(Received for publication, November 30, 1995; and in revised form, January 5, 1996)
From the
P-selectin glycoprotein ligand-1 (PSGL-1), a sialomucin on human
leukocytes, mediates rolling of leukocytes on P-selectin expressed by
activated platelets or endothelial cells under shear forces. PSGL-1
requires both tyrosine sulfate and O-linked glycans to bind
P-selectin. Electron microscopy of rotary-shadowed PSGL-1 purified from
human neutrophils indicated that it is a highly extended molecule with
an extracellular domain that is 50 nm long. Both individual PSGL-1
molecules and rosettes composed of several molecules presumably
attached at their transmembrane segments were observed. The
extracellular domain of PSGL-1 has 318 residues, including a signal
peptide from residues 1-18 and a propeptide from residues
19-41. Using bacterially expressed fusion proteins and synthetic
peptides derived from the extracellular domain, we mapped the epitopes
for two IgG anti-PSGL-1 monoclonal antibodies, PL1 and PL2. PL2 bound
to a region within residues 188-235 that is located in a series
of decameric consensus repeats. PL1, which blocks binding of PSGL-1 to
P-selectin, recognized an epitope spanning residues 49-62. This
sequence overlaps the tyrosine sulfation sites at residues 46, 48, and
51 that have been implicated in binding of PSGL-1 to P-selectin. Our
results demonstrate that PSGL-1 is a long, extended molecule and
suggest that the P-selectin binding site is located near the N
terminus, well above the membrane. This location may facilitate
interactions of PSGL-1 with P-selectin under shear stress.
The selectins are a family of Ca-dependent
lectins that mediate rolling adhesion of leukocytes on the vessel wall
during inflammation (reviewed in (1) and (2) ).
L-selectin is expressed on leukocytes, whereas E- and P-selectin are
expressed on activated endothelial cells or platelets. All three
selectins bind sialylated and fucosylated oligosaccharides such as
sialyl Lewis x, and L- and P-selectin also recognize many sulfated
glycans(3) . However, the selectins bind with high affinity or
avidity to only a few cell surface glycoproteins (1, 2, 3) .
P-selectin glycoprotein
ligand-1 (PSGL-1) ()is the major high affinity/avidity
ligand for P-selectin on human leukocytes(4) . PSGL-1 also
interacts with
E-selectin(5, 6, 7, 8) . As assessed
by SDS-PAGE, PSGL-1 is a disulfide-linked homodimer with two 120-kDa
subunits(4) . Each subunit has few N-linked glycans
but has many clustered, sialylated O-linked glycans that
render the protein susceptible to digestion with O-sialoglycoprotein endopeptidase(4, 9) .
Some of the O-linked glycans have polylactosamine terminating
in sialyl Lewis x(5) . PSGL-1 is a type 1 membrane protein with
an extracellular domain rich in serines, threonines, and prolines,
including a series of decameric repeats (15 in HL-60 cells and 16 in
human leukocytes) (8, 10, 11) . Following an
18-residue signal peptide, there is a putative propeptide extending
from residues 19 to 41(8) . If the propeptide is cleaved, the
extracellular domain begins at residue 42 and extends to residue 318 of
PSGL-1 on human leukocytes. The sequence concludes with a 25-residue
transmembrane domain and a 69-residue cytoplasmic tail.
PSGL-1 must
be sialylated and fucosylated to interact with both P- and
E-selectin(4, 8) . Recombinant PSGL-1 expressed on CHO
cells binds P- and E-selectin when it is co-expressed with an
1,3-fucosyltransferase and with core 2
1,6-N-acetylglucosaminyltransferase, a
glycosyltransferase that creates core 2 O-linked glycans (12) . This indicates that PSGL-1 requires sialylated and
fucosylated core 2 O-linked glycans to bind both P- and
E-selectin. However, the interactions of PSGL-1 with P- and E-selectin
are not identical. Fab fragments of PL1, an IgG mAb to PSGL-1, abolish
binding of purified PSGL-1 to P-selectin, suggesting that PL1
recognizes an epitope that overlaps a specific binding site for
P-selectin on PSGL-1 (10) . In contrast, PL1 only partially
inhibits binding of PSGL-1 to E-selectin, suggesting that PSGL-1 has an
additional binding site(s) for E-selectin(13) . PL2, another
IgG mAb to PSGL-1, has no effect on binding to either P- or
E-selectin(10, 13) . Treatment of leukocytes with O-sialoglycoprotein endopeptidase prevents binding of PL1 but
not PL2, indicating that the PL1 epitope on PSGL-1 is located farther
from the membrane than the PL2 epitope(10) . PSGL-1 has three
potential tyrosine sulfation sites near the N terminus(8) .
Enzymatic removal of sulfate from tyrosine and mutational analysis
indicate that sulfation of at least one of these tyrosines is required
for binding of PSGL-1 to P-selectin but not to
E-selectin(12, 14, 15, 16) .
Collectively, these data demonstrate that N-terminal tyrosine sulfate
functions with core 2 sialylated and fucosylated O-linked
glycans to mediate high affinity binding of PSGL-1 to P-selectin.
PSGL-1 is localized on the tips of microvilli of leukocytes(10) , where it is positioned to interact optimally with P-selectin under flow(17) . Projection of the P-selectin-binding site well above the cell surface might also facilitate rapid interactions with P-selectin. Mucins usually have extended structures(18, 19) , and rotary-shadowed electron micrographs of CD43, another mucin-like protein on leukocytes, confirm that it has a long extracellular domain(20) . Here we employ electron microscopy to demonstrate that PSGL-1 from human neutrophils is also highly extended. Using bacterially expressed fusion proteins and synthetic peptides, we have mapped the epitopes for PL1 and PL2 on PSGL-1. The epitope for PL1 is located near the N terminus, further supporting a specific membrane-distal location for the P-selectin-binding site.
Each construct in PQE-42 was transformed into
M15[pREP4] competent cells (Qiagen), and the resultant fusion
protein was expressed as described(23) . In brief, overnight
cultures were diluted 1:5 in fresh medium and incubated for 30 min at
37 °C. Isopropyl-1-thio--D-galactopyranoside (2
mM) was then added, and the cells were incubated for an
additional 4 h at 37 °C. The cells were then pelleted and lysed,
and the fusion protein was purified on nickel-nitrilotriacetic acid
resin (Qiagen) according to the instructions of the manufacturer.
Aliquots were boiled in SDS sample buffer for analysis by SDS-PAGE.
Much better specimens were obtained after centrifuging PSGL-1 through a glycerol gradient in ammonium bicarbonate free of detergent. The protein eluted as a fairly sharp peak at about 6 s. Rotary-shadowed specimens prepared from the peak fractions showed single molecules and rosettes (Fig. 1). Most were single molecules, identified by a uniform thickness and length. The molecules were all aligned in some areas of the grid, probably by shear flow as the droplet moved on the mica. These straight molecules were selected for length measurements, giving an average length of 54 ± 0.7 nm (n = 41). The 54-nm length should represent the ectodomain plus some portion of the transmembrane and cytoplasmic domains. These images demonstrate that PSGL-1 is a thin, highly extended molecule.
Figure 1: Electron micrographs of rotary-shadowed PSGL-1 molecules. The top two rows show rosettes, and the bottom row shows single molecules aligned by flow on the grid. The bar indicates 100 nm.
Rosettes consisted of two to five molecules
emanating from a central globular hub. These rosettes are typical of
membrane proteins, which are thought to aggregate at their hydrophobic,
transmembrane-spanning ends to form the
hubs(20, 21, 28, 29) . The specimen
areas showing the best rosettes were away from those where molecules
were aligned by shear, and the protruding ectodomains were usually
bent, making length measurements more difficult. The longest and
straightest molecules had a length of 50 nm from the edge of the
hub to the end of the molecule. Therefore, the ectodomain of PSGL-1 was
estimated to be 50 nm long.
Figure 2: Bacterially expressed fusion proteins containing fragments of the extracellular domain of PSGL-1 used to map the epitopes for mAbs PL1 and PL2. A, a schematic structure of PSGL-1 is shown at the top. Below are shown the sequences of PSGL-1 fragments expressed as fusion proteins with murine DHFR. B and C, sequences of the smaller PSGL-1 fusion proteins used to further localize the epitopes for PL1 or PL2. Also shown are the reactivities of the fusion proteins, determined by Western blotting, with the polyclonal anti-19-37 or anti-42-56 sera and with PL1 or PL2.
Figure 3: Western blot analysis of PSGL-1-DHFR fusion proteins shown in Fig. 2A. The left panel shows a Coomassie Blue-stained gel of the PSGL-1-DHFR fusion proteins, following SDS-PAGE under reducing conditions. In the other four panels, identical aliquots of the proteins were electrophoresed under the same conditions, transferred to Immobilon membranes, and probed with the mAbs PL1 or PL2, or with the polyclonal anti-19-37 or anti-42-56 sera. DHFR with no attached PSGL-1 sequence was analyzed as a negative control. A neutrophil membrane protein fraction enriched in PSGL-1 was also analyzed.
Polyclonal antisera generated against residues 19-37 (in the putative propeptide) and 42-56 (at the N terminus immediately following the propeptide) both bound to the 19-78 fusion protein (Fig. 3). The anti-42-56 serum recognized native PSGL-1, as observed previously(5, 10) . In contrast, the anti-19-37 serum did not bind native PSGL-1 on the Western blot, suggesting that the propeptide, which includes residues 19-37, had been removed.
To further localize the epitope for PL1, we expressed a series of smaller fusion proteins containing sequences within residues 19-78 (Fig. 2B). Western blotting revealed that PL1 bound to a fusion protein containing residues 51-63, but not to fusion proteins containing residues 42-56 or 56-63 (Fig. 4). The epitope for PL2 was studied less thoroughly. However, PL2 did not bind a fusion protein containing residues 235-266 (Fig. 2C and 4), indicating that the PL2 epitope requires residues located between positions 188 and 235.
Figure 4: Western blot analysis of PSGL-1-DHFR fusion proteins shown in Fig. 2, B and C. Proteins were resolved by SDS-PAGE under reducing conditions, followed by Coomassie Blue staining or Western blotting as in Fig. 3.
Figure 5: Reactivity of PL1 with octamer synthetic peptides derived from residues 19 to 77 of PSGL-1. Solid-phase overlapping octamer peptides beginning at the indicated N-terminal residue number were synthesized on polyethylene pins in a microtiter plate format. The reactivity of the pins with PL1 was determined by enzyme-linked immunosorbent assay. At the top is shown the sequence of the complete PL1 epitope, spanning residues 49-62. Below this are shown the sequences of all octamer peptides that reacted with PL1, with their relative reactivities listed as +, ++, or +++. Virtually identical results were obtained in a second independent experiment.
Figure 6:
Western blot analysis of native PSGL-1 and
recombinant PSGL-1 expressed in CHO cells. Lysates of a neutrophil
membrane protein fraction enriched in PSGL-1 or of CHO cells
co-expressing PSGL-1 with core 2
1,6-N-acetylglucosaminyltransferase and
fucosyltransferase IV were resolved by SDS-PAGE under reducing
conditions and then probed with the anti-42-56 or the
anti-19-37 sera. The anti-19-37 serum bound to PSGL-1
expressed in CHO cells, indicating that the propeptide had not been
removed from PSGL-1 in these cells. Lysates of CHO cells that did not
express PSGL-1 did not react with either
antiserum.
PSGL-1 is a sialomucin on human leukocytes that binds both P- and E-selectin. Using electron microscopy, we have established that the extracellular domain of PSGL-1 is highly extended, like other heavily O-glycosylated proteins. We have also mapped protein-dependent epitopes on PSGL-1 for two IgG mAbs, one of which completely blocks binding of PSGL-1 to P-selectin.
The rotary-shadowed images of PSGL-1 reported here are remarkably similar to those described previously for CD43, another sialomucin on leukocytes(20) . Both human PSGL-1 and rat CD43 are visualized as thin, highly extended molecules. Both proteins also form rosettes; these are assumed to result from interactions of the transmembrane domains to form the central hubs, with the extracellular domains extending outward from the hubs(20, 21, 28, 29) . The extended structures of the ectodomains of PSGL-1 and CD43 are thought to result from interactions of the Ser/Thr-linked GalNAc residues with adjacent amino acids in the peptide core(18, 19) . We estimate that the ectodomain of PSGL-1 is 50 nm long, which is only slightly longer than the 45-nm length estimated for the ectodomain of CD43(20) . The ectodomain of rat CD43 (assuming cleavage of the signal peptide) has 224 amino acids or 4.98 residues/nm. The ectodomain of human PSGL-1 (assuming cleavage of the signal peptide and propeptide) has 276 amino acids or 5.52 residues/nm. Therefore, the extracellular domain of PSGL-1 appears to be slightly more compact than that of CD43. The ectodomain of PSGL-1 has 72 serines and threonines, whereas the ectodomain of CD43 has 85 serines and threonines. Although it is not known how many of these residues are O-glycosylated, the ectodomain of CD43 may have a higher percentage of O-glycosylated amino acids than that of PSGL-1, accounting for its more extended structure relative to the total number of residues.
The individual molecules of PSGL-1 and CD43 appear to be monomers;
this seems to contradict the apparent disulfide-linked homodimeric
structure of PSGL-1 observed by SDS-PAGE(4) . It is possible
that disulfide bonding of PSGL-1 occurs only during preparation of
samples for SDS-PAGE; however, the dimers are still observed when
samples are alkylated with iodoacetamide before they are added to SDS
denaturing buffer. ()Because PSGL-1 is so extended, two
closely associated subunits in a dimer might be visualized as an
apparent monomer. If so, it is possible that CD43 forms noncovalently
associated dimers that are also visualized as monomers.
We found that the IgG mAbs PL1 and PL2 bind to bacterially expressed fusion proteins and/or synthetic peptides containing portions of the extracellular domain of PSGL-1. Thus, these mAbs clearly recognize protein-dependent epitopes, as do many IgG mAbs to CD43 (20) . Unlike the binding of some IgG mAbs to CD43(20) , however, the binding of PL1 or PL2 to PSGL-1 is not obviously affected by glycosylation. IgM mAbs to PSGL-1 have also been described(30) . Binding of these antibodies is eliminated when PSGL-1 is treated with sialidase. Like other IgM antibodies, these antibodies probably recognize epitopes that include carbohydrate components. If so, each IgM mAb may bind to more than one site on PSGL-1 and may also cross-react with carbohydrate epitopes on proteins other than PSGL-1.
The PL2 epitope includes residues within positions 188-235, located in decameric repeats 7-12. Therefore, the epitope is still positioned a considerable distance from the membrane. When PSGL-1 in solution is treated with O-sialoglycoprotein endopeptidase, it is digested into very small fragments that cannot be detected by SDS-PAGE (9) . In contrast, when leukocytes are treated with O-sialoglycoprotein endopeptidase, a relatively large fragment of PSGL-1 that includes the PL2 epitope is retained on the cell surface(10) . This suggests that, on the intact cell, O-sialoglycoprotein endopeptidase cannot cleave PSGL-1 at sites between the membrane and the PL2 epitope because of steric hindrance or other mechanisms.
PL1 blocks binding
of PSGL-1 to P-selectin, suggesting that the PL1 epitope overlaps the
P-selectin-binding site(10) . The PL1 epitope is located near
the extreme N terminus of PSGL-1 (Fig. 7). The complete epitope
spans residues 49-62, but PL1 binds strongly to octamer peptides
sharing the LPETE sequence at residues 54-58. The complete
epitope includes Tyr-51 and is adjacent to Tyr-46 and Tyr-48. Sulfation
of one or more of these residues is required for PSGL-1 to bind
P-selectin(12, 14, 15, 16) , and a
cobra venom protease that cleaves PSGL-1 between Tyr-51 and Asp-52
eliminates binding to P-selectin(31) . Because PL1 blocks
binding of PSGL-1 to P-selectin, it clearly recognizes
tyrosine-sulfated forms of PSGL-1. However, the interaction of PL1 with
synthetic peptides demonstrates that sulfation is not required for
binding. Furthermore, PL1 recognizes a recombinant form of PSGL-1 in
which the three tyrosines at residues 46, 48, and 51 are replaced with
phenylalanines(12) . The LPETE sequence in the PL1 epitope
includes Thr-57, a potential site for O-glycosylation. An Ig
chimera containing only residues 42-60 of PSGL-1, when
co-expressed with an 1,3-fucosyltransferase in COS cells, binds
P-selectin, although not as well as longer PSGL-1
constructs(16) . Substitution of Thr-57 with alanine in the
short construct eliminates binding. However, binding is only partially
reduced when Thr-44 and Thr-57 are substituted with alanine in longer
PSGL-1 constructs that are co-expressed with an
1,3-fucosyltransferase in COS cells(15) . The next closest
potential O-glycosylation sites are Thr-69 and Thr-70, which
may also be masked by binding of PL1. There is a potential N-linked glycosylation site at Asn-65, but enzymatic removal
of N-linked glycans does not obviously affect binding of
PSGL-1 to P-selectin(4) . More distant O-linked
glycans might participate in P-selectin recognition, but may be
incapable of supporting binding if PL1 masks the critical N-terminal
recognition site.
Figure 7: Localization of the PL1 epitope on PSGL-1. The amino acid sequence of the propeptide and the N-terminal region following the propeptide are shown. The complete PL1 epitope comprises residues 49-62. PL1 also binds strongly to all octamer peptides containing the LPETE sequence (underlined). Listed in bold are the three consensus tyrosine sulfation sites at residues 46, 48, and 51, as well as Thr-57, a potential site for attachment of an O-linked glycan that interacts with P-selectin. Underlined are the nearest other potential O-glycosylation sites at Thr-44, Thr-69, and Thr-70. A potential N-linked glycosylation site (NST) at residues 65-67 is italicized.
De Luca et al.(31) reported that
rabbit polyclonal antibodies raised to a peptide encoding residues
42-56 (1-15 if the sequence is renumbered after cleavage of
the propeptide) blocks binding of P-selectin to neutrophils. In
agreement with this result, we observed that our anti-42-56 serum
blocks binding of P-selectin to purified PSGL-1 or to human HL-60
cells. However, De Luca et al.(31) also
reported that rabbit polyclonal antibodies to a peptide spanning
residues 50-64 (9-23 in the new numbering system) have no
effect on binding of P-selectin to neutrophils. Since the sequence of
the peptide used to raise these polyclonal antibodies is almost
identical with the sequence of the PL1 epitope, it is difficult to
explain why the polyclonal antibodies did not inhibit binding. No data
were provided to confirm that the polyclonal anti-50-64
antibodies actually bind to intact PSGL-1 or to neutrophils (31) . Perhaps these antibodies are of low titer and/or low
affinity.
Residues 19-41 of PSGL-1 (Fig. 7) comprise a
putative propeptide that might be cleaved by proteases in the trans-Golgi network of some cells(8) . We found that a
polyclonal antiserum to residues 19-37 does not bind native
PSGL-1 from human neutrophils. This suggests that human myeloid cells
do cleave the propeptide, in agreement with conclusions made using a
different immunologic assay(32) . In contrast, the
anti-19-37 serum binds strongly to recombinant PSGL-1 expressed
in CHO cells, indicating that these cells do not cleave the propeptide.
PSGL-1 co-expressed in CHO cells with core 2
1,6-N-acetylglucosaminyltransferase and an
1,3-fucosyltransferase interacts with both P- and
E-selectin(12) . Therefore, cleavage of the propeptide is not
required for binding to either P- or E-selectin. The propeptide might
facilitate tyrosine sulfation or terminal O-glycosylation,
modifications that are needed for optimal interaction with P- or
E-selectin. However, deletion of the propeptide from PSGL-1 constructs
expressed in COS cells with an
1,3-fucosyltransferase does not
prevent binding to P-selectin(15) . Thus, the function of the
PSGL-1 propeptide remains unclear.
Like PSGL-1, P-selectin is an
extended molecule, with an ectodomain that is 38 nm
long(21) . The PSGL-1-binding site of P-selectin includes the
membrane-distal lectin domain, and the P-selectin-binding site of
PSGL-1 includes the N-terminal, membrane-distal region. The interaction
sites of both molecules, therefore, are projected well above their
respective cell surfaces, which may optimize the opportunities for
contact under shear forces. Indeed, shortening P-selectin by deletion
of its internal short consensus repeats markedly diminishes its ability
to mediate PSGL-1-dependent rolling of neutrophils on transfected CHO
cells under shear forces(33) . Most of the O-linked
glycans of PSGL-1 may function indirectly by extending the N-terminal
P-selectin-binding site far above the membrane, rather than by
interacting directly with P-selectin.