From the Division and Central Laboratory of
Hematology, Centre Hospitalier Universitaire Vaudois, Bugnon 46, 1011 Lausanne, Switzerland, the § Ludwig Institute for Cancer
Research, Lausanne Branch, chemin des Boveresses 155, Epalinges, and
the ¶ Swiss Institute for Bioinformatics, chemin des Boveresses
155, Epalinges, Switzerland
Received for publication, May 3, 2002, and in revised form, October 10, 2002
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ABSTRACT |
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Interactions between the leukocyte adhesion
receptor L-selectin and P-selectin glycoprotein ligand-1 play an
important role in regulating the inflammatory response by mediating
leukocyte tethering and rolling on adherent leukocytes. In this study,
we have examined the effect of post-translational modifications of PSGL-1 including Tyr sulfation and presentation of sialylated and
fucosylated O-glycans for L-selectin binding. The
functional importance of these modifications was determined by
analyzing soluble L-selectin binding and leukocyte rolling on CHO cells expressing various glycoforms of PSGL-1 or mutant PSGL-1 targeted at
N-terminal Thr or Tyr residues. Simultaneous expression of core-2
Selectins play a major role in regulating leukocyte migration in
inflammatory lesions by mediating leukocyte rolling along vascular wall
at site of inflammation (1-8). L-selectin is expressed by most
leukocytes whereas P-selectin and E-selectin expression is induced on
activated platelets and/or endothelial cells (1, 2, 4, 5, 7, 9). E-,
P-, and L-selectin function at different although overlapping phases of
the inflammatory reaction (10). P-selectin interacts with its major
ligand P-selectin glycoprotein ligand-1
(PSGL-1)1 and supports
leukocyte rolling along postcapillary venules at the earliest phase of
inflammation (11-13). Several studies have indicated that L-selectin
mediates both primary leukocyte-endothelial interactions (14, 15) and
secondary interactions between circulating and adherent leukocytes,
which both participate in leukocyte recruitment in inflammatory
lesions. Secondary interactions are mainly supported by the interaction
of PSGL-1 with L-selectin (16-18).
PSGL-1 is a mucin-like glycoprotein expressed as a homodimer on
leukocyte microvilli (4, 19-21). P-selectin binds with relatively high
affinity (Kd ~300 nM) (22) to PSGL-1
by reacting with N-terminal tyrosine sulfate residues and with the
sLex tetrasaccharide determinants presented by core-2
O-glycans attached to Thr-57 (23-33). Binding studies
performed with glycosulfopeptides indicated a contribution of each
tyrosine sulfate residue in supporting P-selectin binding with a
predominant role of Tyr-48 (34). The molecular contacts between
P-selectin and PSGL-1 were identified by the analysis of the crystal
structure of P-selectin co-complexed with the N-terminal peptide of
PSGL-1 (23). These studies revealed the involvement of Tyr-48 and -51, but no interaction was observed between P-selectin and Tyr-46 (23).
Previous observations indicated the involvement of O-glycans
attached to Thr-57 and tyrosine sulfate residues in supporting L-selectin- and P-selectin-mediated rolling (35). In the present study,
we characterized the PSGL-1 determinants that interact with L-selectin.
Adhesion studies indicated that O-glycosylation of Thr-57
and sulfation of Tyr-46 and -51 play a critical role in supporting
recombinant L-selectin binding to PSGL-1 and leukocyte rolling on
PSGL-1. In addition, these determinants were shown to play a major role
in stabilizing rolling velocity, a key feature for the regulation of
leukocyte exposure to chemotactic stimuli that lead to cell arrest and
firm adhesion. By contrast, Tyr-48 played only a minor role in
supporting L-selectin-mediated adhesion whereas it was shown to be
critical for P-selectin-mediated interactions with PSGL-1 (23, 34).
Antibodies and Chimeric Selectins--
The anti-L-selectin
monoclonal antibodies (mAbs) LAM1-3, LAM-14 (36), HECA-452 (ATCC HB
11485), and CSLEX-1 (ATCC number: HB-10135) were purified from
hybridoma culture medium. mAbs PL1 and PL2 were purchased from Coulter
Immunotech (Marseille, France) and KPL1 from BD Bioscience PharMingen
(Heidelberg, Germany). L-selectin/IgM heavy chain
(L-selectin/µ) and CD4/µ chimera were produced by transient
transfection of COS-7 cells using the DEAE method (37, 38).
cDNAs--
PSGL-1 cDNA was a gift from Genetics
Institute (Boston, MA) (39), fucosyltransferase VII (FucT-VII) cDNA
from J-B Lowe (Howard Hughes Institutes, Ann Harbor, MI) and core-2
Targeted point mutations were introduced in the N-terminal region of
PSGL-1 using the QuickChange site-directed mutagenesis kit (Stratagene,
Woodinville, WA), according to the manufacturer instructions. Sequences
of the forward and reverse primers used to generate wild-type and
mutant PSGL-1 shown in Fig. 1 were
CAGGCCACCGAATATGAGTATCTAGATTTTGATTTCCTGCC and
GGCAGGAAATCAAAATCTAGATACTCATATTCGGTGGCCTG for PSGL-1
Y46F/Y48F/ Y51F; CAGGCCACCGAATATGAGTATCTAGATTTTGATTTCCTGCC and
GGCAGGAAATCAAAATCTAGATACTCATATTCGGTGGCCTG for PSGL-1 Y48F/Y51F;
CAGGCCACCGAATATGAGTTTCTAGATTTTGATTTCCTGCC and
GGCAGGAAATCAAAATCTAGAAACTCATATTCGGTGGCCTG for PSGL-1 Y46F/Y51F; CAGGCCACCGAATTTGAGTATCTAGATTTTGATTTCCTGCC and
GGCAGGAAATCAAAATCTAGATACTCAAATTCGGTGGCCTG for PSGL-1 Y46F/Y51F;
CAGGCCACCGAATTTGAGTTTCTAGATTATGATTTCCTGCC and
GGCAGGAAATCATAATCTAGAAACTCAAATTCGGTGGCCTG for PSGL-1 Y46F/Y48F; GATTTCCTGCCTGAGGCGGAGCCTCCAGAAATGCTGAGG and
CCTCAGCATTTCTGGAGGCTCCGCCTCAGGCAGGAAATC for PSGL-1 T57A;
CGGGACCGGAGACAGGCTGCAGAATATGAGTACCTAGAT and
ATCTAGGTACTCATATTCTGCAGCCTGTCTCCGGTCCCG for PSGL-1 T44A.
Polymerase chain reactions (PCR) were performed using Pfu
Polymerase (Stratagen), and PCR products were cloned into pCR-Blunt
vector (Invitrogen) after digestion of methylated cDNAs with
DpnI. Sequences of the constructs were verified by dideoxynucleotide sequencing.
Cells and Transfections--
Heparinized blood samples were
obtained from healthy donors. Lymphocytes were isolated by blood
centrifugation on Ficoll-Hypaque and monocyte depletion by adherence on
plastic (43). Neutrophils were isolated from Ficoll-Hypaque pellets by
dextran sedimentation and erythrocyte hypotonic lysis.
CHO/dhfr Immunophenotypic Analysis--
One or two color flow cytometry
analysis was carried out by incubating cells with appropriate unlabeled
mAbs, FITC-, PE-conjugated mAbs (10 µg/ml) or soluble adhesion
receptors (L-selectin/µ or CD4/µ chimera at 50 µg/ml) (37, 38).
When required goat anti-mouse IgG-FITC (Tago
BIOSOURCE Europe S.A., Nivelles, Belgium), goat anti-mouse IgM-FITC (Tago BIOSOURCE), or rabbit
anti-rat IgM-FITC were used as secondary antibodies (Dako).
L-selectin/µ and CD4/µ chimeric proteins were suspended in
phosphate-buffered saline containing 1% bovine serum albumin and 1 mM CaCl2. Cell surface binding of chimeric
proteins was detected using a polyclonal FITC-conjugated rabbit
anti-human IgM heavy chain antibody (Dako). The specificity of
L-selectin/µ binding to PSGL-1 was indicated by the abrogation of
L-selectin/µ binding in presence of 5 mM EDTA or 100 µg/ml anti-L-selectin mAb LAM1-3 or 10 µg/ml anti-PSGL-1 mAb KPL1.
In experiments performed to evaluate the role of sialic acid residues in supporting neutrophil rolling on PSGL-1, CHO-PSGL-1 cells, co-expressing C2GnT and FucT-VII, were cultured for 30 min at 37 °C
in PBS containing 0.1 units/ml Vibrio cholerae neuraminidase (Roche Diagnostics). The reactivity of the mAb CSLEX-1 with
sLex was abrogated by this treatment. Flow cytometry was
performed with a Epics XL-MCL cytofluorimeter (Coulter Electronics,
Hialeah, FL). A total of 5000 cells were analyzed in each experiment.
Cell Adhesion Assays--
A laminar flow was generated in a
parallel plate flow chamber (GlycoTech Corp Rockville, MD) mounted on a
glass coverslip (Polylabo SA, Plan-les-Ouates, Switzerland) covered
with a confluent monolayer of transfected CHO cells. Neutrophils
suspended in RPMI 1640/1% fetal calf serum at 0.5 × 106/ml, lymphocytes (106/ml), 300.19 pre-B
cells stably expressing L-selectin (300.19-L-selectin cells, 0.5 × 106/ml) or K-562 cells cells stably expressing
P-selectin (K562 P-selectin cells, 0.5 × 106/ml), were perfused through the chamber using a syringe
pump (Harvard Apparatus, Indulab AG, Gams, Switzerland) for 5 min, at room temperature, under a constant shear stress. Leukocyte
interactions with CHO cells were visualized using a phase
contrast microscope (Leica Leitz DM IL, Renens, Switzerland) and a high
resolution Sony CCD-IRIS videocamera (Japan). Images were recorded
on an S-VHS-recorder (Panasonic MD 830, Telecom Lausanne,
Switzerland). Sequential images of leukocyte interactions with
transfected CHO cells were analyzed using a digital image analysis
system (Mikado software, GPL SA, Martigny, Switzerland) and a
Power-Macintosh 8600/200 workstation equipped with a Scion LG-3 board
(Scion, Frederick, MD) (38). Cell-cell interactions were analyzed from
videotapes at 2-4 min of perfusion. Leukocyte interactions with CHO
cells, in 0.27-mm2 fields, were considered for the analysis
when the interaction time was
Lymphocytes, 300.19-L-selectin cells and K-562 P-selectin cells did not
significantly arrest on CHO-PSGL-1 (6 ± 6 lymphocytes/min/mm2) whereas neutrophils occasionally
firmly adhere to CHO-PSGL-1 cells. Arrested cells were defined as cells
which did not move during an observation period Model of PSGL-1 Interactions with L-selectin and
P-selectin--
P-selectin and L-selectin interactions with PSGL-1
were compared using a L-selectin homology model based on the crystal
structure of P-selectin co-complexed with PSGL-1 (PDB Data Bank ID:
1G1S) (23). 66% sequence identity and 85% sequence similarity were disclosed between P-selectin and L-selectin using a dynamic programming method implemented in the MODELLER program (45). With this sequence homology, the probability that both L-selectin and P-selectin share the
same fold is very high (46-48). A root mean-squared deviation (RMSD)
of 0.67 Å was calculated for all C
The mobility of P-selectin loops was studied in detail with the Swiss
PDB viewer (49) and the MOLMOL programs (52) using the crystal
structure of P-selectin co-complexed with its ligands PSGL-1 (1G1S),
sLex (1G1Q), and the uncomplexed form (1G1R) (23).
PSGL-1-sulfated Tyr-48 was previously reported to interact with a first
loop constituted of P-selectin amino acids 42-48 and with a second
loop constituted of amino acids 108-114. PSGL-1-sulfated Tyr-51
interacts with a third loop constituted of amino acid 64-89 (23). The
mobility of each loop was assessed by calculating with the MOLMOL
program the local RMSDs of the lectin domain amino acid backbone atoms between the different P-selectin structures (52-54). Regions
constituted of residues with local RMSDs higher than the mean local
RMSD (1.3 Å) were defined as mobile.
Statistical Analysis--
Analysis of variance and the
Bonferroni multiple comparison test or the Kruskal-Wallis non
parametric ANOVA test were used to assess statistical significance of
differences between groups. The Mann-Whitney test was used to compare
the medians of two unpaired groups. p values of <0.05 were
considered as significant.
Sialylated, Fucosylated, Core-2 O-Glycans Are Essential to Support
Leukocyte Rolling on PSGL-1--
The requirement in
sLex/CLA and core-2 O-glycans to support
L-selectin-mediated interactions with PSGL-1 was examined by analyzing L-selectin/µ chimera binding and leukocyte rolling on CHO cells co-transfected with PSGL-1 cDNA in pcDNA3.1 vector and/or
FucT-VII cDNA in pZeo SV and/or C2GnT and FucT-VII cDNAs in
pIRES Zeo SV vector. Five different transfectants containing cDNA
sequences of (1) FucT-VII alone, (2) C2GnT and FucT-VII, (3) PSGL-1 alone, (4) PSGL-1 and FucT-VII, or (5) PSGL-1, C2GnT and FucT-VII were
obtained. CHO cells stably expressed similar levels of PSGL-1 and/or
sLex/CLA as ascertained by determination of antibody
binding site density (mean ± S.D.: 247 ± 19 PL2 binding
sites/µm2) and immunostaining of CHO cell monolayers with
mAbs PL2 (anti-PSGL-1), CSLEX-1 (anti-sLex), or HECA-452 (anti-CLA)
(Fig. 2a).
L-selectin/µ chimera weakly interacted with CHO cells
expressing FucT-VII cDNA alone or co-expressing C2GnT and FucT-VII
cDNAs (Fig. 2b, upper panels). L-selectin/µ
bound to a much higher percentage of CHO cells when PSGL-1 was
co-expressed with both FucT-VII and C2GnT (Fig. 2b,
lower right panel).
Neutrophil rolling was studied under flow conditions at a constant
shear stress of 1.25 dyn/cm2. Mock-transfected CHO cells or
CHO cells transfected with PSGL-1 cDNA alone did not support
neutrophil rolling (mean number of rolling cells/min/mm2 ± S.E.: 1 ± 0.3, n = 4 (not illustrated)
versus 1 ± 0, n = 4). Although CHO
cells transfected with the pIRES vector containing C2GnT and FucT-VII
cDNA sequences expressed sLex/CLA (Fig. 2a)
and bound L-selectin/µ (Fig. 2b), the expression of C2GnT
and FucT-VII was not sufficient to confer to CHO cells the ability to
support neutrophil rolling (3 ± 2 rolling
cells/min/mm2, n = 4, Fig.
3a).
Interestingly, neutrophil rolling was observed on CHO cells
co-transfected with PSGL-1 and FucT-VII cDNAs even in the absence of C2GnT expression (64 ± 18 rolling cells/min/mm2,
n = 5, p = 0.02, Fig.
3a, shaded box). The presentation of
sLex/CLA residues at the termini of core-2
O-glycans attached to PSGL-1 strongly increased neutrophil
recruitment at the surface of CHO cells. Neutrophil rolling on CHO
cells co-expressing PSGL-1, C2GnT, and FucT-VII cDNA sequences was
increased by 6-fold over the data obtained without C2GnT expression
(416 ± 45 rolling neutrophils/min/mm2
versus 64 ± 48 rolling cells/min/mm2,
n = 5, p = 0.0008, Fig.
3a, black box). This observation emphasizes the
key role played by core-2 O-glycans in supporting PSGL-1
binding to L-selectin. The L-selectin specificity of this interaction was indicated by the complete inhibition of neutrophil rolling in
presence of LAM1-3 mAb (98 ± 1% of inhibition,
n = 6). The adhesion blocking PL-1 mAb inhibited
neutrophil rolling by 91 ± 3% (n = 6). The
non-blocking anti-L-selectin mAb LAM1-14 and anti-PSGL-1 mAb PL2 had
no significant inhibitory effect.
Sialylation of PSGL-1 Is Required to Support Neutrophil
Rolling--
The importance of sialic acid residues in supporting
L-selectin interactions with PSGL-1 was evaluated by analyzing
neutrophil rolling on CHO cells, co-expressing PSGL-1, C2GnT, and
FucT-VII, treated with V. cholerae neuraminidase (0.1 units/ml) for 30 min at 37 °C. Sialidase treatment inhibited
neutrophil rolling by 93 ± 3% (145 ± 26, n = 8 versus 9 ± 4 rolling
neutrophils/mm2/min, n = 24;
p = 0.0001, not illustrated) indicating that sialic acid residues play a key role in regulating
L-selectin-dependent rolling on PSGL-1.
Regulation of Neutrophil-rolling Velocity by Core-2
O-Glycans--
Neutrophil-rolling velocity on CHO-PSGL-1/FucT-VII or
CHO-PSGL-1/C2GnT/FucT-VII cells was analyzed under a constant shear stress of 1.25 dyn/cm2 (Fig. 3b). Significantly
lower rolling velocities were observed on CHO-PSGL-1/C2GnT/FucT-VII
cells (median-rolling velocity: 39 µm/s, range: 3.4-257 µm/s,
P25 = 24 µm/s, P75 = 61 µm/s,
p < 0.0001, Fig. 3b, black
curve) than on CHO-PSGL-1/FucT-VII cells (median-rolling velocity:
113 µm/s, range: 2.4-348 µm/s, P25 = 73 µm/s,
P75 = 177 µm/s, Fig. 3b, gray
curve). These observations emphasized the critical role played by
core-2 O-glycans, decorated by sLex/CLA
residues, in the regulation of neutrophil-rolling velocity on
PSGL-1.
O-Glycans Attached to Thr-57 Are Essential to Support
L-selectin Binding and Neutrophil Rolling on PSGL-1--
The role
of core-2 O-glycans attached to Thr-57 was evaluated by
replacing Thr-57 by Ala (PSGL-1 T57A, Fig. 1). Similarly, Thr-44,
another potential site of O-glycosylation, was replaced by
Ala (PSGL-1 T44A). CHO cells stably expressed similar levels of PSGL-1
(mean ± S.D.: 115 ± 37 PL2 binding
sites/µm2), sLex, and CLA. L-selectin/µ
strongly reacted with wild-type PSGL-1 or PSGL-1 T44A (Fig.
4a, left and
center panels) whereas it only weakly bound to PSGL-1 T57A
(Fig. 4a, right panel). L-selectin/µ binding was not
completely inhibited by the replacement of Thr-57 by Ala indicating
that additional structures support L-selectin/µ binding to PSGL-1.
The N-terminal tyrosine sulfation consensus is the most likely
alternate potential binding site. Sialyl Lex/CLA
determinants expressed at the surface of CHO cells may also play a role
(Fig. 2b, upper panels).
Replacement of Thr-44 by Ala did not impair neutrophil rolling on
CHO-PSGL-1 T44A cells (335 ± 28, n = 4 versus 322 ± 58 rolling neutrophils/mm2/min, n = 9) whereas Thr-57
replacement by Ala decreased neutrophil rolling by 97 ± 1%
(335 ± 28 versus 7 ± 2 rolling
neutrophils/mm2/min, n = 8;
p < 0.001). These observations indicate that
O-glycans attached to Thr-57 play a major role in supporting
neutrophil tethering and rolling on PSGL-1 (Fig. 4b).
Similar results were obtained with peripheral blood lymphocytes. The
replacement of Thr-57 by Ala decreased lymphocyte recruitment on
CHO-PSGL-1 cells by 74 ± 3% (1596 ± 177 versus
412 ± 43 rolling lymphocytes/mm2/min,
n = 4, p < 0.001, not shown).
Regulation of L-selectin Interaction with PSGL-1 by N-terminal
Tyrosine Su1fate Residues--
Point mutations were introduced in
PSGL-1 cDNA to exchange Tyr-46, -48 and -51 by Phe and Thr-57 by
Ala. Five constructs (PSGL-1 Y46F/Y48F; PSGL-1 Y46F/Y51F, PSGL-1
Y48F/Y51F, PSGL-1 Y46F/Y48F/Y51F, and PSGL-1 Y46F/Y48F/Y51F/T57A; Fig.
1) were stably co-expressed in CHO cells with pIRES Zeo SV vector
containing C2GnT/FucT-VII cDNA sequences. CHO cells exhibited
similar levels of PSGL-1 (mean ± S.D.: 115 ± 37 PL2 binding
sites/µm2), sLex, and CLA (not shown).
L-selectin/µ chimera strongly bound to CHO cells co-expressing
C2GnT/FucT-VII and wild-type PSGL-1 cDNAs (Fig.
5a, upper left
panel). The interaction of L-selectin/µ chimera was reduced by
the replacement of two Tyr residues by Phe (Fig. 5a,
lower panels). Interestingly, L-selectin/µ bound more
efficiently to mutant PSGL-1 expressing a single tyrosine residue at
position 46 or 51 (Fig. 5a, lower left and
right panels) than at position 48 (Fig. 5a,
lower central panel).
The N-terminal Tyrosine Sulfation Consensus of PSGL-1 Regulates
Neutrophil and Lymphocyte Recruitment at CHO Cell Surface--
The
role of Tyr-46, -48, -51 in supporting neutrophil and T-lymphocyte
rolling was assessed under laminar flow conditions using a constant
shear stress of 1.25 dyn/cm2. Replacement of Tyr-46, -48, and -51 by Phe decreased by 82 ± 8% neutrophil rolling on
CHO-PSGL-1/C2GnT/FucT-VII cells. Thus, 409 ± 36 neutrophils/min/mm2 rolled on wild-type PSGL-1
(n = 7) whereas only 74 ± 32 neutrophils/min/mm2 (n = 5, p < 0.0001) rolled on CHO-PSGL-1 Y46F/Y48F/Y51F cells (p < 0.0001, Fig. 5b). Tyrosine replacement
by Phe leaving a single Tyr residue also strongly affected neutrophil
recruitment. Neutrophil rolling decreased: 1) by 56 ± 13% in the
absence of Tyr-48 and -51 (180 ± 50 neutrophils/min/mm2, n = 5, p < 0.0001); 2) by 72 ± 8% after replacement of
Tyr-46 and -51 by Phe (115 ± 29 neutrophils/min/mm2,
n = 5, p < 0.0001); and 3) only by
35 ± 8% after exchange of Tyr-46 and -48 by Phe (266 ± 29 neutrophils/min/mm2, n = 5, p < 0.01). These results suggest, like observations
made with L-selectin/µ chimera, that Tyr-51 plays a predominant role in regulating L-selectin interactions with PSGL-1.
Similar results were obtained with peripheral blood lymphocytes, which
rolled efficiently on wild-type PSGL-1 (1594 ± 74 lymphocytes/min/mm2, n = 3, Fig. 5c).
Lymphocyte rolling was reduced by 32 ± 5% on mutants expressing
Tyr-51 as single sulfated Tyr residue (PSGL-1 Y46F/Y48F: 1085 ± 67 lymphocytes/min/mm2, n = 3, p < 0.01), by 69 ± 3% on mutants expressing
Tyr-48 (PSGL-1 Y46F/Y51F: 481 ± 45 lymphocytes/min/mm2, n = 3, p < 0.001) and by 52 ± 4% on mutants expressing
Tyr-46 (PSGL-1 Y48F/Y51F: 754 ± 52 lymphocytes/min/mm2, n = 3, p < 0.001, Fig. 5c). These observations
confirm: 1) that the presence of a single Tyr residue is less efficient
than three Tyr residues to support leukocyte rolling on PSGL-1 and 2)
that Tyr-51 plays a predominant role in supporting lymphocyte
interactions with PSGL-1.
Additional experiments were performed to examine whether the
predominant role of Tyr-51 in supporting L-selectin interaction with
PSGL-1 was dependent on wall shear stress. At all tested shear stress
(1.0, 2.0, and 3.0 dyn/cm2), the recruitment of 300.19 cells on PSGL-1 Y46F/Y51F, which express Tyr-48, was lower than on
mutants expressing Tyr-51 (number of cells/min/mm2 that
rolled on PSGL-1 Y46F/Y51F at 1.0 dyn/cm2 (mean ± S.E.): 138 ± 12; at 2.0 dyn/cm2: 171 ± 19; at
3.0 dyn/cm2: 201 ± 29). The number of rolling cells
was significantly higher on PSGL-1 Y46F/Y48F (number of rolling cells
at 1.0 dyn/cm2: 290 ± 23; at 2.0 dyn/cm2:
374 ± 20; at 3.0 dyn/cm2: 627 ± 35, p < 0.001). These observations confirmed that Tyr-51 plays a predominant role in mediating L-selectin-dependent
rolling on PSGL-1 and indicate that this property is not dependent on shear stress. In contrast, a predominant role for Tyr-46 was observed only at 1.0 dyn/cm2 (number of rolling
cells/min/mm2 on PSGL-1 Y48F/Y51F at 1 dyn/cm2:
205 ± 18, p < 0.05 versus 138 ± 12 on PSGL-1 Y46F/Y51F). These results indicated that tyrosine residues
distinctly contribute to support L-selectin-dependent
rolling. Since experiments performed with PSGL-1 glycosulfopeptides
previously showed that tyrosine residues distinctly contribute to
support P-selectin binding (34), we compared side-by-side the role of
tyrosine sulfate residues in supporting L-selectin and
P-selectin-dependent rolling. Adhesion studies were
performed with 300.19-L-selectin cells and K-562 P-selectin cells under
a constant shear stress of 2.0 and 3.0 dyn/cm2. Results
were expressed as percentage of rolling cells. Under a constant shear
stress of 2.0. dyn/cm2, 476 ± 35 (mean ± S.E.)
300.19-L-selectin cells (% of rolling cells: 100 ± 6) and
433 ± 7 K-562-P-selectin cells (100 ± 10%), rolled on
wild-type PSGL-1 (Fig. 5, d and e). Tyrosine
replacement by Phe leaving Tyr-48 as single tyrosine residue strongly
decreased L-selectin-dependent rolling (% of rolling
cells: 33 ± 3, p < 0.001, n = 3, Fig. 5d) whereas P-selectin-mediated rolling was not
significantly reduced (96 ± 8%, n = 3, Fig.
5e). Similar results were obtained under a shear stress of
3.0. dyn/cm2 (% of 300.19-L-selectin-rolling cells on
PSGL-1 Y46Y/Y51F: 30 ± 3, p < 0.001, n = 3 versus 82 ± 6 K-562-P-selectin-rolling cells, n = 3) confirming that
tyrosine sulfate residues distinctly contribute to support L-selectin
and P-selectin-dependent rolling, Tyr-51 playing a major
role in supporting L-selectin-dependent rolling whereas
Tyr-48 has an essential role in mediating
P-selectin-dependent rolling (Fig. 5, d and
e).
Leukocyte Rolling Velocity on PSGL-1 Is Regulated by N-terminal
Tyr Sulfate Residues and O-Glycans Attached to
Thr-57--
Neutrophils rolled significantly faster on PSGL-1
Y46F/Y48F/Y51F (median-rolling velocity: 80 µm/s, P25: 29 µm/s;
P75 = 155 µm/s; n = 3, Fig.
6a) than on wild-type PSGL-1
(median: 44 µm/s, P25 = 23 µm/s; P75 = 69 µm/s;
n = 3, p < 0,001, Fig. 6a)
emphasizing the key role played by tyrosine residues in supporting
neutrophil rolling. Higher rolling velocities were also observed on
mutants expressing a single N-terminal tyrosine residue (Fig.
6a). Among these mutants, lower rolling velocities were
observed on PSGL-1 Y48F/Y51F and PSGL-1 Y46F/Y48F presenting Tyr-46 or
-51 as single sulfated tyrosine residue (median-rolling velocity on
Tyr-46: 43 µm/s, P25 = 18 µm/s, P75 = 92 µm/s;
p < 0.001, n = 3; median-rolling velocity on Tyr-51: 47 µm/s, P25 = 22 µm/s, P75 = 92 µm/s; p < 0.01, n = 3, Fig.
6a) than on PSGL-1 Y46F/Y51F expressing only Tyr-48
(median-rolling velocity: 61 µm/s, P25 = 25 µm/s, P75 = 132 µm/s, p < 0.001, n = 3).
Lymphocytes rolled faster than neutrophils on wild-type PSGL-1 (median
rolling velocity of lymphocytes: 58 µm/s, P25 = 48 µm/s,
P75 = 71 µm/s, n = 4 versus 44 µm/s, P25 = 23 µm/s, P75 = 69 µm/s for neutrophils,
n = 5, p < 0.0001). The replacement of
all three Tyr by Phe strongly increased lymphocyte-rolling velocity
(median-rolling velocity on PSGL-1 Y46F/Y48F/Y51F: 163 µm/s, P25 = 135 µm/s, P75 = 198 µm/s, n = 4, p < 0,0001, Fig. 6b). The replacement of 2 N-terminal Tyr residues by Phe also significantly increased
lymphocyte-rolling velocities on PSGL-1 (p < 0.0001, n = 4, Fig. 6b). Lower rolling velocities
were observed on PSGL-1 mutants expressing Tyr-51 or -46 than on
mutants expressing only Tyr-48 (p < 0.001, n = 4, Fig. 6b). Lymphocyte-rolling
velocities on mutants expressing Tyr-51 (PSGL-1 Y46F/Y48F,
median-rolling velocity: 123 µm/s, P25 = 99 µm/s, P75 = 150 µm/s, n = 4) or Tyr-46 (PSGL-1 Y48F/Y51F, median:
116 µm/s, P25 = 92 µm/s, P75 = 145 µm/s) were not
statistically different. On the other hand, higher rolling velocities
were observed on PSGL-1 Y46F/Y51F, which expressed Tyr-48
(median-rolling velocity 150 µm/s, P25 = 119 µm/s, P75 = 190 µm/s, n = 4, p < 0.001).
Additional experiments were performed with 300.19 cells expressing
L-selectin to show that the predominant role of Tyr-51 in regulating
L-selectin-dependent rolling velocity was not cell
type-specific. The rolling velocity of 300.19-L-selectin cells was
significantly lower on PSGL-1 Y46F/Y48F (median-rolling velocity: 89 µm/s; P25: 75 µm/s; P75 = 109 µm/s) than on PSGL-1 Y46F/Y51F
(median-rolling velocity: 173 µm/s; P25: 135 µm/s; P75 = 195 µm/s, p < 0.001) or PSGL-1 Y48F/Y51F (median-rolling
velocity: 121 µm/s; P25: 98 µm/s; P75 = 159 µm/s,
p < 0.05) confirming that Tyr-51 plays a key role in
regulating L-selectin-dependent rolling on PSGL-1 whereas
Tyr-48 has a less important role. The distinct contribution of Tyr
sulfate residues in regulating cell rolling velocity was not dependent
on shear stress. Thus, 300.19 cells rolled faster on PSGL-1 Y46F/Y51F
than on PSGL-1 Y46F/Y48F at 1.0, 2.0 and 3.0 dyn/cm2
(median cell rolling velocity on PSGL-1 Y46F/Y51F versus
PSGL-1 Y46F/Y48F at 1.0 dyn/cm2: 140 µm/s
versus 92 µm/s; at 2.0 dyn/cm2: 173 µm/s
versus 88 µm/s; at 3.0 dyn/cm2: 174 µm/s
versus 92 µm/s; p < 0.001).
The contribution of tyrosine sulfate residues in regulating L-selectin-
and P-selectin-dependent rolling velocity was examined in
experiments comparing side-by-side 300.19-L-selectin and
K-562-P-selectin rolling under a constant shear stress of 2.0 dyn/cm2. Rolling velocities of 300.19-L-selectin cells on
mutant PSGL-1 was strongly increased by the replacement of Tyr-46 and
-51 by Phe, leaving Tyr-48 as single tyrosine residue (median-rolling velocity on wild-type versus PSGL-1 Y46F/Y51F: 41 µm/s
(P25: 30 µm/s; P75 = 80 µm/s) versus 174 µm/s
(P25: 135 µm/s; P75 = 195 µm/s), p < 0.001, n = 3, Fig. 6c). In contrast, rolling
velocity of K-562-P-selectin cells was not significantly increased on
PSGL-1 Y46F/Y51F (median-rolling velocity on wild-type
versus PSGL-1 Y46F/Y51F: 8 µm/s (P25: 4 µm/s; P75 = 18 µm/s) versus 11 µm/s (P25: 5 µm/s; P75 = 20 µm/s); n = 3, Fig. 6d) whereas
significantly higher rolling velocity were observed on PSGL-1 Y48F/Y51F
(median-rolling velocity: 19 µm/s (P25: 12.5 µm/s; P75 = 26 µm/s); n = 3, p < 0.001, Fig.
6d). The key role played by Tyr-48 in regulating
P-selectin-dependent cell-rolling velocity contrasts with
its minor role in supporting L-selectin-dependent rolling
on PSGL-1. These differences indicate that N-terminal tyrosine sulfate
residues are distinctly involved when they support L-selectin or
P-selectin interactions with their common ligand.
O-glycans presented by Thr-57 played an essential role in
regulating lymphocyte-rolling velocity. The replacement of Thr-57 by
Ala strikingly increased rolling velocities (median-rolling velocity on
PSGL-1 T57A: 176 µm/s, P25 = 137 µm/s, P75 = 238 µm/s, n = 4, p < 0.0001, Fig.
6b). Surprisingly, lymphocyte rolling at very high
velocities was still observed on PSGL-1 Y46F/Y48F/Y51F/T57A mutants,
which lack core-2 O-glycans attached to Thr-57 and
N-terminal tyrosine residues (PSGL-1 Y46F/Y48F/Y51F/T57A: 202 µm/s,
P25 = 153 µm/s, P75 = 280 µm/s, n = 4, p < 0.001, Fig. 6b).
Additional analysis was performed to examine the frame-by-frame
velocity of 300.19-L-selectin cells rolling on PSGL-1 mutants under a
constant shear stress of 1.25 dyn/cm2. The velocity of
tracked cells was determined by measuring cell displacements within
successive video frames (0.032 s) in the flow direction. Each increase
in velocity is represented by a peak and each decrease by a valley
(Fig. 7a). The replacement of
tyrosine sulfate residues by Phe strongly increased the variations of
cell-rolling velocity indicated by higher irregularity in
"peaks" and "valleys", as illustrated in Fig.
7a. The observed instability of cell rolling was quantified
by calculating the S.D. of the mean velocity of each tracked cell. The
pooled data obtained from the whole cell population were used to
determine the mean S.D. of rolling velocities on wild-type and each
PSGL-1 mutant. More irregular rolling velocities were observed on
PSGL-1 mutants devoid of tyrosine sulfate residues or expressing Tyr-48
as single tyrosine sulfate residue (mean ± S.D. of
300.19-L-selectin cell-rolling velocities on PSGL-1 Y46F/Y48F/Y51F: 170 µm/s versus 163 µm/s on PSGL-1 Y46F/Y51F,
n = 5, p < 0.001) than on wild-type
PSGL-1 (mean ± S.D.: 60 µm/s, n = 5, p < 0.001, Fig. 7a). The stability of
rolling velocity was less affected on mutant PSGL-1 expressing Tyr-46
(mean ± S.D. on PSGL-1 Y48F/Y51F: 111 µm/s, n = 5) or Tyr-51(mean ± S.D. on PSGL-1 Y46F/Y48F: 117 µm/s,
n = 6) than Tyr-48 (Fig. 7a).
The distribution of travel distances illustrated in Fig. 7b
was assessed by measuring cell displacements within successive video
frames (0.032 s; 891-1477 determinations for each cell category). Data
obtained for each cell category were pooled and illustrated in Fig.
7b. The replacement of tyrosine sulfate residues strongly affected cell displacements. A significantly higher percentage of
300.19-L-selectin cells rolled > 4.1 µm on PSGL-1
Y46F/Y48F/Y51F, within a video frame, than on wild-type PSGL-1 (53.2 versus 15.7%, p < 0.004, Fig.
7b). A broader range of travel distances was observed on
mutants expressing Tyr-48 as single sulfated tyrosine. Thus, the
300.19-L-selectin cells more frequently traveled > 4.1 µm on
PSGL-1 Y46F/Y51F than on PSGL-1 Y48F/Y51F (37.3 versus
11.0%, number of observed events:
The analysis of the variation of velocity and travel distances of
300.19-L-selectin cells on PSGL-1 T57A lead to similar observations. In
the absence of O-glycans attached to Thr-57 (PSGL-1 T57A), 300.19-L-selectin cells exhibited a very unstable rolling velocity (mean ± S.D. of rolling velocities on PSGL-1 T57A: 198 µm/s
(n = 7) versus 60 µm/s on wild-type PSGL-1
(n = 5), p < 0.001, Fig. 8a). In contrast, the
replacement of Thr-44 by Ala had no effect (mean ± S.D. on PSGL-1
T44A: 77 µm/s, n = 7). Similarly to observations made
on PSGL-1 T57A, the rolling velocity of 300.19-L-selectin cells was very unstable in the absence of sLex/CLA
presentation by core-2 O-glycans (mean ± S.D. on CHO
cells co-transfected with PSGL-1 and FucT-VII cDNAs without C2GnT
cDNA: 162 µm/s, n = 11, p < 0.001, not illustrated). A higher percentage of cells rolled on longer
distances on PSGL-1 T57A than on wild-type PSGL-1 (no. of cells that
rolled >4.1 µm: 64.9 versus 20.2%, no. of
determinations: n Lack of Inhibition of L-selectin-dependent Rolling on
PSGL-1 by CSLEX-1 and HECA-452 mAbs.--
The anti-CLA mAb HECA-452
was reported to inhibit by >90% L-selectin-dependent
lymphocyte rolling on the human vascular endothelial cell line EA hy926
transfected with FucT-VII cDNA suggesting that CLA, a
sLex determinant, is a major determinant of endothelial
L-selectin ligand(s) (55). In contrast to these observations,
neutrophil rolling was not significantly reduced by HECA-452 mAb (no.
of neutrophils rolling on PSGL-1 in presence of HECA-452 mAb
versus control mAb, mean ± S.E.: 239 ± 21 versus 213 ± 49 neutrophils/min/mm2,
n = 6).
Molecular Modeling of L-selectin Interactions with PSGL-1--
The
RMSD calculated between all P-selectin structures (23) for each amino
acids allowed for the identification of the regions of high and low
mobility (52). Loops 42-48 and 108-114 of L-selectin and P-selectin,
which interact with PSGL-1-sulfated Tyr-48, have low mobility and are
surrounded by low mobility regions. In contrast, loop 64-89 of
L-selectin and P-selectin, which interacts with PSGL-1-sulfated Tyr-51,
exhibits high mobility. Thus, we analyzed only the interactions of
loops 42-48 and 108-114 of L-selectin with sulfated Tyr-48 of PSGL-1.
Hydrogen-bonding pattern between L-selectin or P-selectin and Tyr-48
was calculated using the HBPLUS program, based on a homology model
obtained with the MODELLER program (45, 50). Hydrogen bonds involved in
the interactions of sulfated Tyr-48 with L-selectin and
P-selectin are indicated in Table
I and Fig.
9. Two potential hydrogen bonds were
disclosed between Ser-47 of L-selectin and the sulfate
group of Tyr-48, whereas P-selectin binding to PSGL-1-sulfated Tyr-48
is supported (1) by 4 hydrogen bonds located between Ser-46, Ser-47,
His-114, and the sulfate group of Tyr-48 and (2) by an additional
hydrogen bond located between the peptidic backbones of Lys-112 and
Tyr-48 (Fig. 9 and Table I). Importantly, the basic residue present at
position 114 of P-selectin (His-114) is absent on L-selectin.
The determinants of PSGL-1 that mediate L-selectin and P-selectin
binding include tyrosine sulfate residues and O-glycans attached to Thr-57 (24, 33, 34, 56-62). The crystal structure analysis
of P-selectin co-complexed with the N-terminal peptide of
PSGL-1 showed that O-glycans terminated by
sLex/CLA as well as Tyr-48 and -51 play an essential role
in supporting P-selectin binding. In addition, although this
interaction had not been highlighted in the crystallographic study
mentioned above, rolling adhesion assays and glycosulfopeptide binding
studies have suggested a role for PSGL-1 Tyr-46 in adhesion to
P-selectin (23, 24, 34, 35). The results presented here show that P-selectin and L-selectin use similar mechanisms to bind to PSGL-1. However, the three tyrosine sulfate residues of PSGL-1 do not contribute in an equal fashion to L- and P-selectin binding.
Specifically, our results indicate that Tyr-48 is of key importance in
supporting P-selectin-mediated rolling on PSGL-1 whereas it only plays
a minor role in mediating L-selectin binding. In addition, the present study shows that: 1) sialylated and fucosylated core-2
O-glycans attached to Thr-57 are essential to allow optimal
L-selectin binding to PSGL-1 and 2) to support and
stabilize leukocyte rolling on CHO-PSGL-1 cells; 3) at least 2 or 3 N-terminal Tyr sulfate residues are required to optimally support
leukocyte recruitment and rolling; and 4) Tyr-51 plays a predominant
role in recruiting and stabilizing leukocyte rolling on PSGL-1.
We have defined here the minimal molecular requirements supporting
L-selectin interactions with PSGL-1. L-selectin/µ weakly bound to
sLex/CLA-expressing CHO cells in the absence of PSGL-1
(Fig. 2b, upper panel). However, this interaction was not
sufficient to support neutrophil rolling (Fig. 3a). This
observation is consistent with the notion that low affinity
interactions between L-selectin and sLex cannot efficiently
support L-selectin-mediated rolling (13). Interestingly, a low number
of neutrophils rolled on CHO-PSGL-1/FucT-VII cells even in the absence
of C2GnT suggesting that the presentation of sLex/CLA by
core-2 O-linked glycans is not essential to support
L-selectin-dependent rolling (Fig. 3a). The
strong reduction in neutrophil rolling and L-selectin/µ binding
observed after the replacement of Thr-57 (but not of Thr-44) by Ala
confirmed the essential role played by Thr-57 in presenting
O-glycans involved in L-selectin binding (Fig. 4).
Interestingly, leukocyte rolling was less affected by the replacement
of the whole tyrosine sulfation consensus by Phe residues, suggesting a
predominant role for core-2 O-glycans (Fig. 5, b
and c). A critical role for sialic acid residues was
indicated by the abrogation of neutrophil rolling on PSGL-1 after
sialidase treatment of CHO cells. Similar observations were previously
reported for P-selectin (22, 34, 37, 63). Sialic acid residues presented by sLex/CLA determinants most likely play a key
role in this interaction.
C2GnT co-expression with FucT-VII and PSGL-1 strongly increased
leukocyte recruitment and decreased rolling velocity (Fig. 3). Similar
observations were made by others who studied PSGL-1 interactions with
P-selectin in vitro using leukocyte isolated from
C2GnT-deficient mice or in vivo in C2GnT-deficient mice (25, 28). Higher rolling velocities on P-selectin were observed in the
absence of C2GnT suggesting that core-2 O-linked glycans
have a major role in regulating leukocyte-rolling velocity. The present study extends these observations to L-selectin. By stabilizing and
reducing leukocyte-rolling velocity on L-selectin or P-selectin, core-2
O-glycans may improve the exposure of rolling cells to cytokines or chemokines at site of inflammation and promote leukocyte arrest and firm adhesion following integrin activation.
Whereas the results of this study confirm that L-selectin interaction
with PSGL-1 is dependent on the expression of O-glycans attached to Thr-57 and of N-terminal tyrosine sulfate residues (Figs. 4
and 5) (35), they also revealed that PSGL-1 mutants exhibiting a single
tyrosine sulfate residue were not as efficient as wild-type PSGL-1 in
supporting leukocyte rolling. Moreover, these results showed that
tyrosine sulfate residues did not contribute equally to L-selectin
interactions with PSGL-1. A predominant role for Tyr-51 was indicated
by: 1) the lower rolling velocities (Fig. 6), 2) the more efficient
recruitment of leukocytes on PSGL-1 Y46F/Y48F expressing Tyr-51 as
single tyrosine sulfated residue (Fig. 5), 3) the more efficient
binding of L-selectin/µ to PSGL-1 Y46F/Y48F (Fig. 5a), and
4) the increased stability of leukocyte-rolling velocity on this mutant
PSGL-1 (Fig. 7).
Experiments performed with glycosulfopeptides previously indicated that
Tyr-48 plays a major role in mediating the interactions of the
N-terminal peptide of PSGL-1 with P-selectin (34). Adhesion studies
performed with K-562-P-selectin cells demonstrated with whole cells
that Tyr-48 plays a predominant role in supporting P-selectin-mediated
rolling interactions with PSGL-1 whereas it had only a minor role in
mediating L-selectin-dependent rolling (Fig. 6).
These observations are in keeping with crystal structure analysis that
revealed a major role for His-114 in mediating P-selectin binding to
PSGL-1 Tyr-48 (23). The analysis of hydrogen-bonding pattern disclosed
additional potential bonds, which may strengthen P-selectin
interactions with Tyr-48 (Fig. 9 and Table I). The absence of a basic
amino acid residue at position 114 of L-selectin and the lower number
on hydrogen bonds may explain why Tyr-48 of PSGL-1 plays only a minor
role in supporting L-selectin binding to PSGL-1 (Fig. 9 and Table I).
Of note, molecular modeling analysis of L-selectin interactions with
Tyr-48 is consistent with results of adhesion studies performed under
flow at various shear stress and validate this model.
L-selectin and P-selectin are likely to bind to PSGL-1 in a similar
fashion because of the conservation of residues within the
sLex binding site and the presence of a basic residue (Lys)
at position 85 (23). Electrostatic interactions most likely play a
major role in supporting the negatively charged Tyr-51 binding to
L-selectin Lys-85 and to P-selectin Arg-85. An important role for
Lys-85 in supporting L-selectin binding to PSGL-1 is
suggested by the elevated partial charge of the ammonium group of
Lys-85 (+0.69) (64). In comparison, a lower charge is associated to the
iminium group of Arg-85 (+0.12) of P-selectin. The absence of a basic amino acid residue at position 114 of L-selectin and the presence of
Lys-85 may explain why sulfated Tyr-51 plays a predominant role in
supporting L-selectin interactions with PSGL-1 whereas sulfated Tyr-48
is less important.
L-selectin/µ binding studies and adhesion assays indicated that
Tyr-46 plays an important role in supporting L-selectin interactions with PSGL-1. Leukocyte rolling was more stable and slower on PSGL-1 Y48F/Y51F than on PSGL-1 Y46F/Y51F (Figs. 6 and 7). Interestingly, the
involvement of Tyr-46 in P-selectin binding was not revealed by crystal
structure analysis (23) whereas binding studies of PSGL-1
glycosulfopeptides to P-selectin (34) and adhesion studies performed
with K-562-P-selectin cells (Figs. 5e and 6d)
indicate that Tyr-46 supports this reaction. The role of Tyr-46 in
mediating L-selectin and P-selectin binding was recently further
supported by the ability of the anti-PSGL-1 mAb PS-4, which reacts with Tyr-46 but not with Tyr-48 or -51, to inhibit
L-selectin-dependent rolling on
PSGL-1.2
Rolling is an important step during which leukocytes are exposed to
chemoattractants at sites of inflammation, a reaction that leads to
integrin activation and leukocyte firm adhesion. Frame by frame
analysis of cell displacements indicates that cell rolling occurs
through a series of steps or jerks that appear to represent
receptor-ligand dissociation events (65-67). Rolling through selectins
is unaffected by alterations in selectin density and hydrodynamic
forces acting on the cell (65). This surprising stability of rolling
has been explained by the ability of leukocyte to reach a dynamic
balance between formation and breakage of bonds between selectins and
their ligands over a wide range of wall shear stress and ligand
densities (66, 67). The stereospecific interactions of tyrosine sulfate
and O-glycans with P-selectin and L-selectin create a high
affinity binding site (23, 27, 34), which contributes to rolling
stabilization. The highly irregular cell rolling observed on PSGL-1
mutants devoid of tyrosine sulfate residues or expressing Tyr-48 as
single tyrosine sulfate residue (Fig. 7a) indicates that
Tyr-46 and -51 play a major role in the ability of PSGL-1 to stabilize
L-selectin-mediated rolling. Similar observations were made for core-2
O-glycans attached to Thr-57 which present
sLex/CLA to L-selectin (Fig. 8a).
Post-translational modifications of PSGL-1 may facilitate endothelium
surveillance for signs of inflammation and thereby represent important
additional levels of regulation of leukocyte traffic.
1,6-N-acetylglucosaminyltransferase and
fucosyltransferase VII was required for optimal L-selectin binding to
PSGL-1. Substitution of Thr-57 by Ala but not of Thr-44, strongly
decreased L-selectin binding and leukocyte rolling on PSGL-1.
Substitution of Tyr by Phe revealed that PSGL-1 Tyr-51 plays a
predominant role in mediating L-selectin binding and leukocyte rolling
whereas Tyr-48 has a minor role, an observation that contrasts with the
pattern seen for the interactions between PSGL-1 and P-selectin where
Tyr-48 plays a key role. Molecular modeling analysis of L-selectin and P-selectin interactions with PSGL-1 further supported these
observations. Additional experiments showed that core-2
O-glycans attached to Thr-57 were also of critical
importance in regulating the velocity and stability of leukocyte
rolling. These observations pinpoint the structural characteristics of
PSGL-1 that are required for optimal interactions with
L-selectin and may be responsible for the specific kinetic and
mechanical bond properties of the L-selectin-PSGL-1 adhesion
receptor-counterreceptor pair.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1,6-N-acetylglucosaminyltransferase transferase (C2GnT)
from M. Fukuda (the Burnham Institute, La Jolla Cancer Research Center,
San Diego, CA). The cDNA sequence encoding the internal ribosome
entry site (IRES) of the encephalomyocarditis virus sequence was a gift
from P. Aebischer (EPFL, Lausanne, Switzerland). The pZeoSV and the
pcDNA3.1 vectors were from Invitrogen (Groningen, The Netherlands).
The IRES sequence was inserted in the multiple cloning sites of the
pZeoSV vector (pIRES Zeo SV vector). C2GnT and FucT-VII cDNAs were
then subcloned in the pIRES Zeo SV vector to permit the translation of
C2GnT and FucT-VII sequences from one mRNA. The expression cassette
was constructed by inserting the C2GnT sequence followed by the IRES
and the FucT-VII cDNA sequences in the multiple cloning site of the
pZeo SV vector. This type of vector allows the expression of the target
gene (C2GnT) and the selection marker (sLex expression
associated to FucT-VII activity) from the same promoter so that
virtually all transfected cells expressing the selection marker also
express the gene of interest (40-42).
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Fig. 1.
N-terminal amino acid
sequences of wild-type and mutant PSGL-1. The first 21 amino acids
of mature PSGL-1 are indicated. Amino acid substitutions at Tyr-46,
-48, and -51 by Phe and at Thr-57 and -44 by Ala are shown in
bold.
cells (ATCC number: CRL 9096) were stably
transfected with cDNAs encoding wild-type or mutant PSGL-1,
subcloned in pCDNA3.1 vector. When indicated, CHO cells were
co-transfected with FucT-VII cDNA subcloned in pZeoSV vector
(Invitrogen) or in the pIRES Zeo SV expression vector, which allows the
simultaneous translation of C2GnT and FucT-VII sequences. Transfections
were performed using LipofectAMINETM Plus (Invitrogen). CHO
dhfr
were cultured in MEM
medium containing
ribonucleotides, deoxyribonucleotides, and 10% fetal calf serum; COS-7
cells were cultured in Dulbecco's modified Eagle's medium/10% fetal
calf serum. Transfectants were selected in medium containing 400 µg/ml G418 (Invitrogen) and, when required, 200 µg/ml Zeocin
(Invitrogen). Individual clones expressing high levels of the various
forms of PSGL-1, C2GnT, and FucT-VII-dependent expression
of sLex and the cutaneous lymphocyte antigen (CLA) were
isolated by limiting dilutions and identified by immunophenotypic
analysis. The expression of PSGL-1, sLex, and CLA was
assessed using, respectively, PL2, CSLEX-1, and HECA-452 mAbs. CHO
cells, which were selected for adhesion studies, expressed similar
levels of PSGL-1 and of sLex/CLA. The expression of C2GnT
and FucT-VII mRNAs in CHO cells expressing wild-type or mutant
PSGL-1 was detected by RT-PCR using the one tube Titan SystemTM (Roche
Diagnostic, Rotkreuz, Switzerland). Sequences of forward and reverse
primers used for C2GnT PCR amplification were
GGCAGTGCCTACTTCGTGGTCA and ATGCTCATCCAAACACTGGATGGCAAA; for FucT-VII, CCCACCGTGGCCCAGTACCGCTTCT and CTGACCTCTGTGCCCAGCCTCCCGT; for
PSGL-1, ATGCCTCTGCAACTCCTCCT and CTGCTGAATCCGTGGACAGGTT. The expression levels of the various forms of PSGL-1 by CHO cells were
determined by the measurement of antigen site density using the DAKO
QIFIKIT® (Dako, Glostrup, Denmark). Antibody binding site
density was calculated (44) using PL2 mAb to assess PSGL-1 expression.
PL2 binding sites were found equal to 115 ± 37 (mean ± S.D.) binding sites/µm2 in CHO cells expressing
constructs used to perform experiments illustrated in Figs. 4-8. CHO
cells used in experiments illustrated in Figs. 2 and 3 expressed higher
levels of PSGL-1 (247 ± 19 PL2 binding sites/µm2).
The determination of sLex and CLA expression indicated that
CHO cells used for L-selectin/µ binding studies and adhesion assays
expressed comparable levels of FucT-VII activity.
1 s and when the distance traveled by
leukocytes during observation periods (20 s) was
1 cell diameter.
These cells were considered as rolling cells. Experiments were
performed in quadruplicates under constant shear stress. Rolling
velocities illustrated in Figs. 3b and 6 were measured in
the direction of flow by tracking individual cells every 0.25 s,
for up to 4 s, using digitized images. 300-800 independent
determinations of cell velocity were measured for each tested
condition. Frame-by-frame velocity data obtained by tracking cells
every 0.032 s are illustrated in Figs. 7a and 8a
and were used to assess the mean velocity ± S.D. of each tracked
cell over 2-5 s observation periods. The mean velocity of
frame-by-frame tracked cells was included between percentile 25-75 of
the velocity of each cell population illustrated in Fig. 6. The S.D.
value of the mean velocity of each tracked cell was then used to
calculate the mean ± S.D. of cell-rolling velocities of each cell
population. The mean ± S.D. was used as an indicator of the
variation of cell-rolling velocity. 373-1477 independent determinations of frame-by-frame velocity were measured for each tested
conditions. The anti-L-selectin mAb LAM 1-3 and the
anti-PSGL-1 mAbs PL1 or KPL1 were used as inhibitors of L-selectin
interaction with PSGL-1. The isotype-matched mAbs LAM1-14 and PL2 were
used as controls. In experiments evaluating the inhibitory effect of HECA-452, a rat IgM mAb was used as control (Dako). Leukocytes expressed similar levels of L-selectin before and at the end of each experiment.
2 s. Neutrophil
arrest was abrogated in presence of the adhesion-blocking anti-CD18 mAb
TS1/18 (Endogen, Woburn, MA).
atoms between the L-selectin model and the P-selectin template with the Swiss PDB viewer program (49). Assuming that PSGL-1 interactions with L-selectin and P-selectin
are highly similar, we used the coordinates of PSGL-1 (PDB Data Bank
ID: 1G1S) and superimposed L-selectin on P-selectin to analyze
L-selectin interactions with PSGL-1. Hydrogen-bonding pattern was
analyzed using the HBPLUS program and standard geometric definitions
considering the distance and the angle between the hydrogen atom and
the acceptor/donor atoms (50). To compute hydrogen bonds, we used, as
criteria, 3,9 Å as maximal distance between heavy atoms and 90° as
minimal angle between the donor (D) atom, the hydrogen (H) atom, and
the acceptor (A) atom (DHA angle), the probability of finding
energetically favorable hydrogen bonds being smaller at longer
distances or smaller angles (51).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Role of core-2 O-glycans and
sLex/CLA in modulating L-selectin interactions with
PSGL-1. a, immunophenotypic analysis of the expression
of sLex and CLA by CHO cells, stably transfected with
C2GnT/FucT-VII/PSGL-1 cDNAs, using mAbs CSLEX-1
(anti-sLex) and HECA-452 (anti-CLA). b,
functional studies examining L-selectin/µ chimera binding to CHO
cells expressing high levels of the indicated cDNAs. Binding of
L-selectin/µ was completely inhibited by the presence of 5 mM EDTA (dotted lines). The proportion of
positive cells is indicated in each histogram. mAbs and L-selectin/µ
did not bind significantly (< 2%) to mock-transfected CHO
cells.
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Fig. 3.
Role of core-2 O-glycans and
sLe/CLA in regulating neutrophil recruitment and velocity
on PSGL-1 expressing cells: a, neutrophils were perfused
under a constant shear stress of 1.25 dyn/cm2 on CHO cells
stably co-transfected with C2GnT/FucT-VII/PSGL-1 cDNAs, as
indicated. Neutrophil rolling was analyzed by videomicroscopy at 2-4
min of perfusion. Results represent the mean ± S.E. of four
experiments. b, velocity of neutrophil rolling on CHO cells
stably co-transfected with C2GnT/FucT-VII cDNAs with or without
PSGL-1 cDNA, after 2-4 min of perfusion. Curves were constructed
using 720 independent determinations of rolling velocity and are
representative of four experiments.
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Fig. 4.
Core-2 O-glycans attached to
Thr-57 mediate L-selectin interactions with PSGL-1. a,
binding of L-selectin/µ to CHO cells stably expressing FucT-VII,
C2GnT and wild-type or mutant PSGL-1 cDNAs. Thr-44 or -57 were
substituted by Ala in PSGL-1 T44A and PSGL-1 T57A. The proportion of
positive cells is indicated in each histogram. b, neutrophil
recruitment on CHO cells stably co-expressing FucT-VII, C2GnT and
PSGL-1 T44A or PSGL-1 T57A cDNAs. Results represent the mean ± S.E. of 4-8 experiments (***, p < 0.001).
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Fig. 5.
Role of N-terminal tyrosine sulfate residues
in mediating L-selectin/µ binding and
neutrophil recruitment on PSGL-1. a, binding of
L-selectin/µ to CHO cells stably expressing FucT-VII, C2GnT, and
wild-type or mutant PSGL-1 cDNAs. Tyr-46 and/or -48 and/or -51 were
substituted by Phe; Thr-57 was substituted by Ala in PSGL-1
Y46F/Y48F/Y51F/T57A. The proportion of positive cells is indicated in
each histogram. b, neutrophil recruitment on CHO cells
stably co-expressing FucT-VII, C2GnT, and mutant PSGL-1 in which the
indicated tyrosine residues were substituted by Phe. Neutrophils were
perfused under a constant shear stress of 1.25 dyn/cm2.
Results represent the mean ± S.E. of five experiments.
c, lymphocyte recruitment on CHO cells expressing wild-type
or mutant PSGL-1. Results represent the mean ± S.E. of three
experiments. d recruitment of 300.19-L-selectin-cells and
e, of K-562-P-selectin cells on wild-type or mutant PSGL-1
cDNAs. Results are expressed as percentage of rolling cells (± S.E.). ***, p < 0.001; **, p < 0.01;
*, p < 0.05.
View larger version (40K):
[in a new window]
Fig. 6.
Regulation of leukocyte rolling velocity by
tyrosine sulfate residues. a, neutrophils were perfused
under a constant shear stress of 1.25 dyn/cm2 on CHO cells
stably co-transfected with C2GnT/FucT-VII and wild-type or mutant
PSGL-1 cDNAs. The rolling velocity of neutrophils was assessed at
2-4 min of perfusion and represent 300-800 independent
determinations. Data are representative of three experiments.
b, rolling velocity of lymphocytes under the conditions
described in a. Data are representative of three
experiments. c, rolling velocity of 300.19-L-selectin cells
and d, of K-562-P-selectin cells under a constant shear
stress of 2.0 dyn/cm2. Data are representative of three
experiments.
View larger version (30K):
[in a new window]
Fig. 7.
Tyrosine sulfate residues stabilize leukocyte
motions. a, frame-by frame rolling velocity of
300.19-L-selectin cells on CHO cells stably expressing C2GnT/FucT-VII
and wild-type or mutant PSGL-1. The velocity of tracked cells was
determined by measuring cell displacements within successive video
frames (0.032 s) in the flow direction, under a shear stress of 1.25 dyn/cm2. Data are representative of 5-11 experiments.
b, distribution of distances traveled by 300.19 cells
rolling on wild-type PSGL-1 or mutant PSGL-1 during successive 0.032-s
observation periods. Data are representative of 891-1477
determinations.
891, p < 0.001)
or PSGL-1 Y46F/Y48F (18.6%, p < 0.003).
440, p < 0.001)
or on PSGL-1 T44A (64.9 versus 16.5%, n
373, p < 0.001, Fig. 8b).
View larger version (20K):
[in a new window]
Fig. 8.
Role of core-2 O-glycans
attached to Thr-57 in stabilizing leukocyte motions. a,
frame-by frame rolling velocity of 300.19-L-selectin cells on CHO cells
stably expressing C2GnT/FucT-VII and wild-type or mutant PSGL-1, under
a shear stress of 1.25 dyn/cm2. The velocity of tracked
cells was determined as explained in the legend of Fig. 7. Data are
representative of 5-11 experiments. b, distribution of
distance traveled by 300.19 cells rolling on wild-type or mutant PSGL-1
during successive 0.032-s observation periods. Data are representative
of 373-1331 determinations.
Hydrogen bonds mediating L-selectin and P-selectin interactions with
sulfated Tyr-48
View larger version (16K):
[in a new window]
Fig. 9.
Models of L-selectin (A) and
P-selectin interactions (B) with PSGL-1-sulfated
Tyr-48. Drawings were made with the LIGPLOT program (68) and
contain the labels of the residues and the atoms involved in the
interactions. The presence of hydrogen bonds is indicated by
dashed lines. The bond lengths are expressed in Å. Carbon
atoms are shown in black, nitrogen atoms in dark
gray, oxygen atoms in light gray, and sulfur atoms in
white. L, L-selectin; P,
P-selectin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Philippe Schneider, Dr. Jean-Daniel Tissot, and the staff of the Centre de Transfusion Sanguine at Lausanne for providing blood samples. We thank Dr. J. Lowe for FucT-VII cDNA and Dr. M. Fukuda for C2GnT cDNA.
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FOOTNOTES |
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* This work was supported by Grant 32-065177.01 from the Swiss National Foundation for Scientific Research.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: Division of
Hematology, University of Lausanne, BH 18-544, 1011-CHUV Lausanne, Switzerland. Tel.: 41-21-314-42-26; Fax: 41-21-314-41-80; E-mail: Olivier.Spertini@chuv.hospvd.ch.
Published, JBC Papers in Press, October 25, 2002, DOI 10.1074/jbc.M204360200
2 S. Giraud, C. Abbal, M. P. Bernimoulin, M. Gikic, A.-S. Rivier, M. Schapira, and O. Spertini, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: PSGL-1, P-selectin glycoprotein ligand-1; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; RMSD, root mean-squared deviation; IRES, internal ribosome entry site.
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