the reactivity of a few
anti-sialyl Lewis X antibodies against human HEV and compared it with
the reactivity pattern of the antibodies against a series of pure
sulfated sialyl Lewis X determinants (15). Our unexpected conclusion
was that the best candidate for the molecular species carried by HEV is
6-sulfo sialyl Lewis X (15), a conclusion quite different from those of
preceding researchers, who suggested the 6'-sulfo sialyl Lewis X
species to be a major determinant (13, 14). In this paper, we expanded
our studies and generated a series of new monoclonal antibodies
directed against 6-sulfo sialyl Lewis X, 6,6'-bissulfo sialyl Lewis X,
and 6-sulfo Lewis X. By using these new reagents, we tried to establish
6-sulfo sialyl Lewis X as the major sialyl Lewis X-like carbohydrate
determinant carried by HEV endothelial cells, and the ligand carrying
this capping structure serves as a ligand for L-selectin in human
HEV.
 |
EXPERIMENTAL PROCEDURES |
Monoclonal Antibodies Directed against Sialyl Lewis X-related
Carbohydrate Determinants--
The anti-sialyl Lewis X antibodies 2H5
(established against a mixture of complex sialyl Lewis X-active
determinants prepared from human cancer cells) and 2F3 (established
against the synthetic sialyl Lewis X glycolipid) were prepared as
described previously (11, 16). The antibody HECA-452 was a kind gift
from Dr. T. K. Kishimoto (Department of Pathology, Stanford
University School of Medicine, Stanford, CA) (17). SNH-3 and FH-6 were
gifts from Dr. S. Hakomori (Pacific Northwest Research Foundation,
Seattle, WA) (7, 18, 19). CSLEX-1 was obtained from American Type Culture Collection (Bethesda, MD) (20). The anti-Lewis X antibody LeuM1
was purchased from Becton Dickinson Labware (Franklin Lakes, NJ); FH-2
was also a gift from Dr. S. Hakomori; and 73-30 and Tx-17 were
established in our laboratory. The antibody SU59 directed against
3'-sulfo Lewis X was a gift from Dr. Akihiro Hino (Nisshin Shokuhin Co.
Ltd., Otsu, Japan). These antibodies are all murine IgM, except for
HECA-452, which is rat IgM.
New hybridoma cells that secrete monoclonal antibodies directed against
sulfated sialyl Lewis X and sulfated Lewis X determinants were
generated according to the method described by Köhler and Milstein (21) and subsequently adopted for anti-carbohydrate antibodies
(22). Briefly, the synthetic 6-sulfo sialyl Lewis X, 6-sulfo Lewis X,
or 6,6'-bissulfo sialyl Lewis X glycolipid was adsorbed to
Salmonella minnesota strain R595 and used for repeated
intraperitoneal immunizations of BALB/c mice on day 0 (5 µg of
glycolipid), day 3 (10 µg), day 7 (15 µg), day 12 (20 µg), day 17 (25 µg), and day 31 (35 µg). Three days after the final
immunization, the spleen cells were harvested and fused with mouse
myeloma P3/X63-Ag8U1. The same glycolipid was used as the antigen in
solid-phase enzyme immunoassays of hybridoma culture supernatants for
the cloning procedures. Two hybridoma clones (G152 and G72) were
obtained by immunization of the 6-sulfo sialyl Lewis X glycolipid, and
four hybridoma clones (G2706, G27011, G27037, and G27039) were obtained
by immunization of the 6,6'-bissulfo sialyl Lewis X glycolipid. The
antibodies AG97 and AG273 were obtained by immunization of the 6-sulfo
Lewis X glycolipid. Monoclonal antibodies obtained in this study were
all of the IgM class.
Preparation of Carbohydrate Determinants, ELISA, and TLC
Immunostaining--
ELISA was performed using glycolipid antigens
immobilized at the bottom of 96-well culture plates by a standard
method described previously (23). Peroxidase-conjugated rabbit anti-rat
IgM (µ-chain-specific; Zymed Laboratories Inc.,
South San Francisco, CA) was used for HECA-452, and
peroxidase-conjugated goat anti-mouse IgM (µ-chain-specific; Cappel
Inc., Malvern, PA) was used for the other antibodies. TLC immunostaining was performed according to the method described previously (23, 24), except that peroxidase-conjugated antibody was
used as the second antibody, and ECL Western blotting detection reagents (Amersham International, Buckinghamshire, United Kingdom) were
used to detect positive reactions instead of 125I-protein
A.
The pure synthetic sialyl Lewis X glycolipid and the synthetic sulfated
glycolipids (6-sulfo sialyl Lewis X, 6'-sulfo sialyl Lewis X,
6,6'-bissulfo sialyl Lewis X, and and 3'-sulfo Lewis X) used in this
study were synthesized as described previously (25-27). For the
structures of these glycolipids, see Table
I. The asialo compounds (6-sulfo Lewis X,
6'-sulfo Lewis X, and 6,6'-bissulfo Lewis X) were prepared by sialidase
treatment of the corresponding synthetic glycolipids using sialidase
from Arthrobacter ureafaciens (Nakarai Chemicals Co. Ltd.,
Kyoto, Japan).
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Table I
Structures of carbohydrate determinants used in this study and summary
of reactivity of monoclonal antibodies
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The oligosaccharide SL2L4 was a generous gift from Dr. Keiichi Yoshida
(Seikagaku Kogyo, Tokyo, Japan) and was prepared from shark cartilage
keratan sulfate by digestion with keratanase II (Bacillus
sp.; Seikagaku Kogyo), followed by anion-exchange chromatography and
gel filtration. The oligosaccharide was coupled to cholesterylaniline through reductive amination (16) by adding the aqueous solution of the
oligosaccharide (200 µl of 5 mg/ml) to 2 ml of cholesterylaniline in
pyridine (180 mg/ml) and incubating for 1 h at 80 °C in the presence of 1.0 mM AlCl3, followed by
additional incubation for 1 h at 80 °C after adding 300 mg of
dimethylamine-borane and 1 ml of acetic acid. AlCl3 was
removed by Dowex 50W-X8 (100-200 mesh, H+ form) column
chromatography from the product SL2L4-cholesterylaniline. Cholesterylaniline had been synthesized by adding 7.2 g of
1,4-p-phenylenediamine (Sigma) in 54 ml of Me2SO
supplemented with 320 µl of pyridine to 1.8 g of cholesteryl
chloroformate (Sigma) in 100 ml of 1,2-dichloroethane and incubating
for 1 h at room temperature. Cholesterylaniline was recovered from
the lower layer after the addition of 46 ml of methanol, 100 ml of
1,2-dichloroethane, and 200 of ml H2O.
Immunohistochemical Examination and Binding Assay of
L-selectin-IgG Chimera on Frozen Lymph Node Sections--
Consecutive
frozen sections of 10-µm thickness were prepared from human axillary
and mesenteric lymph nodes and other tissues and organs for
immunohistochemical examination using anti-sialyl Lewis X and newly
generated anti-sulfo sialyl Lewis X antibodies. The avidin-biotin
complex technique for immunohistochemical study was performed as
described in the instructions for the kits (Vectastain) provided by
Vector Labs, Inc. (Burlingame, CA). When indicated, the sections were
pretreated with sialidase from A. ureafaciens.
For the binding assay of recombinant L-selectin-IgG chimera, frozen
sections prepared from freshly obtained human lymph nodes were fixed
with 0.5% paraformaldehyde at 4 °C for 10 min. The sections were
pretreated for 30 min at room temperature with phosphate-buffered saline containing 20% goat serum. To this, a mixture (1:1:1, v/v/v) of
the recombinant human L-selectin-IgG chimera (20 µg/ml; kindly provided by Drs. H. Kondo and Y. Inoue, New Drug Research Labs, Kanebo
Ltd., Osaka, Japan), affinity-purified biotinylated goat anti-human IgG
antibody (30 µg/ml), and peroxidase-labeled avidin (30 µg/ml) was
overlaid and incubated for 60 min at 4 °C. The reaction was
visualized with 3,3'-diaminobenzidine tetrahydrochloride, followed by
after-staining with 1% methyl green. For the inhibition study,
sections were incubated with anti-carbohydrate antibodies (50-100
µg/ml) for 120 min at 4 °C prior to the addition of the recombinant human L-selectin-IgG chimera/peroxidase mixture.
Adhesion of L-selectin-transfected Cells to 6-Sulfo Sialyl Lewis
X--
A 96-well plate was coated with 6-sulfo sialyl Lewis X by
adding ethanol solution containing glycolipid (200 ng/well), egg yolk
phosphatidylcholine (1 µg/well), and cholesterol (500 ng/well), followed by air-drying at 37 °C. After blocking with 5% bovine serum albumin in phosphate-buffered saline overnight at 4 °C, the
plate was incubated with appropriate inhibitor antibodies (40 µg/well) for 30 min at room temperature. BCECF-AM (Molecular Probes,
Inc., Eugene, OR)-labeled HULT-7 cells (2 × 105/well), the human L-selectin-transfected cells (15),
were added and incubated for 60 min at 37 °C. The number of adherent
cells was estimated by measuring fluorescence at 490 nm excitation and 530 nm emission on a fluorometer (Fluoroscan II, Labosystems, Tokyo).
 |
RESULTS |
Detection of the Sialyl Lewis X-like Determinant on HEV Endothelial
Cells Using Anti-sialyl Lewis X and Anti-Lewis X
Antibodies--
Endothelial cells of HEV in human peripheral lymph
nodes were reactive to only a part of anti-sialyl Lewis X antibodies as shown in Fig. 1 (a-f). The
antibodies 2F3 and HECA-452 were strongly reactive to the HEV
endothelial cells as well as the 2H5 antibody as reported previously
(11). The same pattern of staining was observed consistently with 14 different lymph node samples prepared from different individuals. This
indicated that there are two groups of anti-sialyl Lewis X
antibodies: one group (2F3, 2H5, and HECA-452) of antibodies that stain
HEV endothelial cells and another group (SNH-3, FH-6, and CSLEX-1) that
are unable to do so. Reactivity of the 2F3, 2H5, and HECA-452
antibodies was eliminated when the sections were pretreated with
sialidase (data not shown). These results suggest that the carbohydrate
determinant(s) carried by HEV endothelial cells have sialylated
structures closely related to sialyl Lewis X, but not genuine sialyl
Lewis X, which would have been readily detected by all six anti-sialyl
Lewis X antibodies.

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Fig. 1.
Immunohistochemical detection of sialyl Lewis
X-like and Lewis X-like determinants on human HEV endothelial cells
using conventional anti-sialyl Lewis X and anti-Lewis X
antibodies. In a-f, immunohistochemical staining
patterns of six anti-sialyl Lewis X antibodies are shown. The
antibodies used were CSLEX-1 in a, SNH-3 in b,
FH-6 in c, 2F3 in d, 2H5 in e, and
HECA-452 in f. In g and h,
immunohistochemical staining patterns of two anti-Lewis X antibodies
are shown. The antibodies used were 73-30 in g and Tx-17 in
h. Similar results as in g and h were
obtained also with the anti-Lewis X antibodies LeuM1 and FH-2 (data not
shown).
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HEV endothelial cells were not reactive to four anti-Lewis X
antibodies, including LeuM1, FH-2, 73-30 and Tx-17 (in Fig. 1 (g and h), the results obtained with 73-30 and
Tx-17 were omitted since they were essentially the same as those
obtained with FH-2 and LeuM1), before and after sialidase treatment.
The only cells positively stained by all four antibodies were
intravascular granulocytes. Staining of monocytes was prominent only
with LeuM1. Upon sialidase treatment, the genuine sialyl Lewis X
determinant ordinarily yields the Lewis X determinant, which is readily
detected by these conventional anti-Lewis X antibodies. This finding
again confirmed that the sialyl Lewis X-like determinant on HEV
was not genuine sialyl Lewis X. The anti-3'-sulfo Lewis X
antibody SU59 failed to stain HEV endothelial cells before and
after sialidase treatment.
Reactivity of Anti-sialyl Lewis X Antibodies to Synthetic Sulfated
Sialyl Lewis X Determinants and Other Related Compounds--
The six
anti-sialyl Lewis X antibodies reacted equally well to the genuine
sialyl Lewis X glycolipid in ELISA (Fig.
2a), and we thought the
difference in the reactivity of anti-sialyl Lewis X antibodies
against HEV endothelial cells stemmed from the different cross-reactivity of the antibodies. To determine whether the
determinants detected by these anti-sialyl Lewis X antibodies were
sulfated sialyl Lewis X determinants, which Hemmerich et al.
(13, 14) proposed were carried by GlyCAM-1 in murine lymph nodes, we
tested the reactivity of the six anti-sialyl Lewis X antibodies against synthetic sulfo sialyl Lewis X determinants. As shown in Fig. 2b, the sialyl Lewis X sulfated at
-Gal (6'-sulfo
sialyl Lewis X) was reactive to an unexpectedly wide variety of
antibodies, including CSLEX-1, SNH-3, 2F3, 2H5, and HECA-452. Only the
antibody FH-6 lost its binding activity upon 6-sulfation at the
-Gal
moiety of the sialyl Lewis X structure.

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Fig. 2.
Reactivity of anti-sialyl Lewis X and
anti-Lewis X antibodies against sulfated carbohydrate determinants as
ascertained by ELISA. a-d show the reactivity of six
anti-sialyl Lewis X antibodies against sialyl Lewis X
(a), 6'-sulfo sialyl Lewis X (b), 6-sulfo
sialyl Lewis X (c), or 6,6'-bissulfo sialyl Lewis X
(d). In a-d, open symbols ( ,
CSLEX-1; , SNH-3; , FH-6) indicate the results obtained with the
antibodies that are not reactive to human HEV endothelial cells, and
closed symbols ( , 2F3; , 2H5; , HECA-452) indicate
those obtained with the antibodies reactive to human HEV endothelial
cells. e-h show the reactivity of four anti-Lewis X
antibodies ( , FH-2; , Tx-17; , LeuM1; , 73-30) against
Lewis X (e), 6'-sulfo Lewis X (f), 6-sulfo Lewis
X (g), or 6,6'-bissulfo Lewis X (h). Serial
dilution of immobilized carbohydrate determinants started from 40 ng/well.
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On the other hand, sialyl Lewis X sulfated at the
-GlcNAc moiety
(6-sulfo sialyl Lewis X) was detected by antibodies 2F3, 2H5, and
HECA-452, but not by antibodies CSLEX-1, SNH-3, and FH-6 (Fig.
2c). This reaction profile was exactly the same as that of
human HEV endothelial cells against these six antibodies, as indicated
in Fig. 1, and suggested that 6-sulfo sialyl Lewis X would be a good
candidate for the sialyl Lewis X-like determinant expressed on HEV
endothelial cells.
The reactivity of disulfated sialyl Lewis X (6,6'-bissulfo sialyl Lewis
X) was essentially the same as that of GlcNAc-sulfated sialyl Lewis X
(6-sulfo sialyl Lewis X). The antibodies 2F3, 2H5, and HECA-452 were
reactive to 6,6'-bissulfo sialyl Lewis X, whereas FH-6 and CSLEX-1 were
not (Fig. 2d). This led us to assume that 6,6'-bissulfo
sialyl Lewis X is another good candidate for the determinant carried by
human HEV endothelial cells. The only difference between 6-sulfo sialyl
Lewis X and 6,6'-bissulfo sialyl Lewis X was that the latter
determinant was weakly reactive to SNH-3.
Next, we tested the reactivity of four anti-Lewis X antibodies,
all of which recognize the Lewis X determinant (Fig. 2e), against the asialo derivatives of the above sulfated sialyl Lewis X
determinants (Fig. 2, f-h). Three out of the four
anti-Lewis X antibodies were found to react to 6'-sulfo Lewis X (Fig.
2f), whereas all of them failed to react to 6-sulfo Lewis X
(Fig. 2g). Since sialidase-treated HEV endothelial cells
were not stained with any of the anti-Lewis X antibodies in
immunohistochemical examination, these findings again suggest that
6-sulfo sialyl Lewis X is actually the sialyl Lewis X-like determinant
expressed on HEV endothelial cells, but 6'-sulfo sialyl Lewis X is not. Only one anti-Lewis X antibody (FH-2) showed a weak cross-reactivity, and the others were not reactive to 6,6'-bissulfo Lewis X (Fig. 2h). None of the 10 antibodies tested was reactive to the
3'-sulfo Lewis X determinant, which had been reported to be present in mucin side chains of human meconium (28) and later found to bind to
recombinant L-selectin (27).
Generation of Monoclonal Antibodies Specific to 6-Sulfo Sialyl
Lewis X--
Since analysis of the reactivity pattern of known
anti-sialyl Lewis X and anti-Lewis X antibodies strongly suggested
6-sulfo sialyl Lewis X is most probably the sulfated sialyl Lewis X
determinant carried by HEV endothelial cells, we generated two
monoclonal antibodies (G152 and G72) by immunizing synthetic 6-sulfo
sialyl Lewis X. The specificity of these new antibodies against
pure carbohydrate determinants in ELISA is shown in Fig.
3. Both antibodies were strongly reactive
to 6-sulfo sialyl Lewis X, but were not reactive to other related
structures, including genuine sialyl Lewis X and 6'-sulfo
sialyl Lewis X, and were only slightly cross-reactive to
6,6'-bissulfo sialyl Lewis X in ELISA (Fig. 3). They did not react to
asialo derivatives such as 6-sulfo Lewis X, 6'-sulfo Lewis X,
6,6'-bissulfo Lewis X, and 3'-sulfo Lewis X. G152 was not at all
reactive, whereas G72 showed significant reactivity against the SL2L4
determinant, which does not contain a fucose residue (Fig. 3).

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Fig. 3.
Specificity of newly generated anti-6-sulfo
sialyl Lewis X antibodies as ascertained by ELISA. a and
b show reactivity of the G152 and G72 antibodies,
respectively, against carbohydrate determinants ( , 6-sulfo sialyl
Lewis X; , 6'-sulfo sialyl Lewis X; , 6,6'-bissulfo sialyl Lewis
X; , 6-sulfo Lewis X; , SL2L4-cholesterylaniline; , sialyl
Lewis X, 6'-sulfo Lewis X, 6,6'-bissulfo Lewis X, or 3'-sulfo Lewis X).
Serial dilution of immobilized carbohydrate determinants started from
40 ng/well. For structures of the carbohydrate determinants, see Table
I.
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The results of TLC immunostaining (Fig.
4) also confirmed the reactivity of the
two antibodies, indicating that G152 detects only 6-sulfo sialyl Lewis
X, whereas G72 detects 6-sulfo sialyl Lewis X and SL2L4.
Cross-reactivity to 6,6'-bissulfo sialyl Lewis X was not detected by
TLC immunostaining (data not shown), suggesting that this was only a
weak and negligible cross-reactivity.

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Fig. 4.
Specificity of newly generated anti-6-sulfo
sialyl Lewis X antibodies as ascertained by TLC immunostaining.
a shows the results of orcinol/H2SO4
staining, which visualizes all glycolipids. b and
c show the immunostaining patterns of the same TLC plates
with G152 and G72 antibodies, respectively. Lane R,
reference laminari-oligosaccharides (a mixture of
laminaribiose-laminariheptaose; Seikagaku Kogyo) coupled to
cholesterylaniline serving as TLC mobility controls; lane 1,
sialyl Lewis X; lane 2, 6-sulfo sialyl Lewis X; lane
3, 6-sulfo Lewis X; lane 4, SL2L4-cholesterylaniline.
For structures of the carbohydrate determinants, see Table I.
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These results indicate that the G152 antibody is highly specific to
6-sulfo sialyl Lewis X; it requires the presence of sialic acid and
fucose residues and 6-sulfo modification at GlcNAc in the antigenic
determinant for binding. The specificity of the G72 antibody was very
similar to the specificity of G152, except that recognition of the
fucose residue by this antibody seemed weaker than that by
G152.
Characterization of the 6-Sulfo Sialyl Lewis X Determinant on HEV
Endothelial Cells--
Both G152 and G72 antibodies strongly stained
HEV endothelial cells in human peripheral lymph nodes in
immunohistochemical examination (Fig. 5).
Frozen sections had to be used in the immunohistochemical detection of
the antigen by these two antibodies, and virtually no positive staining
was obtained when Formalin-fixed sections were used. As summarized in
Table II, these antibodies stained HEV
endothelial cells in peripheral lymph nodes, tonsils, Peyer's patches,
and appendices. The staining of HEV in Peyer's patches and appendices
was significantly weaker than in lymph nodes and tonsils. No blood
vessels in other organs including the spleen, thymus, gastrointestinal
tract, lung, and kidney were stained by the two antibodies, which is in
line with the distribution of L-selectin ligands. This distribution of
the antigen also coincides with that detected by the 2H5 antibody as
reported earlier (11).

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Fig. 5.
Immunohistochemical detection of 6-sulfo
sialyl Lewis X on HEV endothelial cells using newly generated
monoclonal antibodies. a (low-power field) and b
(high-power field) show immunohistochemical staining of HEV endothelial
cells in human peripheral lymph nodes with anti-6-sulfo sialyl Lewis X
antibody (G152). c shows staining of a human tonsillar
lymphoid tissue with another anti-6-sulfo sialyl Lewis X antibody
(G72). d shows G152 staining of Peyer's patches, showing a
weaker expression of 6-sulfo sialyl Lewis X compared with HEV in
peripheral lymph nodes. In d, arrows indicate
antigen-positive HEV, and arrowheads indicate
antigen-negative HEV.
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Table II
Summary of reactivity of anti-6-sulfo sialyl Lewis X and
anti-sialyl Lewis X antibodies against endothelial cells in various
human organs and tissues
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A human lymphoid leukemia cell line transfected with human L-selectin
(HULT-7 cells) underwent significant adhesion to a 96-well plate coated
with the synthetic 6-sulfo sialyl Lewis X glycolipid (Fig.
6a). The HULT-7 cells adhered
also to 6'-sulfo sialyl Lewis X and 6,6'-bissulfo sialyl Lewis X as
well as to genuine sialyl Lewis X. A 50-60% increase in binding was
observed with 6-sulfo sialyl Lewis X and 6,6'-bissulfo sialyl Lewis X
compared with the binding to genuine sialyl Lewis X. Adhesion to the
6-sulfo sialyl Lewis X glycolipid was strongly inhibited by the
addition of the G152 or G72 antibody in vitro, as shown in
Fig. 6b. The antibody CSLEX-1, which does not have a binding
activity against the carbohydrate determinant, failed to inhibit the
adhesion.

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Fig. 6.
Inhibition of adhesion of
L-selectin-transfected human lymphoid cells to 6-sulfo sialyl Lewis
X-coated plates by the G152 and G72 antibodies. a shows
adhesion of BCECF-AM-labeled HULT-7 cells expressing human L-selectin
to 96-well plates coated with the synthetic genuine sialyl Lewis X,
6'-sulfo sialyl Lewis X, 6-sulfo sialyl Lewis X, or 6,6'-bissulfo
sialyl Lewis X glycolipid. In b, adhesion of
BCECF-AM-labeled HULT-7 cells to 96-well plates coated with 6-sulfo
sialyl Lewis X was almost completely inhibited by the presence of the
G152 or G72 antibody. Adherent cells are expressed as a percentage of
the number of cells added to the wells. Statistical significance was
calculated according to Student's t test. For experimental
details, see "Experimental Procedures." N.S., not
significant.
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The recombinant human L-selectin-IgG chimera bound to HEV endothelial
cells in frozen sections prepared from human lymph nodes (Fig.
7a), and this was inhibited by
sialidase treatment of frozen sections (Fig. 7b), indicating
that a sialic acid-containing carbohydrate determinant is involved in
the binding. The G152 and G72 antibodies also clearly inhibited the
binding of recombinant human L-selectin-IgG chimera to HEV endothelial
cells (Fig. 7, c and d). Although the possibility
of steric hindrance cannot be fully excluded, these results strongly
suggest that 6-sulfo sialyl Lewis X constitutes a major carbohydrate
capping group of the L-selectin ligand on human HEV endothelial cells.
The antibody 2H5, the anti-sialyl Lewis X antibody having a heavy
cross-reactivity to 6-sulfo sialyl Lewis X, also inhibited the binding
(Fig. 7e), whereas CSLEX-1, the anti-sialyl Lewis X antibody
with no reactivity to 6-sulfo sialyl Lewis X, did not (Fig.
7f).

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Fig. 7.
Inhibition of binding of the recombinant
human L-selectin-IgG chimera to HEV endothelial cells in peripheral
lymph nodes by anti-6-sulfo sialyl Lewis X antibodies. Binding of
the recombinant human L-selectin-IgG chimera to HEV endothelial cells
was studied using frozen sections of human peripheral lymph nodes.
a shows that the recombinant human L-selectin-IgG chimera
significantly bound to HEV endothelial cells, which was eliminated when
the sections were sialidase-treated as in b. c
and d show that the binding was almost completely inhibited
when the sections were pretreated with the anti-6-sulfo sialyl Lewis X
antibodies G152 and G72, respectively. A similar inhibition of binding
was observed when the sections were pretreated with the 2H5 antibody
(e), but not with CSLEX-1 (f). For experimental
details, see "Experimental Procedures."
|
|
Detection of 6,6'-Bissulfo sialyl Lewis X Using Newly Generated
Specific Monoclonal Antibodies--
Next, we generated antibodies
directed against 6,6'-bissulfo sialyl Lewis X since the determinant
seemed to be another candidate for the sulfated L-selectin ligand
expressed in human lymph nodes. Four monoclonal antibodies (G2706,
G27011, G27037, and G27039) were obtained by the immunization of the
synthetic 6,6'-bissulfo sialyl Lewis X glycolipid. These antibodies
showed strong reactivity to the immunogen 6,6'-bissulfo sialyl Lewis X
and were moderately cross-reactive to its desialylated structure,
6,6'-bissulfo Lewis X, in ELISA (Fig. 8,
a-d). None of these antibodies reacted to 6-sulfo sialyl
Lewis X, genuine sialyl Lewis X, or other related structures, including
SL2L4-cholesterylaniline, but some of them showed a weak
cross-reactivity to 6'-sulfo sialyl Lewis X.

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Fig. 8.
Specificity of newly generated 6,6'-bissulfo
sialyl Lewis X and anti-6-sulfo Lewis X antibodies as ascertained by
ELISA. a-d show reactivity of the anti-6,6'-bissulfo sialyl
Lewis X antibodies G2706, G27011, G27037, and G27039, respectively, and
e and f show reactivity of the anti-6-sulfo Lewis
X antibodies AG97 and AG273, respectively, against carbohydrate
determinants ( , 6-sulfo sialyl Lewis X; , 6'-sulfo sialyl Lewis
X; , 6,6'-bissulfo sialyl Lewis X; , 6-sulfo Lewis X; ,
6'-sulfo Lewis X; , 6,6'-bissulfo Lewis X; , genuine sialyl Lewis
X, Lewis X, or 3'-sulfo Lewis X). Serial dilution of immobilized
carbohydrate determinants started from 40 ng/well. For structures of
the carbohydrate determinants, see Table I.
|
|
In TLC immunostaining, the antibodies appeared to be quite specific to
6,6'-bissulfo sialyl Lewis X (Fig.
9a), and cross-reactivity to
other structures weakly detected by ELISA did not show up on TLC
immunostaining. For reasons yet unknown, the G27037 antibody showed
multiple positive bands in 6,6'-bissulfo sialyl Lewis X (lane
6).

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Fig. 9.
Specificity of newly generated
anti-6,6'-bissulfo sialyl Lewis X and anti-6-sulfo Lewis X antibodies
as ascertained by TLC immunostaining. a shows reactivity of
the anti-6,6'-bissulfo sialyl Lewis X antibodies G2706, G27011, G27037,
and G27039, respectively, against carbohydrate determinants (lane
1, sialyl Lewis X; lane 2, 6'-sulfo sialyl Lewis X;
lane 3, 6'-sulfo Lewis X; lane 4, 6-sulfo sialyl
Lewis X; lane 5, 6-sulfo Lewis X; lane 6,
6,6'-bissulfo sialyl Lewis X; lane 7, 6,6'-bissulfo Lewis
X). b shows reactivity of the anti-6-sulfo Lewis X
antibodies AG97 and AG273 against carbohydrate determinants (lane
1, 6-sulfo sialyl Lewis X; lane 2, 6-sulfo Lewis X;
lane 3, 6,6'-bissulfo sialyl Lewis X; lane 4,
6,6'-bissulfo Lewis X). The orcinol panels show results from
TLC plates developed with orcinol/H2SO4
reagent, indicating the TLC mobility of each glycolipid. For structures
of the carbohydrate determinants, see Table I.
|
|
When applied to immunohistochemical examination of HEV endothelial
cells in human peripheral lymph nodes, these four antibodies all failed
to react to HEV endothelial cells (Fig.
10, e-h), in clear contrast
to the strong staining with the G152 and G72 antibodies observed on the
serial frozen sections prepared from the same lymph node (Fig. 10,
a and b). These findings suggest that the major
molecular species of the sulfated sialyl Lewis X determinant on HEV
endothelial cells is 6-sulfo sialyl Lewis X and that expression of the
6,6'-bissulfo sialyl Lewis X determinant is negligible.

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Fig. 10.
Immunohistochemical detection of
6,6'-bissulfo sialyl Lewis X and 6-sulfo Lewis X on HEV endothelial
cells using newly generated monoclonal antibodies. a-d show
immunohistochemical staining with the anti-6-sulfo sialyl Lewis X
antibodies G152 (a and c) and G72 (b
and d) of untreated (a and b) and
sialidase-treated (c and d) frozen sections of
human peripheral lymph nodes, serving as staining controls.
e-h show immunohistochemical staining with the
anti-6,6'-bissulfo sialyl Lewis X antibodies G2706, G27011, G27037, and
G27039, respectively, of untreated frozen sections of peripheral lymph
nodes. i-l show immunohistochemical staining with the
anti-6-sulfo Lewis X antibodies AG97 (i and k)
and AG273 (j and l) of untreated (i
and j) and sialidase-treated (k and l)
frozen sections of lymph nodes.
|
|
Detection of 6-Sulfo Lewis X in Sialidase-treated HEV Endothelial
Cells--
By immunizing sialidase-treated 6-sulfo sialyl Lewis X, we
further established two monoclonal antibodies directed against the
corresponding asialo compound, 6-sulfo Lewis X. The antibodies (AG97
and AG273) thus generated had a strong reactivity to 6-sulfo Lewis X in
ELISA, but were not reactive to other related asialo structures,
including Lewis X, 6'-sulfo Lewis X, and 3'-sulfo Lewis X (Fig. 8,
e and f). Of course, these antibodies lacked reactivity to the sialylated determinants such as 6-sulfo, 6'-sulfo, 6,6'-bissulfo, and genuine sialyl Lewis X. Weak reactivity was noted
against 6,6'-bissulfo Lewis X in ELISA, but this reactivity was not
detected in TLC immunostaining (Fig. 9b, lane 4),
suggesting that it was only a weak cross-reactivity.
These two antibodies did not stain intact HEV endothelial cells in
human lymph nodes in immunohistochemical examination, but strongly
labeled the cells when the sections were pretreated with sialidase
(Fig. 10, i-l). This was in a striking contrast to the strong staining of intact HEV by G152 and G72 antibodies, which was
nearly completely eliminated after sialidase treatment (Fig. 10,
a-d). These findings indicate that 6-sulfo sialyl Lewis X
was abundantly present, whereas there was little or none of its asialo form (6-sulfo Lewis X) on intact HEV endothelial cells. The latter determinant was produced only when 6-sulfo sialyl Lewis X in the frozen
sections was hydrolyzed with sialidase.
 |
DISCUSSION |
The aberrant reaction of anti-sialyl Lewis X antibodies against
HEV endothelial cells in human lymph nodes has long puzzled researchers
(11, 29). Some anti-sialyl Lewis X antibodies react to HEV and inhibit
the binding of L-selectin to HEV, yet others do not. Our preliminary
study (Ref. 15 and this study), using a series of hitherto known
anti-sialyl Lewis X and anti-Lewis X antibodies, suggested that 6-sulfo
sialyl Lewis X and 6,6'-bissulfo sialyl Lewis X, but not 6'-sulfo
sialyl Lewis X, are possible candidates for the complex sialyl Lewis
X-like determinants present on HEV endothelial cells. 6-Sulfo sialyl
Lewis X seemed to be the most predominant molecular species of sulfated
sialyl Lewis X-related determinants on human HEV endothelial cells,
judging from their reactivity pattern to hitherto known anti-sialyl
Lewis X and anti-Lewis X antibodies. The results of this study using a
series of new monoclonal antibodies directed against the respective sulfated determinants provided strong evidence corroborating the predominant expression of 6-sulfo sialyl Lewis X on HEV endothelial cells. The anti-6-sulfo sialyl Lewis X antibodies G152 and G72 strongly
labeled HEV in immunochemical examination, and the anti-6-sulfo Lewis X
antibodies AG97 and AG273 intensely stained HEV after sialidase
treatment.
If a significant amount of conventional sialyl Lewis X were expressed
on HEV, the reactivity of HEV against anti-sialyl Lewis X antibodies
should have been
CSLEX-1+SNH-3+FH-6+2H5+2F3+HECA-452+
instead of
CSLEX-1
SNH-3
FH-6
2H5+2F3+HECA-452+.
Moreover, the Lewis X determinant should have been detected by all
conventional anti-Lewis X antibodies after an appropriate sialidase
treatment, but it was not. Similarly, if a considerable amount of
6'-sulfo sialyl Lewis X were present on HEV, the reactivity of HEV
against anti-sialyl Lewis X antibodies should have been CSLEX-1+SNH-3+FH-6
2H5+2F3+HECA-452+,
and its asialo form determinant (6'-sulfo Lewis X), which is detectable
by a majority of conventional anti-Lewis X antibodies (Fig. 2), should
have been detected after sialidase treatment. Concerning the disulfated
determinant, we could not obtain any positive evidence to support the
presence of 6,6'-bissulfo sialyl Lewis X in HEV of human peripheral
lymph nodes, although we generated as many as four monoclonal
antibodies specific to the determinant.
Our results also indicated that 6-sulfo sialyl Lewis X accounts
for the majority of the carbohydrate capping group of the L-selectin
ligand on HEV endothelial cells as far as human HEVs are concerned,
judging from the nearly complete inhibition of recombinant L-selectin
binding to HEV endothelial cells. These results are at variance with
the previous notion that nearly equal amounts of 6'-sulfo and 6-sulfo
sialyl Lewis X are expressed on murine HEV (13, 14). This might be
partly due to the species difference since we could not detect any
significant reactivity of our anti-6-sulfo sialyl Lewis X antibodies
(G152 and G72) against HEV endothelial cells in murine and rat lymph
nodes.2 It is well known that
sialyl Lewis X, the putative ligand for E- and P-selectins, is barely
detectable on murine and rat leukocytes using most of the conventional
anti-sialyl Lewis X antibodies that readily detect the determinant on
human leukocytes, and the carbohydrate ligand on HEV for L-selectin in
mice and rats could well be different from that in humans.
An isoenzyme of
1
3-fucosyltransferase (fucosyltransferase VII)
has been shown to be present on HEV and is suggested to be mainly
involved in the synthesis of the L-selectin ligand on HEV (30). In line
with this, we found only a disproportionally small amount of the asialo
species of the 6-sulfo sialyl Lewis X determinant (i.e.
6-sulfo Lewis X) on human HEV in this study. This implies the
contribution of fucosyltransferase VII in the synthesis of the
determinant since fucosyltransferase VII is the only
1
3-fucosyltransferase isoenzyme that lacks the synthetic ability
of the asialo species of the corresponding determinant (31, 32).
Malý et al. (33) studied the substrate specificity of
fucosyltransferase VII and concluded that the enzyme has an ability to
transfer fucose residues onto 6-sulfated lactosamine, but not onto
6'-sulfated lactosamine. Habuchi et al. (34) recently
studied the substrate specificity of a cloned 6'-sulfotransferase and
reported that the enzyme preferred Gal
moieties in partially
sulfated polylactosamine much more than those in non-sulfated
polylactosamine as an acceptor of sulfate residues. On the other hand,
6-sulfation has recently been suggested to occur on the terminal
GlcNAc
moiety even before the transfer of the Gal
moiety, and
6-sulfolactosamine has been suggested to be formed afterward by the
action of
1
4-galactosyltransferase on 6-sulfo-GlcNAc
(35).
These findings imply that sulfation at the 6'-position occurs mainly
after the formation of 6-sulfolactosamine and may not proceed without
6-sulfo-GlcNAc
during the synthesis of sulfated substrates for
fucosyltransferases. This could explain our results showing that
6-sulfo sialyl Lewis X is strongly expressed, whereas the 6'-sulfo
species is virtually undetectable in human HEV endothelial cells.
P-selectin glycoprotein ligand-1 (PSGL-1) has been reported to be
necessary for the high affinity binding of sialyl Lewis X-positive
leukocytes not only to P-selectin, but also to L-selectin (36-39).
This finding was reproducible in our laboratory regarding the
leukocyte-leukocyte interaction. The sulfated tyrosine residue near the
N terminus of PSGL-1 was proposed to be responsible for the observed
high affinity binding to L-selectin. The problem with the
lymphocyte-HEV interaction was that we could not detect PSGL-1 in human
HEV endothelial cells using well characterized specific antibodies in
our preliminary experiments. When tested in the binding assay using
recombinant soluble human L-selectin (40) and in the cell adhesion
assay using human L-selectin-transfected cells (Fig. 6a),
the synthetic 6-sulfo sialyl Lewis X glycolipid exhibited a 30-60%
increase in L-selectin binding when compared with the genuine sialyl
Lewis X glycolipid. It is not clear if this increase is comparable to
the enhancing effect of PSGL-1 on L-selectin-mediated cell adhesion.
Sanders et al. (41) recently studied the binding activity of
several synthetic sulfated Lewis X determinants and suggested that even
a subtle difference in the binding activity of the 6-sulfo species
observed in the monovalent binding assay would possibly exert a
profound effect on the polyvalent binding in intercellular
adhesion.
Although this would provide one possible explanation, the sulfate
residue at the penultimate GlcNAc in 6-sulfo sialyl Lewis X
nevertheless may not substitute for the sulfate residue near the N
terminus of PSGL-1 in the high affinity binding to L-selectin. An
alternative explanation would be the presence of an amount of PSGL-1
too small to be detected by immunohistochemical examination or the
presence of PSGL-1-like molecule(s) on HEV endothelial cells. Although
we failed to detect it on normal human HEV endothelial cells, PSGL-1 is
reportedly expressed on microvascular endothelial cells in some
pathologic tissues (42).
 |
ACKNOWLEDGEMENTS |
We thank Drs. T. K. Kishimoto, S. Hakomori, A. Hino, K. Yoshida, H. Kondo, and Y. Inoue for the gifts of
monoclonal antibodies, oligosaccharides, and recombinant selectins.
 |
FOOTNOTES |
*
This work was supported in part by a grant-in-aid for the
second term comprehensive ten-year strategy for cancer control from the
Ministry of Health and Welfare, Japan, and by Grants-in-aid for
Scientific Research 09281238 and 09672371 from the Ministry of
Education, Science, Sports, and Culture, Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Program of Experimental
Pathology, Research Inst., Aichi Cancer Center, 1-1 Kanokoden, Chikusaku, Nagoya 464-8681, Japan. Fax: 81-52-763-5233; E-mail: rkannagi{at}aichi-cc.pref.aichi.jp.
1
The abbreviations used are: HEV, high
endothelial venule(s); ELISA, enzyme-linked immunosorbent assay;
BCECF-AM, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester; PSGL-1, P-selectin glycoprotein ligand-1.
2
M. Sawada-Kasugai, C. Mitsuoka, M. Izawa, and R. Kannagi, unpublished observation.
 |
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Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.