From the
Department of Biochemistry and Molecular
Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma
Health Sciences Center and the
Cardiovascular
Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City,
Oklahoma 73104
Received for publication, April 5, 2003 , and in revised form, May 2, 2003.
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ABSTRACT |
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INTRODUCTION |
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PSGL-1 is a dimeric, mucin-type glycoprotein ligand originally identified
as a ligand for P-selectin
(14), but PSGL-1 also
interacts with L- and E-selectin
(15,
16). To date, however,
detailed biochemical binding studies have only been carried out for P-selectin
and PSGL-1. These studies have shown that P-selectin binds to the extreme N
terminus of PSGL-1 by interacting stereospecifically with clustered tyrosine
sulfates () and a nearby core 2
O-glycan with a sialyl Lewis x (sLex;
Sia
23Gal
14(Fuc
13)GlcNAc
1-R)
epitope (C2-O-sLex)
(1719).
The use of synthetic glycosulfopeptides modeled after the N-terminal region of
PSGL-1 was a key factor in elucidating the molecular requirements for
P-selectin binding (17,
18,
20). By contrast, the
interaction between L-selectin and PSGL-1 has been studied less directly with
blocking monoclonal antibodies and site-directed mutagenesis of recombinant
PSGL-1.
Early studies with blocking monoclonal antibodies indicated that
L-selectin, like P-selectin, bound to the extreme N terminus of PSGL-1
(1012,
21). This region contains
sulfate on tyrosine residues but no sulfate on glycans. However, L-selectin
does bind to sulfated carbohydrate ligands on various HEV mucins, such as
6-sulfo-sLex
(Sia23Gal
14(Fuc
13)(6-sulfo)GlcNAc
1-R),
an epitope recognized by the mAb MECA-79
(8,
22,
23). Surprisingly, a recent
study of cells expressing recombinant PSGL-1 suggested that PSGL-1 requires
6-sulfo-sLex determinants rather than tyrosine sulfation to support
L-selectin-dependent leukocyte rolling
(24). This contrasts with
earlier observations that L-selectin binds poorly to recombinant PSGL-1 in
which the tyrosines are replaced with phenylalanines
(25,
27). It has also been proposed
that L-selectin recognizes nonsulfated sLex on both core 2 and
extended core 1 O-glycans
(26). Mutagenesis studies have
suggested that Thr-57 is the key O-glycosylation site on PSGL-1 for
binding to L-selectin (25,
27).
The present study was designed to directly measure the importance of
tyrosine sulfation and O-glycosylation at Thr-57 for binding of
L-selectin to PSGL-1, utilizing synthetic glyco-(sulfo)peptides (GSPs) modeled
after the N terminus of human PSGL-1, and to measure the binding affinity
between the GSPs and L-selectin. To this end, we synthesized a set of GSPs
containing one, two, three, or no sulfated tyrosine residues and
C2-O-sLex at Thr-57. This approach not only allowed us to
study the role of tyrosine sulfation but also the stereospecific contribution
of individual residues for
binding to L-selectin. We also synthesized GSPs containing three
residues and a modified
O-glycan at Thr-57 to study the role of specific monosaccharide
residues of C2-O-sLex, of sialylated polyfucosylated
polylactosamine O-glycan and of sialyl Lewis x on extended core 1
O-glycan (C1-O-sLex) for binding to L-selectin.
The interaction of L-selectin with GSPs was studied using multiple approaches,
including a fluorescence-based solid phase assay, equilibrium gel filtration,
and in vitro rolling experiments. Our results demonstrate that
L-selectin binds with relatively high affinity to GSPs that contain sulfate on
all three tyrosines and that present sLex on a core 2 rather than
on an extended core 1 O-glycan.
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EXPERIMENTAL PROCEDURES |
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Recombinant Selectin-Ig ChimerasThe vectors that were used
to express soluble P- and L-selectin-Ig chimeric proteins were a gift from Dr.
Ajit Varki (University of California, San Diego)
(28). P- and L-selectin-Ig
chimeric proteins were expressed in 293 cells and purified from the media
using Protein A-Sepharose as described
(29). The purity and
homogeneity of the purified selectin-Ig chimeras were analyzed by reducing and
non-reducing SDS-PAGE, followed by Coomassie Blue staining and Western
blotting. All preparations of P- and L-selectin-Ig chimeras were found to be
>90% pure and homogeneously dimeric, showing a molecular weight of
190,000.
Biotinylation of Glyco(sulfo)peptides and Fluorescence-based Solid Phase AssayBiotinylation of the C-terminal Cys of each GSP was performed using biotin-HPDP (N-(6-[biotinamido]hexyl)-3'-(2-pyridyldithio)propionamide) (Pierce) as described (20). Biotinylated GSPs were dissolved in 20 mM MOPS, pH 7.5, containing 150 mM NaCl, and the concentration of each peptide solution was determined by UV absorbance at 215 nm of a sample subjected to HPLC.
Fluorescence-based solid phase assay was performed essentially as described (20). Briefly, streptavidin-coated black 96-well microtiter plates (Pierce) were washed 3 times with 200 µl of 20 mM MOPS, pH 7.5, containing 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.02% NaN3 (buffer A) or 20 mM MOPS, pH 7.5, containing 150 mM NaCl, 5 mM EDTA, 0.02% NaN3 (buffer B) and coated for 1.5 h with 130 pmol of GSPs in 50 µl of buffer A or B. The wells were then incubated for 1 h with 50 µl of P-sel-Ig chimera (0.510 µg/ml), L-sel-Ig chimera (130 µg/ml), or anti-PSGL-1 mAb PL1 (2 or 5 µg/ml) (30) in buffer A or B containing 0.05% Tween 20 and 1% BSA. The wells were subsequently incubated for 1 h with 50 µl of 1050 µg/ml Alexa FluorTM 488 goat anti-human IgG (H+L) or with 50 µlof5or10 µg/ml Alexa FluorTM 488 goat anti-mouse IgG (H+L) (Molecular Probes, Inc., Eugene, OR) in buffer A or B containing 0.05% Tween 20 and 1% BSA. After a final washing, 100 µl of buffer A or B was added to each well and the fluorescence was measured using a Victor2 (Wallac, Turku, Finland) or Tecan Ultra384 (Tecan U.S., Durham, NC) microtiter plate reader with excitation wavelength at 485 nm and emission wavelength at 535 nm. Peptide coating and all incubations were performed at room temperature, and the wells were washed 3 times using buffer A or B containing 0.05% Tween 20. The assays of Figs. 2 and 3 were performed in duplicate, of Figs. 6 and 7 in triplicate, and the results represent averages of two or three determinations, respectively. Background fluorescence reading without peptide coating was subtracted from each sample in each experiment.
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Equilibrium Gel Filtration ChromatographyHummel-Dreyer equilibrium gel filtration experiments (31, 32) with [3H]GSP-6 and L-sel-Ig/P-sel-Ig were conducted in a 2-ml Sephadex G-100 column (0.5 x 10 cm) at physiological salt concentration (150 mM NaCl) or subphysiological salt concentration (50 mM NaCl) in 20 mM MOPS, pH 7.5, containing 2 mM CaCl2, 2 mM MgCl2, 0.02% NaN3 as described (18). Various concentrations of L-sel-Ig or P-sel-Ig were applied to the column equilibrated with [3H]GSP-6 (1.3 or 5.6 pmol/ml).
Rolling of Neutrophils on Glyco(sulfo)peptidesA 30-µl drop of streptavidin (50 µg/ml) was placed into a demarcated area on a 35-mm tissue culture plate (Corning, Corning, NY) and incubated at 4 °C overnight. The area was washed twice with Hanks' balanced salt solution and then blocked with Hanks' balanced salt solution containing 1% human serum albumin at room temperature for 2 h. Biotinylated GSPs (GSP-6, GSP-1, GP-6, and GP-4) were captured to the adsorbed streptavidin by incubation at 4 °C for 1 h. Site densities of GSPs were determined by binding of radiolabeled anti-PSGL-1 mAb PL1 (33).
Human neutrophils were isolated from healthy donors (34). Neutrophils (106/ml in Hanks' balanced salt solution containing 0.5% human serum albumin) were perfused over GSPs on 35-mm plates in a parallelplate flow chamber. After 5 min, the accumulated number of rolling cells was measured with a video microscopy system coupled to a digitized image analysis system (Inovision) (27, 33). In some experiments, neutrophils were perfused in the presence of 20 µg/ml anti-PSGL-1 mAb PL1 or PL2 (35), anti-L-selectin mAb DREG-56 or isotype mouse IgG1 mAb. Anti-human L-selectin mAb DREG-56 was purified from hybridomas obtained from American Type Culture Collection (ATCC). An isotype control mouse IgG1 was purchased from BD Pharmingen.
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RESULTS |
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L-selectin Binds to Immobilized GSP-6 and Binding Affinity Is Dependent on the Ligand DensityWe first used a newly developed and sensitive fluorescence-based solid phase assay to compare the relative binding affinities of L-selectin and P-selectin to GSP-6. Biotinylated GSP-6 (0.2510 pmol/well) was first captured quantitatively on streptavidin-coated 96-well plates. Different amounts of L-sel-Ig or P-sel-Ig were incubated in the wells, and bound L-sel-Ig and P-sel-Ig were detected with fluorescently labeled anti-human IgG. At the highest GSP-6 coating density (10 pmol/well), binding of L-sel-Ig to GSP-6 increased linearly with increased concentration of L-sel-Ig, reaching a plateau at 1030 µg/ml of L-sel-Ig (Fig. 2A). However, at lower GSP-6 densities, no plateau was observed even at the highest concentrations of L-sel-Ig. This shows that the affinity of L-sel-Ig for immobilized GSP-6 is dependent on the ligand density. By contrast, P-sel-Ig generated a saturated binding curve with all GSP-6 coating densities used (Fig. 2B), indicating that the affinity of P-sel-Ig for GSP-6 is less dependent on the density of the immobilized ligand.
L-selectin Binds to Immobilized GSP-6 with 10-fold
Reduced Affinity Compared with P-selectin at Physiological Salt
ConcentrationBinding affinities of L-sel-Ig and P-sel-Ig for
immobilized GSP-6 were first compared using the fluorescence-based solid phase
assay. Biotinylated GSP-6 was immobilized on streptavidin-coated plates at
different densities, and fixed concentrations of L-sel-Ig (10 µg/ml) and
P-sel-Ig (5 µg/ml) were incubated with wells containing varying amounts of
GSP-6. Binding of L-sel-Ig to increasing densities of GSP-6 formed a
semi-sigmoidal binding curve (Fig.
3A), suggesting that L-sel-Ig binding to immobilized
GSP-6 may be cooperative. By contrast, P-sel-Ig bound to GSP-6 forming a
typical rectangular hyperbola binding curve
(Fig. 3B). Comparison
of the GSP-6 densities that give half-maximal binding for L-sel-Ig (
10
pmol) and P-sel-Ig (
1 pmol) indicates that L-sel-Ig has
10-fold
lower affinity for immobilized GSP-6 than P-sel-Ig.
L-selectin Binds Free GSP-6 with 24-fold Reduced Affinity
in Comparison to P-selectin at Subphysiological Salt
ConcentrationSelectins bind to their ligands with higher affinity
at subphysiological salt concentration than at physiological salt
concentration (28). Our
earlier studies using equilibrium gel filtration have shown that recombinant,
monomeric soluble P-selectin binds to GSP-6 with a
Kd of
650 nM under physiological
conditions and with a Kd of
76 nM
under reduced salt concentration (50 mM NaCl)
(17). Thus, the difference
between subphysiological and physiological Kd for
P-selectin is
8.5-fold. Here we determined if the difference between
subphysiological and physiological Kd is the same
using recombinant P-sel-Ig and GSP-6 in equilibrium gel filtration. P-sel-Ig
bound to GSP-6 with a Kd of
26 nM
at subphysiological salt concentration
(Fig. 4A) and with a
Kd of
200 nM at physiological
salt concentration (Fig.
4B). Thus, the difference between subphysiological and
physiological Kd is
7.7-fold for P-sel-Ig
and GSP-6, which is very close to the difference in
Kd determined for soluble P-selectin and GSP-6.
We then determined the binding affinity of L-sel-Ig to GSP-6 in
subphysiological salt concentration using equilibrium gel filtration. L-sel-Ig
bound to GSP-6 with a Kd
628 nM
in 50 mM NaCl (Fig.
5). This is a
24-fold reduced affinity in comparison to
P-sel-Ig and GSP-6 under low salt concentration. We measured the binding of a
single concentration of L-sel-Ig (400 pmol) to GSP-6 in equilibrium gel
filtration under physiological salt concentration. The amount of
L-sel-Ig·GSP-6 complex formed was
10-fold less than the amount of
P-sel-Ig·GSP-6 complex under the same conditions
(Fig. 5, inset). Thus,
we conclude from the equilibrium gel filtration studies that the affinity of
L-sel-Ig for GSP-6 is between 10- and 25-fold lower than the affinity of
P-sel-Ig for GSP-6. This is in good agreement with the results from the solid
phase assay which indicates that L-sel-Ig binds to immobilized GSP-6 with
10-fold lower affinity than P-sel-Ig
(Fig. 3A). Taken
together, the results of the solid phase and equilibrium gel filtration
binding experiments demonstrate that L-sel-Ig binds to GSP-6 with relatively
high affinity (Kd between 2 and 5
µM) under physiological conditions.
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The Binding of L-selectin to GSP-6 Is Highly Dependent on Tyrosine
SulfationThe role of tyrosine sulfation and the positional
importance of residues of GSP-6
for binding of L-selectin to GSP-6 was studied using the fluorescence-based
solid phase assay. Equimolar amounts of different biotinylated GSPs (10
pmol/well in experiments with L-sel-Ig and 1 pmol/well with P-sel-Ig) were
immobilized on streptavidin-coated 96-well plates. Fixed concentrations of
L-sel-Ig (5 µg/ml) and P-sel-Ig (1 µg/ml) were incubated in the wells in
the presence of either Ca2+ or EDTA, and bound L-sel-Ig
and P-sel-Ig were detected with fluorescently labeled anti-human IgG. L-sel-Ig
showed high affinity binding to GSP-6 containing three
residues and very weak binding
to nonsulfated GP-6 (Fig.
6A). L-sel-Ig did not show clear preferential binding to
any isomer of the monosulfated GSPs, and the affinity of L-sel-Ig to
monosulfated GSPs was
611% relative to GSP-6
(Fig. 6A). In
contrast, binding to the isomers of disulfated GSPs was not equal, and
L-sel-Ig preferred binding to GSP(48,51)-6 with
50% affinity relative to
GSP-6. Binding to the other two isomers of disulfated GSPs was weaker,
1519% relative to GSP-6. In agreement with earlier data
(17,
19), P-sel-Ig preferred to
bind to GSPs containing tyrosine sulfate at position 48
(Fig. 6B). Moreover,
P-sel-Ig bound more strongly to disulfated GSPs than to monosulfated GSPs,
which is in agreement with earlier equilibrium gel filtration data
(Fig. 6B and Ref.
17). Binding of L-sel-Ig and
P-sel-Ig to all GSPs was strictly Ca2+-dependent,
because 5 mM EDTA completely inhibited binding
(Fig. 6). Taken together, these
results indicate that GSP-6 requires multiple sulfated tyrosine residues to
bind with high affinity to L-selectin. L-selectin binds to mono- and
disulfated GSPs with reduced affinity but binding to one disulfated isomer,
GSP(48,51)-6, is stronger than to other disulfated GSPs or monosulfated
GSPs.
The Binding of L-selectin to GSP-6 Is Highly Dependent on Fucose and
Sialic Acid ResiduesThe role of fucose and sialic acid residues of
GSP-6 for binding to L-sel-Ig and P-sel-Ig was studied using the
fluorescence-based solid phase assay. L-sel-Ig bound to desialylated GSP-6
(DS-GSP-6) and nonfucosylated GSP-5 with 36% affinity relative to
GSP-6 (Fig. 6A). This
low residual binding of the L-sel-Ig is likely toward the sulfated peptide
backbone of DS-GSP-6 and GSP-5, because L-sel-Ig showed similar binding to
GSP-1 compared with both DS-GSP-6 and GSP-5. By comparison, P-sel-Ig bound to
DS-GSP-6 with
7% affinity relative to GSP-6, whereas binding to GSP-5 was
undetectable (Fig.
6B). This confirms our earlier results showing that
fucose is more important than sialic acid for P-selectin binding to GSP-6
(17). Our present results show
that fucose and sialic acid residues of GSP-6 are equally important for
L-selectin binding.
L-selectin Binds to GSPs Containing Sialylated Polyfucosylated
Polylactosamine O-Glycan with Low AffinityMost of the fucosylated
O-glycans on PSGL-1 from human HL-60 cells contain a core 2-based
sialylated polyfucosylated polylactosamine (PFPL). We recently showed that
P-selectin binds very poorly to GSPs containing the PFPL structure. Instead, a
monofucosylated and monosialylated core 2 O-glycan is required for
high affinity recognition by P-selectin
(20). However, it is possible
that L-selectin might prefer such PFPL-containing O-glycans. The
potential recognition of sialylated PFPL O-glycans by L-selectin was
studied using a fluorescence-based solid phase assay and the constructs
GSP-6' and GSP-6''. Both GSP-6' and GSP-6'' contain
three residues, but they differ
in the length of the PFPL chains, which contain two
(C2-O-Lex-sLex) or three
(C2-O-Lex-Lex-sLex) fucosylated lactosamine
repeats, respectively, at Thr-57. L-sel-Ig showed weak but detectable affinity
for GSP-6' and GSP-6'' (812% relative to GSP-6)
(Fig. 6A). Binding to
GSP-6' was slightly better than to GSP-6''. Binding of P-sel-Ig to
GSP-6' was very weak and to GSP-6'' undetectable under these
conditions, confirming our earlier results that P-selectin binds to
GSP-6' and GSP-6'' with very low affinity
(Fig. 6B and Ref.
20).
L-selectin Binds with Low Affinity to a Glycosulfopeptide Presenting
sLex on an Extended Core 1 O-GlycanThe role of the
O-glycan core structure of GSPs for binding to L-selectin was
evaluated using the fluorescence-based solid phase assay. Fully sulfated
C1-GSP-6 and nonsulfated C1-GP-6 containing the sLex epitope on an
extended core 1 O-glycan (C1-O-SLex)at Thr-57
were immobilized on streptavidin-coated microtiter plates. The interactions of
L-sel-Ig and P-sel-Ig with C1-GSP-6 and C1-GP-6 were compared with the
isomeric structures GSP-6 and GP-6 containing the sLex epitope on a
core 2 O-glycan (C2-O-SLex) at Thr-57
(Fig. 7). L-sel-Ig showed weak
but detectable affinity for fully sulfated C1-GSP-6 (6.4% relative to
GSP-6) but undetectable binding to nonsulfated C1-GP-6 as well as to GP-6 and
GSP-1 (Fig. 7A). By
comparison, P-sel-Ig did not bind detectably to C1-GSP-6, C1-GP-6, and GSP-1
and bound only weakly to GP-6 (Fig.
7B). The poor interaction of P-selectin with C1-GSP-6 is
consistent with our earlier results employing affinity chromatography
(18). These data show that
L-selectin binds only weakly to C1-GSP-6. This weak binding is dependent on
tyrosine sulfation of the glycopeptide.
Rolling of Neutrophils on Glyco(sulfo)peptidesA recent study reported that L-selectin-expressing cells rolled much better on COS cells expressing recombinant PSGL-1 if the latter cells also expressed a sulfotransferase for carbohydrate 6-sulfation. Under these conditions, mutation of the three N-terminal tyrosines of PSGL-1 did not inhibit rolling. The authors concluded that tyrosine sulfation of PSGL-1 was not required to support rolling interactions with L-selectin (24). To directly address the molecular requirements for PSGL-1 to support L-selectin-dependent leukocyte rolling under flow, we perfused human neutrophils over streptavidin-captured, biotinylated GSP-6 in a parallel flow chamber at different wall shear stresses. Neutrophils required a minimum wall shear stress to roll, with peak accumulation at 1 dyn/cm2 (Fig. 8A). This shear threshold for L-selectin-dependent rolling closely resembled the shear threshold observed on other L-selectin ligands (27, 36). Anti-L-selectin mAb DREG-56 or anti-PSGL-1 mAb PL1, but not control mAbs, inhibited rolling, demonstrating the specificity of the interactions (Fig. 8B). Rolling neutrophils did not accumulate on similar densities of GP-6, which lacks sulfate on tyrosines, or of GSP-1, which contains sulfated tyrosines but not C2-O-sLex (Fig. 8B), although neutrophils formed some transient tethers to these structures (data not shown). These results demonstrate that optimal L-selectin-dependent neutrophil rolling on GSP-6 requires both tyrosine sulfation and C2-O-sLex.
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DISCUSSION |
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The extreme N terminus of human PSGL-1 contains Thr residues at positions 44 and 57, both potential sites for O-glycan addition, which are near three potential sites for tyrosine sulfation at positions 46, 48, and 51 (37, 38). Enzymatic removal of sulfate from Tyr residues in intact PSGL-1 (39) or Tyr replacement by site-directed mutagenesis of recombinant PSGL-1 (37, 40) demonstrated that at least one of the Tyr residues at positions 46, 48, or 51, is required for binding of recombinant PSGL-1 to P-selectin. Similarly, site-directed mutagenesis indicated that the Thr at position 57, but not 44, is essential for PSGL-1 recognition by P-selectin (41). Using synthetic glycosulfopeptides, we previously demonstrated that all three Tyr-SO3 and a core 2-based O-glycan with a sLex determinant at Thr-57 are required for high affinity binding to P-selectin (18). Site-directed mutagenesis revealed that PSGL-1 requires at least one Tyr sulfate residue to bind to L-selectin (25, 27). These studies strongly suggested, but did not directly prove, that tyrosine sulfation of PSGL-1 is required for significant binding to L-selectin.
To more precisely explore the specificity and binding affinity of
L-selectin for determinants within the extreme N-terminal domain of PSGL-1, we
synthesized a large set of glycosulfopeptides modeled after that domain that
varied in degree and position of sulfation of tyrosine and in the type and
structure of the O-glycan Thr-57. Our studies indicate that three
Tyr-SO3 residues at positions 46, 48, and 51
and a sLex determinant on a core 2 but not a core 1
O-glycan are required for high affinity binding to L-selectin. The
observed Kd of binding of the optimal GSP-6 to
L-selectin was 5 µM. This represents a relatively high
affinity interaction and compares very favorably to the high affinity binding
of this glycopeptide to P-selectin (Kd
650
nM) (17). Very weak
binding of L-selectin was observed to glycopeptides lacking Tyr-SO3
and to glycosulfopeptides containing a single Tyr sulfate residue at any
position. We did not observe preferential binding of any of the monosulfated
glycosulfopeptides to L-selectin. This agrees with a previous study in which
the different single Tyr forms of recombinant PSGL-1 supported similar
L-selectin-dependent cell rolling
(24). These combined results
do not support the suggestion that PSGL-1 preferentially uses Tyr-51 to
support L-selectin-dependent rolling
(25). Interestingly, an
isomeric disulfated GSP containing Tyr-SO3 at positions 48
and 51 bound more strongly than other disulfated isomers. By contrast,
sulfation of Tyr-48 contributes relatively more than sulfation of Tyr-46 or
Tyr-51 to binding of glycosulfopeptides to P-selectin
(17,
19).
Engineered Chinese hamster ovary cells expressing the recombinant
6-sulfotransferase that generates the GlcNAc-6-O-sulfate within the
sLex determinant (6-sulfo-sLex) bind to L-selectin
independently of PSGL-1 (24).
This study of transfected cells confirms the ability of L-selectin to bind to
the 6-sulfo-sLex determinant on mucins expressed by lymph node HEV
(42,
43). However, human leukocytes
lack the 6-sulfotransferase and do not express appreciable amounts of
6-sulfo-sLex determinants
(44). Moreover, sulfate
residues within human PSGL-1 are expressed primarily in tyrosine sulfate
rather than on sulfated glycans
(39,
45). Thus, tyrosine sulfation
is critically important for leukocyte PSGL-1 to interact with L-selectin. Our
results with sulfated and nonsulfated glycopeptides modeled after PSGL-1
definitively demonstrate the importance of tyrosine sulfation for L-selectin
binding in a system that lacks 6-sulfo-sLex glycan determinants.
Direct binding studies reveal that human L-selectin binds to GSP-6 with a
Kd in the range of 5 µM, which
is especially interesting in light of previous studies showing that monomeric
L-selectin binds to immobilized GlyCAM-1 with a much lower
Kd of
108 µM
(46). HEV mucin selectin
ligands such as GlyCAM-1 and CD34 contain multiple copies of the
6-sulfo-sLex epitopes that appear to increase avidity, which may
compensate for the low affinity of such determinants for L-selectin. We also
observed that binding of L-selectin, but not P-selectin, to the immobilized
glycosulfopeptides is affected by ligand density (Figs.
2 and
3). While it is premature to
speculate on the biological significance of this observation at present, the
differential effects of ligand density might relate to differential
association and dissociation kinetics of L-selectin binding compared with
P-selectin. The kinetics of L-selectin binding to the glycosulfopeptides could
be very rapid and it may be advantageous for L-selectin to bind cooperatively
to the multiple determinants within glycosulfopeptides to stabilize relatively
labile interactions. Whether this binding feature of L-selectin relates to its
role in shear-dependent rolling of leukocytes as observed in
Fig. 8 is not known. It will be
interesting in the future to prepare homogeneous glycopeptides modeled after
PSGL-1 and other mucins known to be bound by L-selectin, in which sulfate
residues are presented in glycan moieties and/or in tyrosine residues, and
directly measure their binding affinity and kinetics to L-selectin. It is
possible that some HEV mucins express Tyr-SO3-containing
glycoconjugates that function as L-selectin ligands, as noted for endoglycan,
an endothelial CD34 family member that contains two potential N-terminal
tyrosine sulfation sites
(47).
We observed that expression of the sLex determinant on a core 2,
but not a core 1, O-glycan is required for high affinity binding of
glycosulfopeptides to L-selectin, as noted earlier for binding to P-selectin
(18). Unlike binding to
P-selectin, small residual binding of C1-GSP-6 to L-selectin was observed
(Fig. 9). Our findings provide
direct support for previous strong but indirect evidence that core 2-based
O-glycans are important for recognition by both P- and L-selectin. To
bind P- or L-selectin with high affinity, recombinant PSGL-1 requires
co-expression with an 1,3-fucosyltransferase and the core 2
1,6-N-acetylglucosaminyltransferase (Core2GlcNAcT-I) that forms
the core 2 O-glycan from the core 1 O-glycan precursor
(25,
45). Mice lacking the gene
encoding Core2GlcNAcT-I have significantly reduced neutrophil rolling on E-,
L-, and P-selectins and reduced neutrophil recruitment to sites of
inflammation (48). A recent
study reported that human neutrophils and lymphocytes expressed low levels of
transcripts encoding
1,3GlcNAcT-3, an enzyme that extends core 1-based
O-glycans (26).
Chinese hamster ovary cells expressing recombinant PSGL-1 and FucT-VII, plus
either
1,3GlcNAcT-3 or Core2GlcNAcT-I, acquired sLex
determinants on O-glycans. Furthermore, cells expressing PSGL-1 and
sLex on extended core 1 O-glycans supported
L-selectin-dependent tethering and rolling of neutrophils and lymphocytes,
although much less well than Chinese hamster ovary cells expressing PSGL-1 and
sLex in core 2-branched O-glycans. Although these results
suggested that sLex in extended core 1 O-glycans can
support L-selectin-dependent rolling, the specific contribution of PSGL-1 to
this rolling was not tested
(26). Regardless, it is
questionable whether extended core 1 O-glycans on leukocytes function
as physiologically relevant selectin ligands. Extended core 1
O-glycans have not been detected in either human leukocytes
(49) or in the human
promyelocytic leukemic cell line HL-60
(50,
51). Furthermore, leukocytes
from Core2GlcNAcT-I-deficient mice have severe defects in rolling on L- and
P-selectin (48). Our finding
that L-selectin binds much better to a PSGL-1-derived glycosulfopeptide with
sLex presented on a core 2 O-glycan rather than on an
extended core 1 O-glycan strongly supports previous structural and
functional evidence for the importance of leukocyte core 2 O-glycans
for binding to L-selectin.
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L-selectin also binds to a subpopulation of PSGL-1 molecules carrying a sulfated polylactosamine, known as the PEN5 epitope, expressed on activated NK cells (52). This binding was considered to be independent of tyrosine sulfation, based on the ability of NK cells to tether and roll on L-selectin after mocarhagin treatment, a snake venom protease that cleaves the extreme N terminus of PSGL-1 and eliminates binding to P-selectin (12, 53). We found that the unsulfated polylactosamine structures in the glycosulfopeptides GSP-6' and GSP-6'' supported only low affinity, albeit detectable, binding to L-selectin. It remains to be determined if 6-O-sulfation of GlcNAc residues in such polylactosamine-containing glycopeptides is able to substitute for tyrosine sulfation of GSP-6' and GSP-6'' or if carbohydrate sulfation together with tyrosine sulfation promote even higher affinity binding of GSPs to L-selectin.
In summary, our results directly demonstrate that both P- and L-selectin
require dual recognition of
residues and sLex expressed on a core 2-based O-glycan at
Thr-57 for high affinity binding to PSGL-1. However, P-selectin binds to the
same glycosulfopeptide (GSP-6) with
10-fold higher affinity than
L-selectin. It is interesting that L-selectin is able to recognize sulfate
residues both within tyrosine-sulfated peptides and in sulfated GlcNAc
residues such as 6-sulfo-sLex. P-selectin also binds to some extent
to 6-sulfo-sLex
(54). The crystallographic
structure of P-selectin complexed with a PSGL-1-derived glycosulfopeptide
indicates that
residues 48 and
51 form direct contacts with the lectin domain of P-selectin
(19). Crystal structures of
L-selectin complexed with the sulfated tyrosine-containing glycosulfopeptides
described here and with glycans containing sulfate in 6-sulfo-sLex
will provide further insights into how L-selectin can bind different forms of
sulfate in cooperation with sLex.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 975 N.E. 10th St., BRC417, Oklahoma City, OK 73104. Tel.: 405-271-2481; Fax: 405-271-3910; E-mail: richard-cummings{at}ouhsc.edu.
1 The abbreviations used are: HEV, high endothelial venules; PSGL-1,
P-selectin glycoprotein ligand-1; L-sel-Ig, L-selectin IgG chimera; P-sel-Ig,
P-selectin IgG chimera; sLex, sialyl Lewis x
(Sia23Gal
14(Fuc
13)GlcNAc
1-R);
6-sulfo-sLex,
Sia
23Gal
14(Fuc
13)(6-sulfo)GlcNAc
1-R;
Lex, Lewis x
(Gal
14(Fuc
13)GlcNAc
1-R); GP, glycopeptide;
GSP, glycosulfopeptide; PFPL, polyfucosylated polylactosamine;
C2-O-sLex, core 2-based O-glycan with
sLex; C2-O-Lex-sLex, core 2-based
O-glycan with Lex-sLex;
C2-O-Lex-Lex-sLex, core 2-based
O-glycan with Lex-Lex-sLex;
C1-O-sLex, core 1-based O-glycan with
sLex;
, tyrosine
sulfate; MOPS, 4-(N-morpholine)propanesulfonic acid; GlyCAM-1,
glycosylation-dependent cell adhesion molecule; mAb, monoclonal antibody;
HPLC, high performance liquid chromatography;
1,3-FucTVI,
1,3-fucosyltransferase VI; BSA, bovine serum albumin.
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ACKNOWLEDGMENTS |
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REFERENCES |
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