(Received for publication, July 5, 1995; and in revised form, August 4, 1995)
From the
P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like
glycoprotein on leukocytes that is a high affinity ligand for
P-selectin. Previous studies have shown that sialylation and
fucosylation of PSGL-1 are required for its binding to P-selectin, but
other post-translational modifications of PSGL-1 may also be important.
We demonstrate that PSGL-1 synthesized in human HL-60 cells can be
metabolically labeled with [S]sulfate that is
incorporated primarily into tyrosine sulfate. Treatment of PSGL-1 with
a bacterial arylsulfatase releases sulfate from tyrosine, resulting in
a concordant decrease in binding to P-selectin. These studies
demonstrate that tyrosine sulfate on PSGL-1 functions in conjunction
with sialylated and fucosylated glycans to mediate high affinity
binding to P-selectin.
P-selectin is a calcium-dependent carbohydrate-binding protein
that is expressed on the surfaces of activated platelets and
endothelium in response to thrombin and other
agonists(1, 2, 3) . Through its binding to
glycoconjugate-based counter-receptors on leukocytes, P-selectin
mediates rolling adhesion of these cells on activated platelets and
endothelium(4, 5) . Both sialic acid and fucose are
components of the P-selectin counter-receptors on
leukocytes(6, 7, 8) . Oligosaccharides
containing sialyl Lewis x (sLe), (
)NeuAc
2-3Gal
1-4[Fuc
1-3]GlcNAc
1-R,
a determinant present on leukocyte surfaces, inhibit adhesion of
leukocytes to P-selectin(9, 10) . However, expression
of sLe
on cell surfaces is not sufficient for high affinity
binding of cells to P-selectin, since non-myeloid cells that express
high levels of sLe
bind poorly to P-selectin compared to
myeloid cells(11) .
Leukocytes express a single high affinity ligand for P-selectin, termed P-selectin glycoprotein ligand-1 (PSGL-1)(5, 7, 8, 12, 13) . PSGL-1 is a homodimeric glycoprotein with two disulfide-bonded 120-kDa subunits (7) . The cDNA-derived sequence for PSGL-1 predicts a type 1 transmembrane protein of 402 amino acids(8) . The extracellular domain has an N-terminal signal peptide from residues 1-18 and a putative propeptide from residues 19-41. Assuming cleavage of the propeptide, the extracellular domain of the mature protein begins at residue 42 and extends to residue 308. The sequence concludes with a 25-residue transmembrane domain and a 69-residue cytoplasmic tail. The extracellular domain is rich in serines and threonines that are potential sites for O-glycosylation. There are also three potential N-glycosylation sites and three potential tyrosine sulfation sites at residues 46, 48, and 51(8) .
PSGL-1 must be
sialylated and fucosylated to bind P-selectin(7, 8) .
Consistent with these observations, PSGL-1 is highly O-glycosylated (12) and contains sialylated and
fucosylated O-linked poly-N-acetyllactosamine,
including some glycans that terminate in sLe(13) .
It is not clear, however, that sLe
or related glycans are
sufficient for high affinity binding of PSGL-1 to P-selectin. For
example, sulfated compounds lacking either sialic acid or fucose can
inhibit adhesion of leukocytes to
P-selectin(14, 15, 16, 17) . These
data suggest that PSGL-1 may require sulfation to bind with high
affinity to P-selectin. We demonstrate that PSGL-1 is sulfated,
primarily on tyrosine residues. Furthermore, tyrosine sulfation of
PSGL-1 is required for high affinity binding to P-selectin.
The specificity of the A. aerogenes arylsulfatase was determined for the sulfated monosaccharides GlcNAc-6-sulfate, Gal-6-sulfate, GalNAc-6-sulfate, and GalNAc-4-sulfate. Each of the sulfated monosaccharides (100 nmol) was treated separately with 1000 milliunits of boiled or active enzyme. After overnight incubation, the reaction mixtures were boiled for 15 min, lyophilized, resuspended in water, and analyzed by high pH anion exchange chromatography with a Dionex CarboPac PA-1 column and PAD detection as described(25) .
Intact S-PSGL-1,
I-PSGL-1, or
H-PSGL-1 was treated with A.
aerogenes arylsulfatase as described above. The
[
S]sulfate released from
S-PSGL-1
by arylsulfatase was quantified by BaSO
precipitation, in
which 100 µl of saturated BaCl
was added to the
reaction mixture with 10 µl of saturated Na
SO
as a carrier(26) . In the graphic representation of the
data, the sulfate released from the sham-treated sample was adjusted
setting the sham-treated sample equal to 0%. The actual percentage of
radioactivity released from the sham-treated samples ranged from
1.3-5.0%.
S-PSGL-1 was treated with
peptide:N-glycosidase F as described(26) .
S-PSGL-1 was denatured by boiling 5 min in 50 µl of 50
mM sodium phosphate, pH 7.5, 50 mM 2-mercaptoethanol,
0.5% SDS. The SDS was then diluted to a final concentration of 0.2%
with 1.5% Nonidet P-40. 10 units of enzyme was added to the denatured
S-PSGL-1 and incubated overnight at 37 °C.
In a
control experiment, I-PSGL-1 was treated with 100
milliunits of A. ureafaciens neuraminidase in 0.05 M sodium acetate, pH 5.0, overnight at 37 °C prior to
application to a CSLEX-1 antibody column. Chromatography on CSLEX-1 was
performed as described previously(13) .
To determine if PSGL-1 is post-translationally sulfated,
human promyelocytic leukemia HL-60 cells were metabolically labeled
with [S]sulfate, and PSGL-1 was purified from
lysates of these cells by affinity chromatography on a P-selectin
column. A [
S]sulfate-labeled protein was
detected that migrated in SDS gels with a relative molecular mass of
120,000 under reducing conditions and 240,000 under nonreducing
conditions (Fig. 1). These mobilities are consistent with the
disulfide-linked homodimeric structure of PSGL-1(7) .
Furthermore, the [
S]sulfate-labeled protein was
immunoprecipitated with a specific rabbit antiserum generated against a
synthetic peptide encoding residues 42-56 of the extracellular
domain of PSGL-1 (13) (Fig. 1). Analysis of the
supernatants showed that anti-42-56 precipitated all of the
S-PSGL-1, whereas NRS precipitated none of the
S-PSGL-1 (data not shown). These results demonstrate that
PSGL-1 is post-translationally sulfated.
Figure 1:
PSGL-1 is post-translationally
sulfated. A, affinity-purified S-PSGL-1 from
HL-60 cells was resolved by SDS-PAGE under reducing (R) and
nonreducing (NR) conditions, followed by autoradiography. B, the purified
S-PSGL-1 was immunoprecipitated
with a specific rabbit antiserum to PSGL-1 (anti-42-56) or with
normal rabbit serum (NRS). The immunoprecipitates were electrophoresed
under nonreducing conditions.
Sulfate can be incorporated
into eukaryotic glycoproteins as tyrosine sulfate (27) or as
sulfated carbohydrates(28, 29) . In preliminary
studies we failed to detect sulfated carbohydrates on PSGL-1, using
techniques that detected such structures on other
glycoproteins(25, 26) . We then considered the
possibility that PSGL-1 might contain tyrosine sulfate. The cDNA
sequence of PSGL-1 predicts four extracytoplasmic tyrosine
residues(8) , three of which are clustered at positions 46, 48,
and 51 within a predicted consensus sequence for tyrosine
sulfation(27) . To determine if PSGL-1 has tyrosine sulfate,
the gel slice containing the 240-kDa S-PSGL-1 was
hydrolyzed with strong base and analyzed by both anion exchange
chromatography and descending paper chromatography. In both systems, a
single radioactive peak was recovered that comigrated with authentic
tyrosine sulfate (Fig. 2, A and B). No
radioactivity was recovered that comigrated with sulfated
monosaccharide standards.
Figure 2:
PSGL-1 contains tyrosine sulfate. S-PSGL-1 was hydrolyzed with strong base as described
under ``Experimental Procedures.'' A, the
hydrolysate was analyzed by anion exchange chromatography using HPLC
with a NaH
PO
, pH 3.0 gradient (dashed
line). The retention times of tyrosine, tyrosine sulfate,
Gal-6-sulfate, and free sulfate are indicated. Other sulfated
monosaccharides (GlcNAc-6-sulfate, GalNAc-6-sulfate, and
GalNAc-4-sulfate) eluted with similar retention times between 14 and 15
min. B, the hydrolysate was analyzed by descending paper
chromatography in the solvent system 1-butanol:acetic acid:water
(125:30:125) for 18 h. The migration distances for tyrosine, tyrosine
sulfate, Gal-6-sulfate, and free sulfate are indicated. The R
values for tyrosine, tyrosine sulfate,
sulfated monosaccharides (Gal-6-sulfate, GlcNAc-6-sulfate,
GalNAc-6-sulfate, and GalNAc-4-sulfate), and free sulfate were 0.5,
0.28, 0.12-0.15, and 0.04,
respectively.
We sought to test whether tyrosine sulfate
is important for binding of PSGL-1 to P-selectin. Although the
functions of tyrosine sulfate within proteins are not
clear(27) , some proteins require this modification for optimal
activity(30, 31) . The usual approach for assessing
the importance of tyrosine sulfate is to prevent sulfation of newly
synthesized proteins with chemical inhibitors or to replace tyrosine
with phenylalanine by site-directed mutagenesis. We developed an
alternative approach in which sulfate was enzymatically removed from
tyrosine on intact PSGL-1. We first tested the ability of an
arylsulfatase from A. aerogenes to release sulfate from
[S]tyrosine sulfate. Treatment of the
hydrolysate of
S-PSGL-1 with 1000 milliunits of
arylsulfatase quantitatively released [
S]sulfate
from [
S]tyrosine sulfate (Fig. 3, A and B). In other experiments, as little as 50 milliunits
of this arylsulfatase also quantitatively released
[
S]sulfate from
[
S]tyrosine sulfate derived from PSGL-1 (data
not shown). Cleavage by arylsulfatase was specific, since 1000
milliunits of the enzyme did not release sulfate from Gal-6-sulfate,
GlcNAc-6-sulfate, GalNAc-4-sulfate, and GalNAc-6-sulfate (data not
shown).
Figure 3:
Tyrosine sulfate from PSGL-1 is sensitive
to arylsulfatase. S-PSGL-1 was hydrolyzed with strong base
as described under ``Experimental Procedures.'' The
hydrolysates were either sham-treated with 1000 milliunits of boiled
arylsulfatase (A) or treated with 1000 milliunits of active
enzyme (B). The hydrolysates were then analyzed by anion
exchange chromatography.
We then examined the ability of the arylsulfatase to release
sulfate from intact PSGL-1 and the effect of this release on rebinding
of PSGL-1 to P-selectin. [S]Sulfate released
from
S-PSGL-1 by arylsulfatase was quantified by
precipitation as insoluble BaSO
. Up to 50% of the
[
S]sulfate on
S-PSGL-1 was released
by 500 milliunits of arylsulfatase; increasing amounts of enzyme did
not release more radioactivity (Fig. 4A). The
functional importance of the tyrosine sulfate on PSGL-1 was assessed by
treating both
H-PSGL-1 from HL-60 cells and
I-PSGL-1 from human neutrophils with arylsulfatase and
measuring the rebinding of the treated ligands to a P-selectin column.
Binding of both arylsulfatase-treated
H-PSGL-1 and
I-PSGL-1 to P-selectin was reduced in a dose-dependent
manner; the decreased binding was inversely related to the amount of
sulfate released from
S-PSGL-1 (Fig. 4A).
The reduced binding of PSGL-1 to P-selectin following arylsulfatase
treatment was not due to general release of sialic acid and/or fucose
by contaminating exoglycosidases, because PSGL-1 treated with 1000
milliunits of arylsulfatase bound quantitatively to an affinity column
containing CSLEX-1, a monoclonal antibody to sLe
(data not
shown).
Figure 4:
Tyrosine sulfate is important for
P-selectin binding. A, arylsulfatase releases
[S]sulfate from intact
S-PSGL-1 and
decreases Ca
-dependent binding of both
I-PSGL-1 and
H-PSGL-1 to P-selectin.
S-PSGL-1 and
H-PSGL-1 purified from HL-60
cells and
I-PSGL-1 purified from human neutrophils were
treated with increasing amounts of arylsulfatase. The
[
S]sulfate released from
S-PSGL-1
was quantified using BaSO
precipitation (dashed
line). Enzyme-treated
I-PSGL-1 and
H-PSGL-1 were analyzed for their ability to rebind to
P-selectin (solid lines). B,
[
S]tyrosine sulfate is present on PSGL-1 that
binds P-selectin but is absent on PSGL-1 that does not bind P-selectin.
Arylsulfatase-treated
S-PSGL-1 and
I-PSGL-1
were applied to a P-selectin column. The unbound(-) and bound
(+) fractions were analyzed by SDS-PAGE under nonreducing
conditions, followed by autoradiography.
Arylsulfatase released 50% of the
[
S]sulfate from
S-PSGL-1 and
decreased rebinding of
H-PSGL-1 and
I-PSGL-1
to P-selectin by the same degree. We considered the possibility that
the fraction of
S-PSGL-1 that rebound to P-selectin
following treatment with arylsulfatase retained critical tyrosine
sulfate residues, whereas the fraction that did not rebind to
P-selectin had lost tyrosine sulfate.
S-PSGL-1 was treated
with 1000 milliunits of arylsulfatase and then applied to a P-selectin
column. The bound and unbound fractions were analyzed by SDS-PAGE (Fig. 4B). A band corresponding to
S-PSGL-1 was observed in the bound fractions, whereas no
band was seen in the unbound fractions. The radioactivity in the
unbound fractions represented free sulfate. When the band of
S-PSGL-1 in the bound fractions was hydrolyzed,
radioactivity was recovered in tyrosine sulfate (data not shown). In
the control experiment,
I-PSGL-1 was treated with 1000
milliunits of arylsulfatase and then applied to the P-selectin column.
SDS-PAGE analysis revealed that intact
I-PSGL-1 was
recovered in both the unbound and bound fractions (Fig. 4B). Thus, arylsulfatase removed sulfate
quantitatively from a subset of PSGL-1, which no longer bound
P-selectin, but the enzyme did not otherwise degrade PSGL-1.
The
tyrosine sulfate remaining in the P-selectin-bound subset of PSGL-1 may
be resistant to arylsulfatase because of its inaccessibility or because
of some other feature of PSGL-1 that blocks action of the enzyme. When S-PSGL was partly deglycosylated by treatment with
peptide:N-glycosidase F and A. ureafaciens neuraminidase, subsequent treatment with arylsulfatase released up
to 70% of the radioactivity as [
S]sulfate (data
not shown). Since neuraminidase also eliminates binding of PSGL-1 to
P-selectin, we could not determine whether the increased removal of
[
S]sulfate further reduced binding of PSGL-1 to
P-selectin. These results suggest that the extensive glycosylation of
PSGL-1 may account for the inaccessibility of some tyrosine sulfate
sites to arylsulfatase.
These results demonstrate that PSGL-1
contains tyrosine sulfate that is required for high affinity binding to
P-selectin. It has been shown previously that PSGL-1 contains the
sLe determinant on O-linked oligosaccharides and
that both sialic acid and fucose are required for binding of PSGL-1 to
P-selectin(7, 8, 12, 13) . Tyrosine
sulfate may be important because it promotes appropriate presentation
of the glycans that bind directly to P-selectin. Alternatively,
tyrosine sulfate may directly interact with P-selectin. This latter
possibility seems more likely, since sulfatide and sulfated
oligosaccharides are known to bind
P-selectin(14, 15, 16, 17) . Sulfate
is also a critical determinant for the binding of GlyCAM-1 to
L-selectin, but GlyCAM-1 contains sulfate in Gal-6-sulfate and
GlcNAc-6-sulfate (32, 33, 34) . In contrast,
PSGL-1 is sulfated primarily on tyrosine rather than on
monosaccharides.
Our results suggest a model in which PSGL-1 presents both carbohydrate and tyrosine sulfate as components of a critical recognition site for P-selectin. We hypothesize that this site is located at the N-terminal, membrane-distal region of PSGL-1, near the three potentially sulfated tyrosine residues. Consistent with this hypothesis, PL1, a monoclonal antibody that recognizes a membrane-distal epitope on PSGL-1, blocks binding of fluid-phase P-selectin to leukocytes and abolishes adhesion of neutrophils to P-selectin under both static and shear conditions (5) . In contrast, PL2, a monoclonal antibody that recognizes a membrane-proximal epitope on PSGL-1, does not inhibit binding to P-selectin(5) . The concept that P-selectin recognizes a localized site on a mucin-like glycoprotein is in contrast to a model in which a selectin recognizes multiple, clustered O-linked glycans attached along the entire polypeptide(35, 36) .