From the W. K. Warren Medical Research Institute,
Departments of
Medicine and § Biochemistry and
Molecular Biology, University of Oklahoma Health Sciences Center, and
the ** Cardiovascular Biology Research Program, Oklahoma Medical
Research Foundation, Oklahoma City, Oklahoma 73104 and the
¶ Department of Immunology, Boehringer Ingelheim Pharmaceuticals,
Ridgefield, Connecticut 06877
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ABSTRACT |
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The major high affinity ligand for P-selectin on human leukocytes is P-selectin glycoprotein ligand-1 (PSGL-1). To bind P-selectin, PSGL-1 must be modified with tyrosine sulfate and sialylated, fucosylated, core-2 O-glycan(s). The required sites for these modifications on full-length PSGL-1 have not been defined. The N-terminal region of mature PSGL-1, which begins at residue 42, includes tyrosines at residues 46, 48, and 51, plus potential sites for Thr-linked O-glycans at residues 44 and 57. We expressed full-length PSGL-1 constructs with substitutions of these residues in transfected Chinese hamster ovary cells. The cells were co-transfected with cDNAs for the glycosyltransferases required to construct sialylated and fucosylated, core-2 O-glycans on PSGL-1. The transfected cells were assayed for their abilities to bind fluid-phase P-selectin and to support rolling adhesion of pre-B cells expressing P-selectin under hydrodynamic flow. In both assays, substitution of Thr-57 with alanine eliminated binding of PSGL-1 to P-selectin without affecting sulfation of PSGL-1, whereas substitution with serine, to which an O-glycan might also be attached, did not affect binding. Binding was not altered by substituting alanines for the two amino acids on either side of Thr-57, or by substituting alanine for Thr-44. Substitution of all three tyrosines with phenylalanines markedly reduced sulfation and prevented binding to P-selectin. However, all constructs in which one or two tyrosines were replaced with phenylalanines bound P-selectin. These results suggest that full-length PSGL-1 requires an O-glycan attached to Thr-57 plus sulfation of any one of its three clustered tyrosines to bind P-selectin.
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INTRODUCTION |
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The selectins are Ca2+-dependent lectins that initiate rolling adhesion of leukocytes on blood vessel surfaces at inflammatory sites (reviewed in Refs. 1 and 2). L-selectin is expressed on leukocytes, and E- and P-selectin are expressed on activated endothelial cells or platelets. The selectins bind weakly to sialylated and fucosylated glycans such as sialyl Lewis x (sLex),1 and L- and P-selectin also recognize many sulfated glycans (3). However, the selectins bind preferentially to only a few cell surface glycoproteins (1). One of these is P-selectin glycoprotein ligand-1 (PSGL-1) (reviewed in Ref. 4). PSGL-1 is the major high affinity ligand for P-selectin on human leukocytes (5). PSGL-1 also binds to E-selectin (6-9) and to L-selectin (10-14). Studies with mAbs indicate that PSGL-1 mediates rolling adhesion of flowing neutrophils on P-selectin (15-17).
Human PSGL-1 is a disulfide-bonded homodimer with two 120-kDa subunits as determined by SDS-PAGE (5). The PSGL-1 subunit is a type 1 membrane protein with an extracellular domain containing many serines, threonines, and prolines, including a series of decameric repeats (15 in promyelocytic HL-60 cells and 16 in leukocytes) (8, 15, 18). Following an 18-residue signal peptide, there is a propeptide spanning residues 19-41 that is removed from PSGL-1 after its synthesis in leukocytes (8, 19, 20). The extracellular domain of the processed mature protein extends from residues 42 to 318, and is followed by a 25-residue transmembrane domain and a 69-residue cytoplasmic tail (Fig. 1). Consistent with its predicted amino acid sequence, each PSGL-1 subunit has no more than three N-glycans, but has many clustered, sialylated O-glycans (5, 21). The extracellular domain of PSGL-1 is also highly extended, a characteristic feature of mucins (19).
The post-translational modifications of PSGL-1 required for selectin
recognition have been studied in human leukocytes and in COS, CHO, or
K562 cells co-transfected with cDNAs encoding PSGL-1 and specific
glycosyltransferases (5, 6, 8, 21-26). The N-glycans of
PSGL-1 are not required for selectin binding (5). Instead, PSGL-1 must
be sialylated and fucosylated on branched core-2 O-glycans
to interact with selectins (25). Almost all of the O-glycans
on PSGL-1 from HL-60 cells have core-2 structures, but only 14% of
these glycans are fucosylated (27). This suggests that Ser/Thr-linked
O-glycans may be modified in a site-specific manner.
The O-glycans of PSGL-1 are not sulfated (22, 27), but PSGL-1 does incorporate sulfate into a cluster of three N-terminal tyrosines located at residues 46, 48, and 51 (22-25). Enzymatic removal of sulfate, blockade of sulfation, or replacement of the three tyrosines with phenylalanines prevents binding of PSGL-1 to P-selectin but not to E-selectin (22-25). PL1, an IgG anti-PSGL-1 mAb that blocks binding to P-selectin (15), recognizes a protein epitope spanning residues 49-62 that overlaps the tyrosine sulfation sites (19). These data indicate that PSGL-1 must be modified with tyrosine sulfate as well as with specific O-glycan(s) to bind P-selectin. An important unresolved issue is which of the three tyrosine residues must be sulfated for PSGL-1 to bind P-selectin.
The location of the O-glycan(s) on PSGL-1 required for
binding to P-selectin is also unclear. An Ig chimera containing only residues 42-60 of PSGL-1 binds to P-selectin when it is co-expressed with an 1,3 fucosyltransferase (FTIII) in COS cells (24) or with
core-2 N-acetylglucosaminyltransferase (C2GnT) and FTVII in
CHO cells (28). This small N-terminal sequence contains two potential
sites for attachment of O-glycans at residues 44 and 57. Substitution of Thr-57 with alanine in this Ig chimera decreases binding to both P- and E-selectin (24). When co-expressed with FTVII in
COS cells, a membrane-anchored form of PSGL-1 with residues 38-57
attached directly to residue 118 of the extracellular domain mediates
cell adhesion to immobilized P-selectin. Replacement of Thr-44 and
Thr-57 with alanines decreases but does not eliminate adhesion (23).
The limited mutational analysis of these truncated chimeras suggests
that O-glycans located at Thr-44 and Thr-57 participate in
binding to P-selectin. However, a key unanswered question is whether
these residues are required for P-selectin recognition in the context
of full-length PSGL-1, in which fucosylated O-glycans
attached to other residues might have equal or greater importance.
In this study we examined how substitutions of the N-terminal clustered tyrosines or the adjacent potential sites for O-glycan addition affected binding of full-length PSGL-1 to P-selectin. We co-expressed each PSGL-1 construct with C2GnT and either FTIII or FTVII in CHO cells. Two complementary assays were used: binding of fluid-phase P-selectin to suspended CHO cells, and rolling of pre-B cells expressing P-selectin on CHO cell monolayers under hydrodynamic flow. Our results suggest that full-length PSGL-1 requires an O-glycan attached at Thr-57 plus sulfation of any one of its three clustered tyrosines to bind P-selectin.
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EXPERIMENTAL PROCEDURES |
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Materials-- Carrier-free [35S]sulfate (1.1-1.6 Ci/mmol) and [3H]GlcNH2 (40 Ci/mmol) were purchased from NEN Life Science Products. Phycoerythrin-streptavidin and protein A-agarose were purchased from Sigma. Biotin hydrazide was obtained from Pierce. LipofectAMINETM and all cell culture reagents were purchased from Life Technologies, Inc. ZeocinTM was from Invitrogen. JM110 competent cells were from Stratagene. Restriction enzymes and T4 ligase were from New England Biolabs. Other chemicals were ACS grade or better; unless stated otherwise, they were obtained from Fisher Scientific.
Antibodies-- The anti-human P-selectin mAbs G1 and S12 were prepared as described (29, 30). G1, but not S12, blocks binding of P-selectin to leukocytes. The anti-human PSGL-1 mAbs PL1 and PL2 were prepared as described (15). PL1, but not PL2, blocks binding of PSGL-1 to P-selectin. These mAbs are all of the IgG1 subclass. The hybridoma secreting the IgM anti-sLex mAb CSLEX-1 (31) was obtained from ATCC. Rabbit antiserum to a peptide corresponding to residues 42-56 of the cDNA-derived amino acid sequence of human PSGL-1 (8) was prepared as described (6). MOPC21, a control nonbinding IgG1 mAb, and MOPC104E, a control nonbinding IgM mAb, were purchased from Cappel-Organon Technika. FITC-conjugated goat anti-mouse IgG/IgM was purchased from Caltag Laboratories. R-phycoerythrin-conjugated goat anti-rabbit IgG was from Molecular Probes.
Proteins-- Human platelet P-selectin and a recombinant soluble form of human P-selectin (sPS, formerly called tPS) were purified as described (32, 33).
cDNAs-- The PSGL-1 cDNA was amplified by the polymerase chain reaction from human leukocyte cDNA (15). The cDNA insert was excised from the pBK-EF vector with ScaI and KpnI and ligated into the mammalian expression vector pZeoSV (Invitrogen) as described (25). The pZeoSV plasmid allows selection for resistance to the antibiotic ZeocinTM in both Escherichia coli and mammalian cells. Mutations in the PSGL-1 cDNA were made by previously described methods (25), with the clarification that the mutant construct in BluescriptTM was excised with XbaI and KpnI and ligated into pBK-EF. The insert was then excised with ScaI and KpnI and ligated into pZeoSV. The sequences of the constructs were confirmed by automated dideoxynucleotide sequencing of both strands of each polymerase chain-reaction cassette. The cDNAs for FTIII and C2GnT were prepared as described (25). The cDNA for human FTVII in the plasmid pCDM8 (34) was a gift from Dr. John Lowe, University of Michigan.
Transfections--
CHO DHFR() cells were maintained in
Dulbecco's modified Eagle's medium containing 10% fetal calf serum,
1% hypoxanthine-thymidine, 0.1 mM nonessential amino
acids, 1% penicillin/streptomycin, and 2 mM glutamine at
37 °C in an atmosphere containing 5% CO2. CHO DHFR(
)
cells were permanently transfected with cDNAs encoding C2GnT and
FTIII as described (25). Portions of these cells were then transfected
with empty pZeoSV, or with wild-type or mutant PSGL-1 cDNA in
pZeoSV using LipofectAMINETM. The cells were selected in medium
containing both G418 (400 µg/ml) and ZeocinTM (200 µg/ml). Individual colonies were expanded and recloned by limited dilution or
by fluorescence-activated cell sorting. In this manner, permanently transfected CHO cells were isolated that expressed wild-type or mutant
PSGL-1 plus C2GnT and FTIII. However, flow cytometric analysis with the
anti-sLex mAb CSLEX-1 indicated that some of the isolated
clones no longer had significant FTIII activity. Furthermore, lysates
of one of the clones (T57S/E61A) no longer had detectable C2GnT
activity. Before P-selectin binding studies, therefore, all clones (at
50-75% confluence) were transiently transfected with C2GnT in
pCDNA3 (Invitrogen) and either FTIII in pRcRSV (Invitrogen) or
FTVII in pCDM8 using LipofectAMINETM. After 48 h the cells reached
confluence. At this time the cells were either harvested for use in the
fluid-phase P-selectin binding assay, or maintained as monolayers for
use in cell adhesion studies under hydrodynamic flow. When cell
adhesion experiments were performed, a fluid-phase P-selectin binding
assay was performed in parallel on CHO cells expressing each
construct.
Flow Cytometry-- Binding of mAbs to intact CHO cells was measured as described (15). Saturable binding of PL1 or PL2 to cells expressing wild-type PSGL-1 was obtained at 10-15 µg/ml; the standard mAb concentration used for all experiments was 50 µg/ml. Binding of polyclonal antibodies was measured similarly, except that bound antibody was detected with FITC goat-anti-rabbit IgG/IgM. Saturable binding of the anti-42-56 serum was obtained at a dilution of 1:20; the standard dilution used for experiments was 1:10.
The previously described flow cytometric assay for measuring binding of fluid-phase platelet-derived P-selectin to intact cells (25, 35) was modified to measure binding of recombinant P-selectin. Soluble recombinant P-selectin (sPS) (33) was biotinylated by the method used to biotinylate mAbs (36). Transfected CHO cells (2 × 106) were washed twice with HBSS plus 1% bovine serum albumin. The cells were then incubated with 16.7 µg/ml (150 nM) of biotinylated P-selectin in 100 µl of HBSS for 30 min on ice. Preliminary experiments revealed that this concentration of biotinylated P-selectin was the minimum required to saturate nearly all the binding sites on transfected cells expressing wild-type PSGL-1. Therefore, impaired binding of soluble P-selectin to a given PSGL-1 mutant was predicted to be readily detected. After centrifugation and removal of the supernatant, the cell pellet was resuspended in 10 µl of phycoerythrin-streptavidin. After a 20-min incubation on ice, the cells were analyzed on a Becton-Dickinson FACscan flow cytometer. In some experiments, the cell pellet was preincubated with 10 µl of mAb PL1 or PL2 (50 µg/ml) for 30 min on ice before addition of soluble P-selectin. Alternatively, 100 µl of P-selectin in HBSS was incubated with 10 µl of mAb G1 or S12 (final concentration of 50 µg/ml) before addition to the cell pellet. In other experiments, the assay was performed in buffer containing 5 mM EDTA.C2GnT Assay--
C2GnT activity in cell lysates was measured as
described (37), with the following minor modifications. Each assay used
100 µg/ml cell protein and 0.5 µCi of [3H]UDP-GlcNAc
(60 Ci/ml) in buffer containing 60 mM sodium cacodylate, pH
6.5. The reaction mixtures were incubated for 4 h at 37 °C. The
reactions were terminated by addition of 1 ml of H2O before isolation of the reaction products. Nontransfected CHO DHFR() cells
were used as a negative control. HL-60 cells were used as a positive
control. All permanently transfected CHO cell lines were analyzed.
Three of these lines (T57S/E61A, T57A, and Y46F/Y48F/Y51F) were also
analyzed 48 h after transient transfection with cDNAs encoding
C2GnT and FTIII.
Metabolic Labeling and Immunoprecipitation of PSGL-1--
CHO
cells were metabolically labeled with 100 µCi/ml
[35S]sulfate or with 100 µCi/ml
[35S]sulfate plus 25 µCi/ml
[3H]GlcNH2 as described (22, 27). Equivalent
amounts of cell lysate (2.5 × 107 cpm) were
immunoprecipitated with rabbit antiserum to the 42-56 peptide sequence
of PSGL-1. The immunoprecipitates were analyzed by SDS-PAGE under
nonreducing conditions, followed by fluorography. The dried gel
containing the immunoprecipitates from cells double-labeled with both
[35S]sulfate and [3H]GlcNH2 was
aligned with the x-ray film. Each 240-kDa band corresponding to
nonreduced PSGL-1 was excised and digested with 10 mg/ml Pronase in 100 mM Tris-HCl, pH 8.0, 1 mM CaCl2 for
16 h at 37 °C. The 3H and 35S
radioactivity in each digest was measured in separate channels in a
Beckman liquid scintillation counter. Spillover of 3H and
35S into each channel was corrected by using isotope
standards. The 35S:3H ratio was determined for
each construct. All ratios were normalized to that of wild-type PSGL-1,
which was assigned a value of 1.0.
Adhesion Measurements under Hydrodynamic Flow-- Adhesive interactions under laminar flow conditions were measured as described previously (15, 17). Confluent monolayers of transfected CHO cells in 35-mm dishes were inserted into a parallel-plate flow chamber. Stably transfected murine L1-2 pre-B cells expressing human P-selectin (38) were resuspended at 5 × 105 cells/ml in Hanks' balanced salt solution supplemented with 1% fetal bovine serum. The cells were perfused through the flow chamber at a wall shear stress of 1 dyn/cm2. Data were acquired after 4 min of perfusion, at which time equilibrium had been reached. The number of L1-2 cells that accumulated on the CHO cell monolayer was quantified using a computer imaging system (Sun Microsystems, Mountain View, CA; Inovision, Durham, NC). For each experiment, the adherent L1-2 cells in 12-18 20× fields were counted. Virtually all adherent cells rolled on the CHO cell monolayer. In some experiments, the CHO cells were preincubated with 20 µg/ml amounts of the anti-PSGL-1 mAb PL1 or PL2, or the L1-2 cells were preincubated with 20 µg/ml anti-P-selectin mAb G1. In all cases, cells were perfused through the chamber in the continued presence of the mAb. To measure resistance to detachment under shear, L1-2 cells were allowed to settle on the CHO cell monolayer. Cell-free buffer was then perfused at 1 dyn/cm2 for 1 min to remove loosely adherent cells. The wall shear stress was then increased every every 30 s, and the percentage of remaining adherent cells was determined (15, 17). The rolling velocities of the L1-2 cells were measured as described (15, 17).
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RESULTS |
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Preparation and Expression of PSGL-1 Constructs-- We made a series of cDNA constructs in which we altered one or more amino acids within residues 42-62 of full-length PSGL-1 (Fig. 1). This sequence represents the N-terminal 21 residues of mature PSGL-1 in leukocytes where the propeptide is proteolytically removed; the propeptide is not removed from transfected CHO cells (25). The sequence includes potential sites for tyrosine sulfation at residues 46, 48, and 51, and potential sites for Thr-linked O-glycans at residues 44 and 57. It also includes the epitope for the mAb PL1. CHO cells permanently transfected with a cDNA for each PSGL-1 construct were transiently transfected with cDNAs for C2GnT and FTIII to construct core-2, sialylated, and fucosylated O-glycans on PSGL-1 (25). Comparable expression of PSGL-1 and FT activity in the transfected CHO cells was confirmed by flow cytometry with mAbs to PSGL-1 (see Figs. 2 and 7) or to the sLex determinant (Table I). Expression of C2GnT activity was confirmed by assays of cell lysates (Table II).
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Alanine Substitution of Thr-57 but Not Thr-44 Blocks Recognition by Soluble P-selectin-- Thr-44 and Thr-57 are the potential sites for attachment of O-glycans in the 42-62 region of PSGL-1. Amino acid substitutions were made to these residues or to adjacent residues to test their importance for binding P-selectin. Flow cytometry was used to measure binding of biotinylated, soluble P-selectin to the surface of the transfected cells. P-selectin bound to cells co-expressing wild-type PSGL-1 with C2GnT and FTIII (Fig. 3B), but not to mock-transfected cells expressing only C2GnT and FTIII (Fig. 3A). P-selectin binding to cells expressing wild-type PSGL-1 was blocked by mAb PL1. In contrast, binding was not blocked by PL2, an IgG anti-PSGL-1 mAb that identifies an epitope within the decameric consensus repeats (15, 19). Binding was Ca2+-dependent and was abrogated by the blocking anti-P-selectin mAb G1, but not by the nonblocking mAb S12 (data not shown). Thus, binding of soluble P-selectin to transfected cells required a specific, Ca2+-dependent interaction between P-selectin and the N terminus of PSGL-1.
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Substitution of All Three Clustered Tyrosines Is Required to Block Recognition by Soluble P-selectin-- Simultaneous substitution of Tyr-46, Tyr-48, and Tyr-51 with phenylalanines blocks binding of PSGL-1 to P-selectin (23-25). To determine which of these residues might be most important for P-selectin recognition, we made constructs that changed only a single tyrosine to phenylalanine (Y46F, Y48F, and Y51F) or changed two of the tyrosines to phenylalanines (Y46F/Y48F, Y48F/Y51F, and Y46F/Y51F). CHO cells co-expressing each construct with C2GnT and FTIII expressed similar levels of PSGL-1 and sLex (Fig. 7 and Table I). Soluble P-selectin bound specifically to cells expressing each of these constructs, and PL1, but not PL2, prevented binding (Fig. 8). By contrast, P-selectin did not bind to cells expressing the Y46F/Y48F/Y51F construct (Fig. 8I), confirming previous results (25). These data demonstrate that PSGL-1 requires sulfation of only one of its three clustered tyrosines to bind P-selectin.
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Rolling of L1-2 Cells Expressing P-selectin on CHO Cells Expressing PSGL-1 Constructs under Shear Stress-- Under hydrodynamic flow, rolling adhesion of leukocytes requires both rapid formation and rapid dissociation of selectin-ligand bonds that have high tensile strength (39, 40). These requirements are met for the PSGL-1-dependent rolling of flowing leukocytes on cells expressing P-selectin (15, 16). We used a parallel-plate flow chamber to determine whether transfected cells expressing P-selectin could roll on PSGL-1 constructs expressed on CHO cell monolayers. At a shear stress of 1 dyn/cm2, L1-2 cells expressing P-selectin tethered to and rolled on CHO cells expressing wild-type PSGL-1 (Fig. 9, A and B). The number of rolling cells at equilibrium was comparable in different experiments and was observed when the CHO cells were transiently transfected with cDNA encoding either FTIII or FTVII. Rolling was dependent on P-selectin on the L1-2 cells, because it was blocked by the anti-P-selectin mAb G1. Similarly, rolling required a specific interaction with PSGL-1 on the CHO cells, because it was blocked by PL1 but not by PL2. In parallel experiments, P-selectin-L1-2 cells also rolled on CHO cells expressing T57S/E61A but not on cells expressing T57A (Fig. 9A). Similarly, P-selectin-L1-2 cells rolled on CHO cells expressing any construct in which two of the three tyrosines were replaced with phenylalanines, but not on cells expressing the construct in which all three tyrosines were replaced with phenylalanines (Fig. 9B). In all cases, rolling was blocked by G1 and by PL1 but not by PL2, confirming the specificities of the interactions. P-selectin-L1-2 cells rolled with comparable adhesive strength on all constructs, as measured by similar resistance to detachment by increasing shear stress (Fig. 9C) and by similar mean rolling velocities at two different shear stresses (Fig. 9D). These data demonstrate that the PSGL-1 constructs that bind soluble P-selectin also support rolling of P-selectin-expressing cells under shear stress.
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DISCUSSION |
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Earlier studies have suggested that P-selectin binds to a small N-terminal region on both native and recombinant PSGL-1 (15, 19, 41). Binding requires at least one sulfate on a group of three clustered tyrosines (22-25) and at least one sialylated, fucosylated, core-2 O-glycan (5, 8, 21, 25). Previous analyses of specific residues required for binding have relied on truncated or chimeric forms of recombinant PSGL-1 (23, 24). As a result, the key sites for tyrosine sulfation and O-glycosylation on full-length PSGL-1 have not been well characterized. We have co-expressed full-length PSGL-1 with C2GnT and either FTIII or FTVII in CHO cells. The recombinant PSGL-1, like native PSGL-1 on leukocytes, is tyrosine-sulfated and carries appropriately modified core-2 O-glycans that promote high affinity binding to P-selectin (25). Here we show that PSGL-1 binds P-selectin when Thr-57 is replaced with serine, but not when it is replaced with alanine, suggesting that PSGL-1 requires an O-glycan attached to residue 57 to bind to P-selectin. We also show that any one of the three clustered tyrosines at residues 46, 48, and 51 is sufficient for binding to P-selectin. The PSGL-1 constructs that bind soluble P-selectin also support rolling of P-selectin-expressing cells under hydrodynamic flow, supporting the physiological relevance of the data.
Residues 42-62 of PSGL-1 represent the first 21 amino acids in the
mature molecule following cleavage of the signal peptide and propeptide
(Fig. 1). This sequence has two threonines at residues 44 and 57 that
are potential attachment sites for O-glycans. Substitution of Thr-44 with alanine had no effect on binding of PSGL-1 to
P-selectin, whereas substitution of Thr-57 with alanine eliminated
binding. Substitution of Thr-57 with serine had no effect on binding,
suggesting that an O-glycan attached to either a serine or
threonine at this position might interact with P-selectin. The
O-glycans of PSGL-1 require 2,3-linked sialic acid and
1,3-linked fucose on a core-2 backbone to mediate interactions with
P-selectin (25). The fucosylated O-glycans of PSGL-1 from
HL-60 cells are a mixture of short disialylated, monofucosylated core-2
heptasaccharides and long monosialylated, trifucosylated core-2 glycans
with polylactosamine, both of which contain a terminal sLex
determinant (27). The structures of the fucosylated
O-glycans on recombinant PSGL-1 may depend on the specific
1,3-FT that is co-expressed in CHO cells (25). Significantly, only a
minority of the O-glycans on PSGL-1 from HL-60 cells are
fucosylated (27). Our data suggest that one of these fucosylated
glycans is attached to Thr-57.
We cannot formally exclude the possibility that loss of P-selectin binding to the T57A construct resulted from indirect effects on glycosylation at other sites. However, P-selectin bound normally to constructs in which the two tandem residues on either side of Thr-57 were replaced with alanines. This indicates that not all amino acid substitutions in this region affect binding. The sequence immediately surrounding Thr-57 is the core of the epitope for PL1, the IgG mAb that blocks binding to P-selectin. Consistent with this feature, PL1 did not detectably bind to the P55A/E56A and E58A/P59A constructs, each of which contains two substitutions in the middle of the core epitope. PL1 also bound less well to other PSGL-1 constructs with mutations in this region (T44A/T57A, T57A, and T57S/E61A).
Previous studies have also examined specific residues on PSGL-1 to which key O-glycans might be attached. When co-expressed with FTIII in COS cells, an Ig chimera containing only residues 42-60 of PSGL-1 bound to P-selectin (24). This sequence has only two potential O-glycosylation sites at Thr-44 and Thr-57. Substitution of Thr-57 with alanine in this Ig chimera markedly decreased binding to P-selectin. These results demonstrated that Thr-57 is required for P-selectin binding in this short construct. However, they did not indicate whether this residue is required for recognition in the context of full-length PSGL-1, in which many serines and threonines are glycosylated. This issue is critical, because PSGL-1 contains several fucosylated O-glycans that could potentially interact with selectins (27).
When co-expressed with FTVII in COS cells, a shortened membrane-anchored form of PSGL-1 with residues 38-57 attached directly to residue 118 of the extracellular domain mediated cell adhesion to immobilized P-selectin (23). Replacement of both Thr-44 and Thr-57 with alanines in the shortened PSGL-1 construct decreased, but did not eliminate, adhesion of transfected COS cells (23). Because this shortened construct fused a small N-terminal fragment of PSGL-1 directly to the decameric consensus repeats, the juxtaposition of the two sequences may have altered local O-glycosylation, accounting for the residual adhesion despite the loss of the putative O-glycan at Thr-57 (23). Furthermore, the site densities of immobilized P-selectin were not reported, and there was significant adhesion of transfected COS cells expressing only FTVII. Perhaps some of the residual adhesion of the T44A/T57A-transfected COS cells was mediated by non-PSGL-1-dependent interactions with high densities of immobilized P-selectin. In any event, the experiments with this chimeric molecule did not address the function of Thr-44 or Thr-57 in the context of full-length PSGL-1.
We found that PSGL-1 constructs containing at least one of the three clustered tyrosines at residues 46, 48, or 51 bound soluble P-selectin and supported rolling of P-selectin-L1-2 cells under shear stress. These results indicate that only one tyrosine on each subunit of the PSGL-1 homodimer is required for binding, and suggest that each tyrosine can be sulfated under some conditions. Whether a single tyrosine sulfate presented on a monomeric form of PSGL-1 is sufficient for binding is unknown. More quantitative assays are required to determine whether the three tyrosines allow wild-type PSGL-1 to bind with higher affinity to P-selectin than do constructs with only one or two tyrosines. P-selectin-L1-2 cells detached slightly more easily from CHO cells expressing the Y46F/Y48F and Y48F/Y51F constructs than from cells expressing wild-type PSGL-1, but this could reflect subtle differences in site density or glycosylation among the constructs.
It is noteworthy that murine PSGL-1 also has an N-terminal consensus sequence for tyrosine sulfation, but it has only two tyrosines rather than the three found in the human sequence (42). Although several features of consensus sites for tyrosine sulfation have been described (43), tyrosine sulfation in some proteins is incomplete (44). Further studies of the substrate specificity of the tyrosine protein sulfotransferase are needed to define the features required for optimal tyrosine sulfation. The current data do not establish whether there is preferential sulfation of specific tyrosines in wild-type PSGL-1. As expected, very little sulfate was attached to the Y46F/Y48F/Y51F construct. The small amount of residual sulfation may represent incorporation of [35S]sulfate into cysteine or methionine residues. Alternatively, the CHO cells may have attached minor quantities of labeled sulfate esters to newly synthesized N- or O-glycans (45).
Our results support a model in which P-selectin binds to a small N-terminal region of PSGL-1 that requires a sialylated and fucosylated, core-2 O-glycan at Thr-57 and a sulfate at Tyr-46, Tyr-48, or Tyr-51. Characterization of a specific glycan structure attached to Thr-57 is required to further support this model. Because any one of the tyrosines is sufficient for binding, there may be variability allowed in the distance between the tyrosine sulfate and the O-glycan. This raises the question as to whether P-selectin binds simultaneously or sequentially to the tyrosine sulfate and the O-glycan.
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ACKNOWLEDGEMENTS |
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We thank Ginger Hampton and Cindy Carter for technical assistance. Oligonucleotides were synthesized by the Molecular Biology Resource Facility of the University of Oklahoma Health Sciences Center. Automated DNA sequencing was performed by the Molecular Biology Core Facility at Oklahoma State University.
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FOOTNOTES |
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* This work was supported by Grant P01 HL 54304 from the National Institutes of Health.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: W. K. Warren
Medical Research Institute, University of Oklahoma Health Sciences Center, 825 N.E. 13th St., Oklahoma City, OK 73104. Tel.: 405-271-6480; Fax: 405-271-3137; E-mail: rodger-mcever{at}ouhsc.edu.
1
The abbreviations used are: sLex,
sialyl Lewis x; CHO, Chinese hamster ovary; C2GnT, core-2
1,6-N-acetylglucosaminyltransferase; DHFR, dihydrofolate
reductase; FITC, fluorescein isothiocyanate; FT, fucosyltransferase;
HBSS, Hanks' balanced salt solution; PSGL-1, P-selectin glycoprotein
ligand-1; mAb, monoclonal antibody; PAGE, polyacrylamide gel
electrophoresis; Ig, immunoglobulin.
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