(Received for publication, October 19, 1995)
From the
P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like ligand
for P- and E-selectin on human leukocytes. PSGL-1 requires sialylated,
fucosylated O-linked glycans and tyrosine sulfate to bind
P-selectin. Less is known about the determinants that PSGL-1 requires
to bind E-selectin. To further define the modifications required for
PSGL-1 to bind P- and E-selectin, we transfected Chinese hamster ovary
(CHO) cells with cDNAs for PSGL-1 and specific glycosyltransferases.
CHO cells synthesize only core 1 O-linked glycans
(Gal1-3GalNAc
1-Ser/Thr); they lack core 2 O-linked glycans
(Gal
1-3(Gal
1-4GlcNAc
1-6)GalNAc
1-Ser/Thr)
because they do not express the core 2
1-6-N-acetylglucosaminyltransferase (C2GnT). CHO
cells also lack
1-3 fucosyltransferase activity. PSGL-1
expressed on transfected CHO cells bound P- and E-selectin only when it
was co-expressed with both C2GnT and an
1-3
fucosyltransferase (Fuc-TIII, Fuc-TIV, or Fuc-TVII). Chromatography of
-eliminated O-linked glycans from PSGL-1 co-expressed
with C2GnT confirmed synthesis of core 2 structures. Tyrosine residues
on PSGL-1 expressed in CHO cells were shown to be sulfated.
Phenylalanine replacement of three tyrosines within a consensus
sequence for tyrosine sulfation abolished binding to P-selectin but not
to E-selectin. These results demonstrate that PSGL-1 requires core 2 O-linked glycans that are sialylated and fucosylated to bind
P- and E-selectin. PSGL-1 also requires tyrosine sulfate to bind
P-selectin but not E-selectin.
The selectins are a group of three
Ca-dependent lectins that mediate rolling adhesion of
leukocytes on the vessel wall during inflammation (reviewed in McEver et al.(1) ). L-selectin, expressed on leukocytes,
binds to constitutively or inducibly expressed carbohydrate ligands on
endothelial cells. E-selectin, expressed on cytokine-activated
endothelium, and P-selectin, expressed on thrombin-activated
endothelial cells and platelets, bind to carbohydrate ligands on
myeloid cells and subsets of lymphocytes. Leukocyte rolling requires
that selectins rapidly associate and then dissociate from their ligands
in a manner largely independent of shear stress(2) . The
selectins interact weakly with sialylated and fucosylated
oligosaccharides such as sialyl Lewis x (sLe
), (
)but bind with higher affinity/avidity to only a few
glycoproteins(1) . Studies with mAbs support a physiologic role
for one of these glycoproteins, P-selectin glycoprotein ligand-1
(PSGL-1). PSGL-1 accounts for all the high affinity binding sites for
P-selectin on human leukocytes(3) . PSGL-1 must interact with
P-selectin in order for neutrophils to roll on P-selectin under
physiologic shear forces(3, 4) , and it also
contributes to the rolling of neutrophils on E-selectin (5) .
PSGL-1 is a membrane glycoprotein with two identical
disulfide-linked subunits(6) . Each subunit has at most three N-linked glycans, but has many clustered, sialylated O-linked glycans(6, 7) , some with
polylactosamine terminating in sLe(8) . PSGL-1 is a
type I membrane protein with an extracellular domain that is rich in
serines, threonines, and prolines(9) . PSGL-1 binds both
E-selectin and
P-selectin(8, 9, 10, 11) . The
structural requirements for binding are not well characterized,
although there is evidence that PSGL-1 does not interact identically
with P- and E-selectin(5, 8) . PSGL-1 must be
sialylated and fucosylated to bind both
molecules(6, 9, 10) . Enzymatic removal of N-linked glycans from human neutrophil PSGL-1 does not affect
binding to P-selectin, suggesting that O-linked glycans are
required for binding(6) . It is not known if O-linked
glycans are required for binding to E-selectin. Fab fragments of PL1,
an IgG mAb to PSGL-1, block binding of PSGL-1 to P-selectin but only
partially inhibit binding to E-selectin(3, 5) . The
PL1 epitope is located near the N terminus (3) , (
)near three clustered tyrosines within a consensus motif
for tyrosine sulfation(9, 12) . PSGL-1 is sulfated on
tyrosine rather than on carbohydrate, and enzymatic removal of sulfate
from tyrosine eliminates binding of PSGL-1 to P-selectin(13) .
These data demonstrate that tyrosine sulfate functions in conjunction
with sialylated and fucosylated glycans to mediate high affinity
binding of PSGL-1 to P-selectin. It is not known if tyrosine sulfation
is required for PSGL-1 to bind E-selectin.
To further characterize
the post-translational modifications that confer binding of PSGL-1 to
P-selectin and E-selectin, we transfected CHO cells, which have well
characterized oligosaccharide structures, with cDNAs for PSGL-1 and
various glycosyltransferases. PSGL-1 was co-expressed with a branching
enzyme for O-linked glycans (core 2
1-6-N-acetylglucosaminyltransferase (C2GnT)),
and/or an
1-3 fucosyltransferase (Fuc-TIII, Fuc-TIV, or
Fuc-TVII). Recombinant PSGL-1 co-expressed in CHO cells with the
appropriate glycosyltransferases bound avidly to both P- and
E-selectin.
A mutant PSGL-1 cDNA was created in which the codons encoding the tyrosines at residues 46, 48, and 51 were replaced with codons encoding phenylalanines. Mutagenesis was performed with a polymerase chain reaction protocol(20) . The PSGL-1 cDNA template was inserted into Bluescript (Stratagene) at the XbaI and KpnI sites. The outside primers for the polymerase chain reaction included a restriction site for XbaI on the 5` end in the Bluescript vector and for a unique StuI site in the coding region of the PSGL-1 cDNA. The amplified product was sequenced to confirm that it contained the expected substitutions. It was then excised with XbaI and StuI and used to replace the corresponding XbaI-StuI fragment of wild-type PSGL-1 in Bluescript. The entire PSGL-1 mutant cDNA was then excised from Bluescript with XbaI and KpnI and inserted into pZeoSV.
The C2GnT cDNA was amplified by the reverse transcriptase polymerase chain reaction from total RNA from human testis (Clontech). The two oligonucleotides used for polymerase chain reaction (5`-GCG GCG GCG TCT AGA CCA CCA TGC TGA GGA CGT TGC TGC GAA G-3` and 5`-CGC GCG CGT CGA CTC ACA GTC AGT GTT TTA ATG TCT CCA AAG C-3`) were designed to match the 5` and 3` ends of the published sequence of the human C2GnT cDNA(21) . The primers also included additional non-complementary sequence that included an XbaI site 5` to the initiating methionine and a SalI site 3` to the stop codon. The amplified product was ligated into the PCRII vector using the TA cloning kit (Invitrogen). The entire insert was sequenced to confirm its identity with the published sequence. The insert was then excised with EcoRI and ligated into the expression vector pcDNA3 (Invitrogen).
The human Fuc-TIII and Fuc-TIV cDNAs were
amplified by the polymerase chain reaction from genomic DNA. ()The amplified products were sequenced to confirm that they
matched the published sequences (22, 23) and were then
ligated into the expression vector pRC/RSV (Invitrogen). The cDNA
encoding human Fuc-TVII in the plasmid pCDM8 (24) was a
generous gift from Dr. John Lowe.
Fuc-TVII cDNA was transiently transfected into CHO cells permanently transfected with PSGL-1 cDNA, or with PSGL-1 cDNA plus C2GnT cDNA. In other experiments, wild-type or mutated PSGL-1 cDNA was transiently transfected into CHO cells permanently transfected with Fuc-TIII cDNA or C2GnT cDNA plus Fuc-TIII cDNA. Transiently transfected CHO cells were analyzed 2 days after transfection.
COS-7 cells were transfected with Fuc-TIII or Fuc-TIV
cDNA using Lipofectamine(TM) and selected in medium containing 400
µg/ml G418 to obtain cells permanently expressing Fuc-TIII or
Fuc-TIV. COS cells expressing Fuc-TIII were transiently
transfected with PSGL-1 cDNA, or PSGL-1 plus C2GnT cDNA using
Lipofectamine(TM). The cells were analyzed 2 days after
transfection.
Microtiter wells (Immulon I Removawell(TM) strips,
Dynatech Laboratories) were coated with soluble P-selectin or soluble
E-selectin (each at 2.5 µg/ml) in 100 µl of HBSS overnight at 4
°C. The wells were then blocked with 1% human serum albumin in HBSS
for 2 h at room temperature. Labeled CHO cells (100 µl, 2
10
cells) were added to each well and incubated on an
orbital shaker for 20 min at room temperature. In some experiments, the
cells were preincubated with 10 µg/ml of the mAbs PL1, PL2, G1, or
S12. After aspirating the unbound cells, the wells were gently washed
three times with HBSS. Each well was then cut out, and the bound
radioactivity was determined by liquid scintillation counting. The
number of bound cells was derived from a standard curve generated with
known concentrations of cells.
The O-linked core
1 disaccharide standard, Gal1-3GalNAcOH, was prepared by
reduction of Gal
1-3GalNAc with
NaB[
H]
. Other
[
H]glucosamine-labeled standards were prepared
from
-eliminated oligosaccharides from radiolabeled glycoproteins
of HL-60 cells. Each O-linked standard was authenticated by
sequential exoglycosidase digestion to yield the
Gal
1-3GalNAcOH structure(28) . Columns were also
calibrated using chitin-derived oligosaccharides
(GlcNAc
) prepared as described
previously(28) .
To determine
whether both C2GnT and an 1-3 fucosyltransferase must modify
PSGL-1 for it to bind P- or E-selectin, we first isolated clones of
permanently transfected CHO cells that expressed various combinations
of PSGL-1, C2GnT, and/or an
1-3 fucosyltransferase (Fuc-TIII
or Fuc-TIV). C2GnT activity was detected only in lysates of CHO cells
that were transfected with the C2GnT cDNA (Table 1). Flow
cytometric analysis with the anti-sLe
mAb CSLEX-1 indicated
that CHO cells expressed no endogeneous sLe
on the cell
surface (Fig. 1B). However, expression of Fuc-TIII or
Fuc-TIV caused appearance of sLe
on the CHO cell surface (Fig. 1, A, C-E, G, H). Clones of CHO cells transfected with PSGL-1 cDNA expressed
comparable levels of the protein, as measured by binding of the
anti-PSGL-1 mAbs PL1 and PL2 (Fig. 1, B-D, G, H). Indeed, expression of PSGL-1 on the CHO cells
was higher than on HL-60 cells (Fig. 1F).
Figure 1:
Expression of PSGL-1 and sLe on HL-60 cells and transfected CHO cells. CHO cells were
permanently transfected with cDNAs for PSGL-1, C2GnT, Fuc-TIII, or
Fuc-TIV as indicated. HL-60 cells and transfected CHO cells were
incubated with the nonbinding mAb MOPC21 (Control), with the
anti-PSGL-1 mAbs PL1 or PL2, or with the anti-sLe
mAb
CSLEX-1. Bound antibody was detected with fluorescein
isothiocyanate-conjugated goat anti-mouse IgG/IgM. The data are
representative of at least three independent
experiments.
To
determine whether PSGL-1 on CHO cells bound P-selectin, we used flow
cytometry to measure binding of fluid-phase P-selectin to the cell
surface (Fig. 2). Using this assay, we previously demonstrated
that PSGL-1 accounts for all the high affinity binding sites for
P-selectin on leukocytes; binding is abolished by the blocking
anti-PSGL-1 mAb PL1, but not by the non-blocking mAb PL2 (3) (Fig. 2F). P-selectin did not bind CHO
cells lacking PSGL-1, even when they expressed both C2GnT and an
1-3 fucosyltransferase (Fig. 2, A and E). P-selectin also did not bind CHO cells expressing PSGL-1
plus C2GnT (Fig. 2B), or CHO cells expressing PSGL-1
plus either Fuc-TIII or Fuc-TIV (Fig. 2, C and G). In contrast, P-selectin bound avidly to CHO cells
expressing PSGL-1 plus C2GnT and either Fuc-TIII or Fuc-2TIV (Fig. 2, D and H). PL1, but not PL2,
completely inhibited the binding, suggesting that P-selectin interacted
with a similar region on PSGL-1 expressed by either CHO cells or
leukocytes. These results establish that CHO cells require both C2GnT
and an
1-3 fucosyltransferase in order for PSGL-1 to bind
P-selectin.
Figure 2: Fluid-phase P-selectin binds PSGL-1 on transfected CHO cells when it is co-expressed with C2GnT and either Fuc-TIII or Fuc-TIV. The cells analyzed in Fig. 1were incubated without platelet P-selectin (Control) or with platelet P-selectin, in the presence or absence of the anti-PSGL-1 mAbs PL1 or PL2. Bound P-selectin was detected with a biotinylated nonblocking anti-P-selectin mAb, S12, followed by phycoerythrin-streptavidin. The data are representative of at least three independent experiments.
We also transiently expressed Fuc-TVII in CHO cell
clones that stably expressed PSGL-1, or PSGL-1 and C2GnT. Like Fuc-TIII
and Fuc-TIV, Fuc-TVII caused expression of sLe on the cell
surface (Fig. 3, A and B). Fuc-TVII also
conferred binding of PSGL-1 to P-selectin, but only when the cells also
expressed C2GnT (Fig. 3, C and D). Thus, CHO
cells express a functional form of PSGL-1 when it is co-expressed with
C2GnT and either of the fucosyltransferases normally expressed in
leukocytes, Fuc-TIV or Fuc-TVII.
Figure 3: Fluid-phase P-selectin binds PSGL-1 on transfected CHO cells when it is co-expressed with C2GnT and Fuc-TVII. CHO cells permanently transfected with cDNAs for PSGL-1, or for PSGL-1 and C2GnT, were transiently transfected with cDNA for Fuc-TVII. Two days after transfection, the cells were analyzed for binding of mAbs as in Fig. 1or for binding of fluid-phase P-selectin as in Fig. 2. The data are representative of three independent experiments.
Figure 4: Fluid-phase P-selectin does not bind PSGL-1 co-expressed with C2GnT and Fuc-TIII on COS cells. COS cells permanently transfected with cDNA for Fuc-TIII (A and C) or C2GnT and Fuc-TIII (B and D) were transiently transfected with cDNA for PSGL-1. Two days after transfection, the cells were analyzed for binding of mAbs as in Fig. 1or for binding of fluid-phase P-selectin as in Fig. 2. In parallel assays, fluid-phase P-selectin bound avidly to HL-60 cells and to CHO cells expressing PSGL-1, Fuc-TIII, and C2GnT as in Fig. 1. The data are representative of at least three independent experiments.
Figure 5: PSGL-1 co-expressed with C2GnT and Fuc-TIII in CHO cells mediates cell adhesion to immobilized P- and E-selectin. Adhesion of CHO cells permanently transfected with the indicated cDNAs was measured in wells containing immobilized P-selectin (A) or E-selectin (C). Adhesion of CHO cells expressing C2GnT, Fuc-TIII, and PSGL-1 to immobilized P-selectin (B) or E-selectin (D) was measured in the presence of the indicated mAb or in buffer containing EDTA. The data represent the mean ± S.D. of triplicate wells and are representative of three independent experiments.
Figure 6:
SDS-PAGE of
[H]glucosamine-labeled PSGL-1 expressed in CHO
cells. A, PSGL-1 was immunoprecipitated from lysates of
[
H]glucosamine-labeled CHO cells that
co-expressed Fuc-TIII, Fuc-TIV, or C2GnT and either Fuc-TIII or
Fuc-TIV. The immunoprecipitates were fractionated by SDS-PAGE under
reducing or nonreducing conditions, in 7.5% or 6% polyacrylamide gels,
respectively, followed by fluorography. For comparison,
I-labeled PSGL-1 from human neutrophils was analyzed in
parallel. B, [
H]glucosamine-labeled
PSGL-1 from CHO cells expressing Fuc-TIV, or C2GnT and Fuc-TIV, was
mock-treated, treated with sialidase, or treated with sialidase and
endo-
-N-acetylgalactosaminidase (endo-
-GalNAcase). The samples were then analyzed by
SDS-PAGE under nonreducing conditions, followed by
fluorography.
To confirm
that co-expression of C2GnT caused addition of core 2 O-linked
glycans to PSGL-1, immunoprecipitated
[H]glucosamine-labeled PSGL-1 was resolved by
SDS-PAGE under nonreducing conditions. The gel slice containing PSGL-1
was excised and subjected to
-elimination. The released
[
H]glucosamine-labeled O-linked glycans
were desialylated and analyzed by amine-adsorption high performance
liquid chromatography. The radiolabeled oligosaccharides from PSGL-1,
when co-expressed only with FucT-III or Fuc-TIV, eluted as the
GalNAcitol and Gal
1-3GalNAcitol standards, consistent with
their predicted core 1 structures (Fig. 7). In contrast, most of
the radiolabeled oligosaccharides from PSGL-1, when co-expressed with
C2GnT plus either Fuc-TIII or Fuc-TIV, co-eluted with the
tetrasaccharide core 2 standard,
Gal
1-3(Gal
1-4GlcNAc
1-6)GalNAcitol (Fig. 7). Some of the radioactivity in cells co-expressing C2GnT
and Fuc-TIV eluted as a pentasaccharide, indicating the presence of a
larger core 2 structure(29) . This peak was no longer observed
after treatment of the labeled glycans from these cells with an
1-3-specific fucosidase (Fig. 7). This suggests that
the larger structure is a core 2 tetrasaccharide modified with a fucose
in
1-3 linkage to the
1-6-linked N-acetylglucosamine. These data confirm that co-expression of
C2GnT results in addition of core 2 O-linked glycans to PSGL-1
in CHO cells.
Figure 7:
Neutral O-linked glycans from
PSGL-1 are modified by C2GnT.
[H]Glucosamine-labeled O-linked glycans
were
-eliminated from PSGL-1 co-expressed in CHO cells with
Fuc-TIII (A) or Fuc-TIV (B), in the presence or
absence of C2GnT. The oligosaccharides were desialylated and then
analyzed by amine-adsorption high performance liquid chromatography. A
portion of the glycans from PSGL-1 co-expressed with C2GnT and Fuc-TIV
was also treated with
1-3 fucosidase prior to analysis. The
radioactivity in each fraction was measured by scintillation counting.
The retention times of the core 1 and core 2 oligosaccharide standards
are shown.
Figure 8:
PSGL-1 expressed in CHO cells is sulfated
on tyrosine. CHO cells expressing PSGL-1, C2GnT, and either Fuc-TIII or
Fuc-TIV were metabolically labeled with
[S]sulfate. A,
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 analyzed by SDS-PAGE under nonreducing
conditions, followed by fluorography. B,
S-PSGL-1
from CHO cells co-expressing C2GnT and Fuc-TIV was hydrolyzed with
strong base. The hydrolysate was analyzed by ion exchange
chromatography using a Na
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.
PSGL-1 has three tyrosines at residues 46, 48, and 51 that are
located within a consensus sequence for tyrosine
sulfation(9, 12) . To test the importance of these
residues for binding P-selectin and E-selectin, we prepared a construct
of PSGL-1 in which all three tyrosines were replaced with
phenylalanines. Wild-type or mutant PSGL-1 cDNA was transiently
transfected into CHO cells that permanently expressed C2GnT and
Fuc-TIII. The use of the same permanently transfected cells for
transient expression of wild-type or mutant PSGL-1 ensured equivalent
levels of C2GnT activity (Table 1) and surface expression of
sLe (Fig. 9). Furthermore, surface levels of
wild-type and mutant PSGL-1 were comparable (Fig. 9). However,
fluid-phase P-selectin bound to CHO cells expressing wild-type PSGL-1,
but not to cells expressing mutant PSGL-1 (Fig. 9). Furthermore,
CHO cells expressing mutant PSGL-1 did not adhere to immobilized
P-selectin, although they did adhere to immobilized E-selectin (Fig. 10). These data demonstrate that PSGL-1 expressed in CHO
cells requires at least one of the three N-terminal tyrosines to bind
P-selectin, presumably because this residue(s) must be sulfated. In
contrast, appropriately glycosylated PSGL-1 does not require the
tyrosines, and therefore tyrosine sulfate, to bind E-selectin.
Figure 9: Fluid-phase P-selectin does not bind CHO cells expressing PSGL-1 in which phenylalanines replace three tyrosines in a consensus sequence for tyrosine sulfation. CHO cells permanently transfected with cDNAs for C2GnT and Fuc-TIII were transiently transfected with cDNA for wild-type or mutant PSGL-1. Two days after transfection, the cells were analyzed for binding of mAbs as in Fig. 1or for binding of fluid-phase P-selectin as in Fig. 2. The data are representative of at least four independent experiments.
Figure 10:
CHO cells expressing PSGL-1 in which
phenylalanines replace three tyrosines do not adhere to immobilized
P-selectin but do adhere to immobilized E-selectin. Aliquots of the
same transfected cells used in the flow cytometry experiment of Fig. 9were used in the cell adhesion assay. As an additional
control, some cells permanently transfected with cDNAs for C2GnT and
Fuc-TIII were not transiently transfected with cDNA for PSGL-1. One day
after transfection, the cells were labeled with
[H]thymidine. Two days after transfection,
adhesion of the CHO cells was measured in wells containing immobilized
P-selectin (A) or E-selectin (B). The data represent
the mean ± S.D. of triplicate wells and are representative of
two independent experiments.
Because their oligosaccharide structures are well
characterized, CHO cells are an excellent model for analyzing the
effects of glycosylation on protein structure and function. CHO cells
synthesize complex-type bi-, tri-, and tetra-antennary N-glycans with type 2 lactosamine (Gal1-4GlcNAc)
and polylactosamine; the only other modification is terminal
sialylation as NeuAc
2-3Gal
-4GlcNAc-R. CHO cells
synthesize O-glycans with simple core 1 structures that can be
mono- or disialylated. Our results establish that PSGL-1 expressed in
CHO cells binds P- and E-selectin only when it is co-expressed with
C2GnT and an
1-3 fucosyltransferase. These enzymes mediate
branching and fucosylation of the O-linked glycans,
modifications that are critical for PSGL-1 to bind P- and E-selectin.
PSGL-1 expressed in CHO cells is also sulfated on tyrosine, a
modification required for binding P-selectin but not E-selectin. These
studies extend previous findings on PSGL-1 from human leukocytes that
1) sialic acids on PSGL-1 are required for binding
P-selectin(6) ; 2) enzymatic removal of N-linked
glycans from PSGL-1 does not affect binding to P-selectin(6) ;
3) PSGL-1 has many clustered, sialylated O-linked
glycans(7) , some with polylactosamine terminating in
sLe
(8) ; and 4) enzymatic removal of sulfate from
tyrosine on PSGL-1 abrogates binding to P-selectin(13) .
We
demonstrated previously that Fab fragments of PL1, a mAb to a
membrane-distal epitope on PSGL-1, block binding of P-selectin to
PSGL-1 on human leukocytes(3) . Here we show that PL1 also
blocks binding of P-selectin to PSGL-1 that is expressed with C2GnT and
an 1-3 fucosyltransferase on CHO cells. This suggests that
P-selectin binds a similar region on native and recombinant PSGL-1. On
leukocytes, the putative N terminus of mature PSGL-1 begins at residue
42, just C-terminal to a consensus propeptide cleavage
site(9) . The PL1 epitope has been mapped to a region within
residues 49-62.
A polyclonal antiserum to residues
42-56 also prevents binding of P-selectin to human leukocyte
PSGL-1 and to PSGL-1 co-expressed with C2GnT and an
1-3
fucosyltransferase in CHO cells.
This region contains three
tyrosines within a consensus sequence for tyrosine
sulfation(12) , and tyrosine sulfation of PSGL-1 on leukocytes
is required for high affinity binding to P-selectin(13) . Here
we show that PSGL-1 expressed in CHO cells is also sulfated on tyrosine
and that mutation of the three tyrosines eliminates binding to
P-selectin. Taken together, the data suggest that P-selectin
preferentially binds an N-terminal region of PSGL-1 that requires at
least one tyrosine sulfate and one sialylated, fucosylated, core 2 O-linked glycan. The precise spatial relationship of the
tyrosine sulfate(s) to the O-linked glycan(s) remains to be
determined.
Although PL1 blocks binding of purified leukocyte PSGL-1 to P-selectin, it only partially inhibits binding to E-selectin(3, 5) . Competitive binding experiments suggest that E-selectin binds with lower affinity than P-selectin to the region that overlaps the PL1 epitope, but also binds an additional site(s) on PSGL-1 from human leukocytes(5, 8) . Here we demonstrate that PL1 fails to inhibit PSGL-1-dependent adhesion of transfected CHO cells to E-selectin. Furthermore, substitution of the N-terminal tyrosines, which eliminates adhesion to P-selectin, does not affect adhesion to E-selectin. These data demonstrate that, like leukocyte PSGL-1, PSGL-1 expressed in CHO cells binds differently to E-selectin than to P-selectin, although binding to both selectins requires sialylated, fucosylated, core 2 O-linked glycans.
The O-glycosylation sites and specific structures of the
core 2 O-linked glycans required for native or recombinant
PSGL-1 to optimally bind P-selectin or E-selectin need further study.
For example, native PSGL-1 has polylactosamine(8) , which can
be extended from core 2 structures(35) , but the actual
structures of the O-linked glycans on native PSGL-1 have not
been determined. It is possible that polylactosamine extension is
required for a terminal sLe structure to interact optimally
with P-selectin or E-selectin. Fucosylation of internal N-acetylglucosamine residues might also be required for
optimal binding(39) . Internal fucosylation renders
polylactosamine resistant to cleavage by endo-
-galactosidase and
may explain why treatment of native PSGL-1 with this enzyme does not
eliminate binding to P-selectin(8) .
Although
fucosyltransferases have been intensively studied with regard to their
actions on small oligosaccharide acceptors and N-linked
glycans(22, 23, 24, 25, 40, 41, 42, 43, 44) ,
little is known about how fucosyltransferases modify O-linked
glycans. We found that PSGL-1 binds P- and E-selectin when it is
co-expressed in CHO cells with C2GnT and either Fuc-TIII, Fuc-TIV, or
Fuc-TVII. However, the data do not demonstrate that these
fucosyltransferases modify specific O-linked glycans in the
same manner. Furthermore, it is not known whether Fuc-TIV and Fuc-TVII
modify PSGL-1 similarly in leukocytes, where the two enzymes are
normally expressed(23, 24, 42) .
Chromatography of the -eliminated glycans of PSGL-1 co-expressed
with C2GnT and Fuc-TIV in CHO cells revealed an oligosaccharide eluting
as a pentasaccharide that was not observed in
-eliminated O-linked glycans from CHO cells co-expressing C2GnT and
Fuc-TIII. Based on the change in elution position after digestion with
an
1-3-specific fucosidase, the pentasaccharide is probably
a core 2 tetrasaccharide with a fucose linked
1-3 to the N-acetylglucosamine. It should be noted that larger O-linked structures with polylactosamine could be present but
not be detected if they do not elute from the amine-adsorption column
under the conditions used. The fucosylated polylactosamine-containing N-linked glycans synthesized by CHO cells expressing Fuc-TIV
are smaller than those synthesized by CHO cells expressing Fuc-TIII,
suggesting that fucosylation by Fuc-TIV competes with polylactosamine
chain extension(37) . Perhaps Fuc-TIV and Fuc-TIII also
differentially modify the core 2 O-linked glycans in CHO
cells. If so, Fuc-TIV might produce a core 2 glycan with a single
terminal sLe
; this structure would be detected as a core 2
pentasaccharide after sialidase treatment, as observed in Fig. 7. Fuc-TIII might preferentially fucosylate core 2 O-linked glycans with longer polylactosamine that are not
readily identified by amine-adsorption high performance liquid
chromatography under the conditions used.
The importance of appropriate core 2 O-linked glycans is underscored by the observation that P-selectin bound better to PSGL-1 co-expressed with C2GnT and Fuc-TIII on CHO cells than to PSGL-1 co-expressed with C2GnT and Fuc-TIII on COS cells. It is possible that PSGL-1 requires modification of only a few specific O-linked glycans to bind optimally to P-selectin. Modifications of other O-linked glycans may ensure optimal binding to E-selectin. PSGL-1 expressed on CHO cells shares several structural and functional features with PSGL-1 on leukocytes. However, detailed characterization of the O-linked structures at specific sites on native and recombinant PSGL-1 is needed to define the requirements for optimal binding to P- and E-selectin.
Note Added in Proof-Since
submission of this manuscript, two other groups have reported that
PSGL-1 must be tyrosine sulfated at its N-terminal region to confer
binding to P-selectin but not to E-selectin (45, 46) .
Both groups studied PSGL-1 that was co-expressed in COS cells with an
1-3 fucosyltransferase. These reports, in conjunction with our
data, indicate that PSGL-1 is tyrosine sulfated in COS cells as well as
in CHO cells and myeloid cells. It is still not known why fluid-phase
P-selectin binds better to PSGL-1 co-expressed with C2GnT and Fuc-TIII
on CHO cells than to PSGL-1 co-expressed with C2GnT and Fuc-TIII on COS
cells.