(Received for publication, September 14, 1995; and in revised form, December 7, 1995)
From the
P-selectin is a vascular cell adhesion molecule that is
expressed on the surface of platelets and endothelial cells in response
to inflammatory stimuli. It is believed to aid in the binding and
recruitment of leukocytes to inflamed tissue. P-selectin adhesion to
leukocytes is mediated by the amino-terminal lectin domain that binds
the sialyl Lewis (sLe
) carbohydrate
(Neu5Ac
2-3Gal
1-4(Fuc
1-3)GlcNAc).
Neither the three-dimensional structure of P-selectin nor the
protein-carbohydrate interactions that mediate the binding of
P-selectin to the sLe
carbohydrate have been determined.
The most closely related protein for which a ligand-bound
three-dimensional structure has been resolved is the rat
mannose-binding protein (Weis, W. I., Drickamer, K., and Hendrickson,
W. A.(1992) Nature 360, 127-134). Using the known
binding interactions that occur between the rat mannose-binding protein
and its ligand (oligomannose) as a template, we have used site-directed
mutagenesis to substitute Ala-77 with lysine. This substitution changed
P-selectin-carbohydrate binding specificity from sLe
to
oligomannose. Further substitution altered the binding preference from
mannose to galactose in a predictable manner. These results indicate
that P-selectin binds sLe
in a shallow cleft that is
similar to the mannose-binding protein saccharide-binding cleft.
Additionally, we present an extensive mutagenic analysis of P-selectin
Lys-113, a residue that has previously been implicated in P-selectin
binding to both sLe
and 3-sulfated galactosylceramide
(sulfatide). Our analysis demonstrates that Lys-113 is probably not
involved in P-selectin binding to either sulfatide or sLe
.
Functionally, it appears that P-selectin has retained a conserved
carbohydrate and calcium coordination site that enables it to bind
carbohydrate in a manner that is quite similar to that which has been
determined for the rat mannose-binding protein.
P-selectin (CD62-P, PADGEM, GMP-140) is a 140-kDa glycoprotein that is typically stored in the secretory granules of platelets and endothelial cells. Various inflammatory stimuli including thrombin and histamine induce the fusion of these storage vesicles with the cell plasma membrane and result in the immediate surface expression of P-selectin(1, 2) . Once present on the cell surface, P-selectin is able to bind to carbohydrate ligands on the surface of circulating leukocytes and aid in the adhesion and subsequent recruitment of leukocytes to areas of injury or inflammation.
The
nucleotide sequence of human P-selectin was reported by Johnston et
al.(3) , who also noted amino acid sequence homology to
several other proteins. Following the signal sequence at the amino
terminus, the protein is composed of a 120-amino acid lectin-like
(lectin or carbohydrate recognition domain), an epidermal growth
factor-like domain, nine short consensus or complement receptor-like
repeats, a membrane-spanning domain, and a cytoplasmic tail containing
35 amino acids.
The amino acid sequence of the P-selectin
lectin domain is similar to a variety of calcium-dependent
carbohydrate-binding proteins known as C-type lectins(4) . E-,
P-, and L-selectins, the asialoglycoprotein receptor, the low affinity
IgE receptor (CD23), the pulmonary surfactant apoprotein SP-A, and the
rat mannose-binding protein (rMBP) ()are some of the members
of the C-type lectin family. The lectin domains of each of these
proteins are related through amino acid sequence similarity that
includes 14 invariant and 17 highly conserved
residues(5, 6) . The conserved amino acids are thought
to be essential for the establishment of a hydrophobic core or scaffold
that is a structural characteristic of each of the members of this
protein class(7) .
E-, P-, and L-selectins are vascular cell
adhesion molecules that share 60-70% identity between the amino
acid sequences of their lectin domains. Each selectin has been
demonstrated to bind the sLe carbohydrate(8, 9, 10, 11) .
However, the binding characteristics of each of the selectins are not
comparable, and many differences in their binding specificities have
been
described(12, 13, 14, 15, 16, 17) and extensively reviewed(18) . One important
binding distinction is that P-selectin is able to bind a second ligand,
3-sulfated galactosylceramide (sulfatide), while E-selectin does
not(19, 20) . Similarly, the adherence of both P- and
L-selectins to the sLe
carbohydrate can be competitively
inhibited by heparin, while E-selectin-sLe
binding appears
to be heparin-insensitive(14) . The individual molecular
components and interactions that are responsible for most of the
observed differences in selectin-carbohydrate adherence remain
undefined. Moreover, the mechanisms that allow each selectin to
discriminate between distinct biological protein ligands have in most
cases not been determined(21, 22) . However, it is
apparent that the physical ligand binding characteristics of one
selectin should not be attributed to another member of the protein
family without definitive experimental evidence.
We previously
predicted that the C-2 and C-3 hydroxyls of the sLe
1-3-linked fucose coordinate in a calcium-dependent
manner with E-selectin residues Glu-80, Asn-82, Asn-105, and Asp-106
and that the remainder of the sLe
carbohydrate is retained
in a shallow pocket that is defined on one side by the E-selectin
5-strand, on a second side by loop 5 (IKREK, residues
95-99), and on a third side by loop 3 (NWAPGE, residues
75-80)(23) . This positioning is analogous to the site
that oligomannose occupies upon binding to
rMBP(24, 25) . We wished to determine if P-selectin
binds sLe
in a similar manner. All previously published
reports that have attempted to identify the P-selectin
sLe
-binding site have predicted that it does not. Instead,
these reports have proposed that the bound sLe
carbohydrate
extends in the opposite direction and that P-selectin binding to
sLe
(20, 27) occurs through the formation
of a charge-paired interaction between P-selectin Lys-113 and the
sLe
sialic acid carboxylate. It has further been
hypothesized that P-selectin Lys-113 also directly coordinates with the
sulfatide sulfate moiety (27) and that sulfatide binding is
therefore able to displace or block sLe
-dependent cellular
adherence (27) because the sulfatide- and
sLe
-binding sites are overlapping.
To determine which of
these two proposed P-selectin-sLe binding models is
correct, we attempted to mutagenically change P-selectin-ligand
recognition by altering the same amino acids that we had previously
determined were essential for E-selectin-ligand recognition and
discrimination. We predicted that if P-selectin mutant binding proved
to be phenotypically identical to that of the E-selectin mutants, it
would serve to confirm the structural conformation of the P-selectin
calcium site. Additionally, if P-selectin-carbohydrate adherence was
similarly affected by these same amino acid substitutions, then the
mutant binding data would support our hypothesis that the
sLe
-binding site is defined by the same shallow grove that
is occupied by oligomannose upon binding to MBP. Alternatively, if the
P-selectin mutants did not display similar binding characteristics, it
would indicate that P-selectin-carbohydrate ligation is fundamentally
different from E-selectin binding and that the noted differences in
sulfatide and heparin affinity merely reflect that basic difference.
It seemed probable that if P-selectin binds sLe in a
manner similar to that which we proposed for E-selectin, many of the
residues predicted to be essential for E-selectin-sLe
binding would be conserved between E- and P-selectins. In this
regard, it is notable that the E-selectin calcium-coordinating ions
(Glu-80, Asn-82, Asn-105, and Asp-106) as determined from the x-ray
crystallographic structure (23) are also present in the
P-selectin primary sequence. For clarification and comparison purposes,
a structural alignment of E-selectin, rMBP, and P-selectin that was
adapted from the work of Graves et al.(24) is shown
in Fig. 1A, and an illustrative model of P-selectin
that was constructed from the E-selectin structural coordinates
(Brookhaven Protein Data Bank accession number 1ESL) is shown in Fig. 1B.
Figure 1:
A,
structural alignment of the P-selectin lectin domain with E-selectin
and the rat mannose-binding protein. Shown is a protein sequence
alignment of human P- and E-selectin and rat mannose-binding protein
lectin domains as deduced from a comparison of the respective crystal
structures(24) . Specific residues discussed in the text have
been labeled with the appropriate amino acid sequence numbers. Shaded residues denote residues conserved among all three
proteins. B, illustration of the approximate positioning of
pertinent P-selectin amino acids. A model of P-selectin was constructed
for illustrative purposes only using the three-dimensional crystal
structure of E-selectin as a template. The amino acid backbone is shown
in green, with the -helices pictured as red
cylinders. The bound calcium is a turquoise sphere.
Pertinent amino acid side chains described in the text are illustrated
as space-filling spheres as follows: Lys-96 is white, Ser-97
is magenta, Pro-98 is turquoise, Ala-77 is gold, and Lys-113 is pink.
As can be seen from the amino acid sequence
alignment (see Fig. 1A), substantial homology is shared
between E- and P-selectins, yet the two proteins still contain a large
number of amino acids that are not identical (30%). Some of these
nonidentical residues comprise the proposed sLe
-binding
site and include two amino acid substitutions in loop 3 (residues
75-80) and three of the five amino acids in loop 5 (residues
95-99; E-selectin residues IKREK as compared with P-selectin
residues IKSPS).
Since the three-dimensional structure of the
P-selectin lectin domain has not been determined, we do not know how
these differing amino acids affect the conformation of the protein or
how the individual amino acid side chains alter or influence
P-selectin-ligand binding. In this regard, it is notable that extensive
evidence derived from mutagenic structure-function analyses of
P-selectin that were performed by Hollenbaugh et al. (26),
Bajorath et al. (27) and Erbe et al.(20) indicate that the P-selectin amino acids in loop 5
may influence P-selectin binding to sLe. Additionally, each
of these groups noted the importance of P-selectin Lys-113, which when
changed to an alanine or arginine, obliterates P-selectin binding to
both sLe
and sulfatide.
To further our understanding of
the protein-carbohydrate interactions that occur when P-selectin binds
to either the sLe oligosaccharide or sulfatide, we have
used site-directed mutagenesis to complete a more extensive mutational
analysis of P-selectin-ligand binding interactions. Unlike the results
previously reported by others (27) , our data indicate that
Lys-113 does not directly contact either the sLe
sialic
acid carboxylate or bound sulfatide in a charge-dependent manner.
Furthermore, in an effort to elucidate the critical contacts that are
made between P-selectin and its carbohydrate ligands, we have used the
same mutagenesis scheme that we described previously for E-selectin (23) and have altered P-selectin amino acids that would be
predicted from homology to the primary amino acid sequences of
E-selectin and rMBP to influence the adherence of P-selectin to
sLe
. The presented evidence suggests that it is likely that
P-selectin binds sLe
in a manner analogous to that in which
the rat mannose-binding protein binds oligomannose and that sulfatide
is bound at an entirely different location.
To
aid in protein stabilization, detection, quantitation, and
purification, this P-selectin cassette was fused to the hinge region of
the mouse IgG heavy chain coding sequence to create a
soluble, secreted protein expressed from a construct similar to that
previously described(10) . The mouse IgG
constant
region coding sequence containing one intron was PCR-cloned from cDNA
made from total RNA isolated from the hybridoma cell line 402C10 (29) (kindly provided by Dr. R. J. Bjercke) using
oligonucleotides GAAAGGTACCAGAGGGCCCACAATC and GAGCAAGCTTACCCGGAGTCCG.
The P-selectin-mouse IgG fusion gene was constructed in the mammalian
expression vector pJB20, the pCMV plasmid described in (30) with several restriction sites removed. This construct
allowed specific mutations in the P-selectin CRD to be made using
site-directed mutagenesis on single-stranded M13 templates. Each mutant
was plaque-purified, sequenced, and subsequently cassetted into the
fusion gene as an
700-base pair HindIII/KpnI
fragment. The E-selectin-IgG construct and mutant proteins were made
and expressed as described previously(23) .
The amount of recombinant protein recovered on the beads was
monitored by ELISA using anti-P-selectin antibody AC1.2 or anti-E-
selectin antibody BBA8 (R& Systems). ELISAs were performed as
follows. Beads (8 10
) were added to duplicate wells
of 96-well flexible assay plates that had been previously blocked with
PBS supplemented with 3% BSA; equivalent amounts of wild-type
E-selectin- or P-selectin-coated beads together with beads prepared
from mock-transfected cell lysates were tested at the same time on the
same plate as positive and negative controls. Using a magnetic
separator to retain the beads inside the wells, the wells were washed
once with 50 µl of PBS and then incubated in a solution of 6%
formaldehyde in PBS for 20 min at room temperature. This was followed
by two sequential PBS washes and a second 20-min incubation in PBS
supplemented with 100 mM ammonium chloride. The beads and
wells were then blocked overnight in PBS supplemented with 3% BSA and
1% rabbit serum.
When the blocking buffer was removed, three
sequential PBS washes were performed, and the primary anti-selectin
antibodies were added at final concentrations of 0.2 µg/ml in PBS
supplemented with 3% BSA and incubated for a minimum of 2 h at 4
°C. The sample wells were then washed three times with PBS and, in
the case of BBA8, incubated with a 1:2000 dilution of alkaline
phosphatase-conjugated streptavidin (Calbiochem, catalog No. 189732).
For AC1.2, a 1:5000 dilution of alkaline phosphatase-conjugated goat
anti-mouse light chain (Caltag Laboratories, catalog No. M33008)
or, for some experiments, a 1:5000 dilution of alkaline
phosphatase-conjugated rabbit anti-mouse IgG
(Cappel,
catalog No. 59569) was added and incubated in a solution of PBS
supplemented with 3% BSA for a minimum of 2 h at 4 °C. The sample
wells were then washed two times with PBS and once with alkaline
phosphatase ELISA buffer solution (pH 9.4) of 10 mM diethanolamine, 0.5 mM MgCl
, and 10 mM NaCl. The alkaline phosphatase colorimetric assays were typically
developed overnight at 4 °C using standard buffers and substrates
(5-bromo-4-chloro-3-indolyl phosphate and nitro blue
tetrazolium)(31) . The absorbance was monitored with a 415-nm
filter in a Bio-Rad model 450 microplate reader.
All of the mutant proteins discussed in this work were readily detectable using these mouse monoclonal antibodies, and the background absorbance generated by the mock control beads was negligible and typically undetectable. Furthermore, the ELISA results (data not shown) indicate that the proteins were synthesized and secreted in similar quantities and that they each have similar stabilities when purified and stored for extended periods of time at 4 °C.
P- and E-selectin-HL-60 cell
binding assays were performed in Falcon 96-well flexible assay plates.
The wells were first blocked by incubating briefly with PBS
supplemented with 3% BSA. After aspirating the blocking buffer, 10
µl of HL-60 cells (10 cells/ml of RPMI 1640 medium
supplement with 10% FCS) that had been fluorescently labeled with
calcein AMC-3099 (Molecular Probes, Inc.) were added to each well,
followed by 10 µl of beads (4
10
/ml). Beads
used in the mock experiments were prepared from COS-1 supernatants that
had been transfected with vector alone. The cells and beads were
incubated together at room temperature for 10 min. If sLe
tetrasaccharide inhibition was being tested, the tetrasaccharide
was added immediately prior to this incubation. Following incubation,
the assay plate was then placed on a magnetic separator and incubated
for an additional 2 min. While the plate remained on the separator,
excess unbound HL-60 cells were removed, and the wells were washed
twice with PBS to remove any remaining unbound cells. The HL-60 cells
remaining bound to the beads were inspected by microscopy and then
lysed by adding 50 µl of a 1% solution of Nonidet P-40 in PBS.
Binding was quantitated fluorometrically using a Millipore Cytofluor
2350 fluorometer.
Figure 3:
A, invertase filter binding assay. The
wild-type P-selectin-IgG fusion protein (unsubstituted) and two
P-selectin mutants containing the A77K mutation bound to Dynabeads were
assessed for their ability to bind to an invertase-coated
nitrocellulose filter. The dark gray areas shown are the
actual protein-bound beads retained on the filter after washing. It is
apparent that each of the P-selectin A77K mutants, like the E-selectin
A77K positive control, bind the oligomannose present on yeast invertase
and are retained on the filter, whereas the unmutagenized wild-type (WT) P- and E-selectins as well as the mock IgG beads are not
retained. B, assay of P-selectin mutant mannose-binding
activity. Stock solutions containing 2.5 mg/ml (lyophilized
weight/volume) yeast invertase, S. cerevisiae (yeast), and BSA
in 25 mM Tris (pH 7.5), 1 mM 2-mercaptoethanol, 0.5%
SDS, and 5% glycerol were coated onto 96-well flexible assay plates.
Beads conjugated with various P- selectin-IgG proteins (2
10
beads) were added to each well. After washing, the
number of beads retained in the wells was determined by ELISA. The
results shown are the average of two independent experiments run in
duplicate.
Figure 2:
A, lysine substitution mutants are able to
bind HL-60 cells. P-selectin mutant proteins were immunoprecipitated
using goat anti-mouse IgG-conjugated magnetic beads. After ELISA
quantitation to ensure that the same amount of recombinant P-selectin
protein was attached to the beads, 4 10
P-selectin-IgG-adsorbed beads were mixed with 10
fluorescently labeled HL-60 cells and incubated together at room
temperature for 10 min. A magnetic separator was used to separate the
bead-bound HL-60 cells. Unbound cells were removed by sequential PBS
washes. The number of cells bound by the wild-type P-selectin-IgG
fusion protein is defined as 100% and that for the mock control wells
is designated 0%. All assays were performed in duplicate, and the
results shown are the average of two independent experiments. Beads
used in the mock experiments were prepared from COS-1 supernatants that
had been transfected with vector alone. Binding was quantitated
fluorometrically using a Millipore Cytofluor 2350 fluorometer. B, shown is P-selectin mutant glycolipid-binding ability.
P-selectin-IgG fusion proteins were assayed for adherence to sLe
glycolipid. Stock solutions containing sLe
glycolipid
were prepared and coated onto 96-well flexible assay plates as
described(32) . Beads conjugated with various P-selectin-IgG
proteins (2
10
beads) were added to each well.
After washing, the number of beads retained in the wells was determined
by ELISA. The results shown are the average of two independent
experiments run in duplicate. C, P-selectin mutant
sulfatide-binding ability. P-selectin-IgG fusion proteins were assessed
for the ability to adhere to sulfatide-coated 96-well flexible assay
plates. Beads conjugated with various P-selectin mutants (2
10
beads) were added to each well. After washing, the
number of beads retained in the wells was determined by ELISA. The
results shown are the average of two independent experiments run in
duplicate.
If P-selectin-sLe binding is indeed similar to
that which we have proposed for E-selectin(23) , the P-selectin
sLe
tetrasaccharide-binding site would be defined by a
cleft located between loop 3 (NWADNE, residues 75-80)), loop 5
(IKSPS, residues 95-99), and the
-strand (WNDE,
residues 104-107) as determined from homology to E-selectin and
rMBP(23, 24, 25) . Lys-113 would not be
expected to be directly involved in adherence to the sLe
tetrasaccharide (Fig. 1B). Since this did not
agree with previous proposals(20, 26, 27) ,
it was important to analyze the precise involvement of this residue by
completing a more extensive mutagenic analysis.
To preserve the
hydrophilic nature of the side chain, K113E and K113Q mutants together
with a K113A control mutant were generated by site-directed
mutagenesis. These mutants were analyzed for sLe binding,
which was assessed using an sLe
-dependent HL-60 cell assay
(see ``Materials and Methods'' for
details)(20, 24, 26, 27) . The
results in Fig. 2A, together with the previously
reported inability of the K113R mutant to bind
sLe
(27) , indicate that the side chain length of
the amino acid at this position may be critical for sLe
binding. However, replacement of this lysine residue with an
uncharged or oppositely charged amino acid does not alter P-selectin
adherence to HL-60 cells, to sLe
glycolipid, or to
sulfatide coated onto polystyrene dishes (Fig. 2, B and C). Therefore, it appears that the Lys-113 side chain probably
does not form a charge-paired electrostatic interaction with either the
negatively charged sialic acid carboxylate of sLe
or the
galactose 3-sulfate group of sulfatide as previously
hypothesized(27) . Instead, it seems likely that the precise
positioning of this amino acid is essential for local secondary
structure or otherwise affects ligand recognition in some as yet
undetermined manner.
As described for Lys-113, the simple
substitution of many amino acid residues with alanine or a seemingly
closely related amino acid (arginine in the case of Lys-113) does not
always yield conclusive data that are easy to interpret. Consequently,
we attempted to mutagenically alter P-selectin-ligand binding
specificity to that of a different carbohydrate to identify structural
or functional features of the protein that are essential for ligand
recognition and adherence. Since it seemed very possible that
P-selectin might bind sLe in the same shallow pocket that
is analogous to the rMBP oligosaccharide-binding
cleft(25, 33) , we attempted to alter
P-selectin-ligand binding specificity to oligomannose as described
previously for E-selectin(23) .
The binding of rMBP to the terminal mannose residue of the N-linked oligosaccharide chain is very specific and appears to be coordinated through several interactions with the protein and the bound calcium atom. However, rMBP interactions with other mannose units in the N-linked oligosaccharide chain are primarily mediated through water molecules, with the exception of rMBP Lys-182, which appears to directly contact the mannose-6 residue of the oligosaccharide(25) .
A major
difference between the putative P-selectin binding cleft and that of
rMBP is the presence of a five-amino acid insertion loop (residues
95-99) that is present in P-selectin. An adjacent residue,
P-selectin Tyr-94, is a valine in rMBP (Val-199). Additionally, three
amino acids located between residues 75 and 79 (rMBP loop 3) are also
not conserved between rMBP and P-selectin. These include rMBP Lys-182,
Lys-183, and Asp-184 and P-selectin Ala-77, Asp-78, and Asn-79. One of
these unconserved amino acids, Lys-182, is known to coordinate with the
4-hydroxyl of the 6-mannose residue on the
Man91-2Man6
1-3Man4 branch of the N-linked chain(25) . It seemed possible that replacing
P-selectin Ala-77 with lysine might enable P-selectin to bind
oligomannose in a similar manner.
If the P-selectin A77K mutant did bind oligomannose, it would provide evidence that the spatial orientation of the putative binding pocket and the calcium-carbohydrate coordination are conserved between rMBP and P-selectin. Additionally, to provide further confirmation that P-selectin A77K mutant binding to oligomannose occurred in the same manner as rMBP-oligomannose binding, a triple mutant with the substitutions A77K, E80Q, and N82D was also generated. The additional mutation of Glu-80 and Asn-82, two of the calcium-coordinating amino acids, was previously shown by Drickamer (34) to increase rMBP binding affinity for galactose and to reduce affinity for mannose. We believed that a similar effect upon P-selectin-saccharide binding would serve to substantiate the proposed carbohydrate binding interactions.
The ability of the P-selectin
A77K mutant to bind oligomannose was assessed using an invertase-coated
filter binding assay(7) . As can be seen by inspection of Fig. 3A, the P-selectin A77K mutant and the A77K triple
mutant (A77K,E80Q,N82D) bind to the filter, whereas wild-type
P-selectin, wild-type E-selectin, and mock control protein do not. The
E-selectin A77K mutant was included in the experiment as a positive
control(23) . The P-selectin A77K mutant proteins can be
released from the filter by washing briefly in 5 mM EDTA or by
incubating the filter with free mannose monomer. These results indicate
that the binding is both Ca-dependent and
saccharide-specific, respectively.
To confirm that these mutants adhere to oligomannose, both A77K single and triple mutants were tested for binding to denatured yeast protein or denatured invertase coated onto polystyrene dishes (Fig. 3B). While none of the P-selectin proteins tested were retained in wells coated with glycated BSA, only the mutants possessing the A77K substitution bound to both denatured yeast protein and invertase. Like the binding to the invertase-coated filter, this adherence could be effectively inhibited by increasing concentrations of free mannose or galactose monomer (Fig. 4, A and B). The binding of the A77K mutant to oligomannose is inhibited by 20 mM mannose and 125 mM galactose monomer. As illustrated in Fig. 4(A and B), the binding of the triple mutant (A77K,E80Q,N82D) is inhibited by much lower concentrations of galactose (25 mM) and higher concentrations of mannose (60 mM). This reversal in binding sensitivity to inhibition by these sugars is consistent with the effect of the original mutations on rMBP binding(6) , evidence that supports the interpretation that changing one P-selectin amino acid residue from alanine to lysine allows P-selectin to bind oligomannose in a manner that is probably quite similar to rMBP.
Figure 4: A, effect of free mannose on mutant invertase binding. The binding of the P-selectin A77K (closed circles) and A77K,E80Q,N82D (open squares) mutants to invertase in the presence of increasing concentrations of free mannose was determined. This assay was performed as described for Fig. 3B, except that serial dilutions of free mannose were included during the 1-h P-selectin incubation. The concentrations of free mannose at which half-maximal binding was observed were 25 mM (A77K) and 45 mM (A77K,E80Q,N82D). The results shown are the average of two independent experiments that were completed in duplicate. Percentages were determined by dividing the number of beads bound in the presence of free mannose by the number of beads bound by each mutant in its absence. B, effect of free galactose on mutant invertase binding. The binding of the P-selectin A77K (closed circles) and A77K,E80Q,N82D (open squares) mutants to invertase in the presence of increasing concentrations of free galactose was determined. This assay was performed as described for Fig. 3B, except that serial dilutions of free galactose were included during the 1-h P-selectin incubation. The concentrations of free galactose at which half-maximal binding was observed were 100 mM (A77K) and 25 mM (A77K,E80Q,N82D). The results shown are the average of two independent experiments that were completed in duplicate. Percentages were determined by dividing the number of beads bound in the presence of galactose by the number of beads bound in its absence.
The
A77K mutants were analyzed further for their effect on P- and
E-selectin binding to sLe and sulfatide using the same
HL-60 cell and sulfatide assays described previously (see
``Materials and Methods'' and the legend to Fig. 2(A and C)). The results of these assays
are shown in Table 1. While substitution of Ala-77 with lysine
did not appreciably alter sulfatide binding, it did abolish binding to
HL-60 cells and to sLe
-containing glycolipids (data not
shown). Taken together with the mannose binding data, these results
indicate that the substituting lysine residue is essential for
P-selectin-mannose binding, interferes with sialyl Lewis
binding, and does not substantially affect sulfatide binding.
We had previously hypothesized that the sLe sialic acid
carboxylate might form a charge-paired interaction with E-selectin
Arg-97(23) . Since this residue is not conserved between E- and
P-selectin and both proteins are believed to require sialic acid for
highest affinity binding(35, 36, 37) , we
also performed a more extensive mutagenic analysis of each of the
positively charged amino acids located in this region of both E- and
P-selectin. The results of these experiments are detailed in Table 1. Substitution of several amino acid residues within the
putative sLe
-binding pocket (amino acid residues
96-99) with amino acids possessing oppositely charged side chains
did not significantly influence selectin binding to HL-60 cells, nor
did these mutations appear to alter the sLe
tetrasaccharide K
of 3 mM. This was also true of
a K112E,K113E double-charge substitution mutant.
We have attempted to identify the amino acid residues
involved in P-selectin-sLe binding by altering
P-selectin-ligand specificity and by determining the effect of amino
acid substitutions on the binding of the protein to its known ligands,
sLe
and sulfatide. In contrast to analyses reported
previously by others, the results presented here do not support a
hypothesized charge-paired interaction between P-selectin Lys-113 and
sulfatide or the sialic acid residue of sLe
. Indeed, the
role of this amino acid in selectin binding appears to be entirely
speculative. We present data that support an alternative binding
hypothesis, that P-selectin binds sLe
in a region that is
analogous to the binding pocket that is occupied by oligomannose when
bound by the closely related rat mannose-binding protein.
The
relationship between L-fucose and D-mannose and its
implication in selectin binding have been
noted(24, 25, 38, 39) . The ability
of the P-selectin A77K mutant to alter P-selectin binding specificity
from sLe to oligomannose indicates that the P-selectin
calcium-coordinating ions are spatially positioned similarly to
E-selectin and rMBP (23, 24, 25) and that the
P-selectin site is also capable of recognizing mannose. This is further
supported by the ability of the P-selectin E80Q and N82D mutations to
predictably alter ligand preference from mannose to galactose, which is
the same effect that the analogous mutations (E185Q and N187D) had upon
rMBP binding(34) .
Perhaps the most compelling argument for
positioning the P-selectin sLe-binding cleft between loop 3
(NWADNE, residues 75-80), loop 5 (IKSPS, residues 95-99),
and the
5-strand (WNDE, residues 104-107) as determined from
homology to E-selectin and rMBP (23, 24, 25) is the loss of sLe
binding that is generated by the replacement of Ala-77 with
lysine. It seems most probable that this loss of binding may be
attributed to steric interference generated by the lysine side chain
extending into the sLe
-binding pocket. A computer-generated
modeling analysis completed using the E-selectin structural coordinates (24) supports this proposal(23) . Although we do not
know the precise manner in which P- and E-selectins contact
sLe
, the A77K mutation does disrupt that interaction.
It
is pertinent that the P-selectin A77K mutant is able to bind sulfatide,
while it has lost the ability to bind sLe. It is possible
that the sulfatide-binding site is located in the same cleft where we
believe the sLe
-binding site to be located and that since
the sulfatide molecule is actually much smaller than sLe
,
the A77K substitution does not sterically interfere with its binding.
Alternatively, the sulfatide-binding site may be completely removed
from the sLe
-binding site, and therefore, the position of
the Lys-77 side chain would not be critical and would not interfere
with P-selectin-sulfatide binding. Although we have not modeled the
proposed sulfatide interaction, one would predict from the mutagenesis
data that P-selectin binding to free galactose occurs via coordination
with the 3`- and 4`-equatorial hydroxyls as has been noted for the
mannose-binding protein(25, 34) . It is sterically
difficult to rationalize this type of interaction with the presence of
the 3`-sulfate residue that comprises the sulfatide molecule.
Alternatively, if the 3`-sulfated galactoceramide is bound identically
to the galactose found in sLe
, one would expect sulfatide
to be excluded from the binding pocket just as sLe
is.
Since sulfatide binding by both the P-selectin A77K and A77K,E80Q,N82D
mutants is not sensitive to competitive inhibition by free mannose or
galactose (data not shown), the preponderance of evidence generated by
this study actually supports the proposal that the sulfatide site is in
fact removed from the sLe
-binding site.
To our
knowledge, with the exception of the noted report that sulfatide
binding is dependent upon Lys-113 and that two anti-P-selectin
antibodies are able to block both sLe and sulfatide
binding(27) , the sulfatide-binding site has not been
identified. Further mutagenesis work performed in this laboratory as a
result of the data described here also indicates that while the
sulfatide-binding site can allosterically influence sLe
binding, there is in fact no overlap between the amino acids
involved in binding sLe
and sulfatide. (
)
We
had previously hypothesized that the sLe sialic acid
carboxylate might form a charge-paired interaction with E-selectin
Arg-97(23) . Since this residue is not conserved between E- and
P-selectins and both proteins are believed to require sialic acid for
highest affinity binding(35, 36, 37) , we
undertook a more extensive mutagenic analysis of each of the positively
charged amino acids located in this region of both E- and P-selectins.
It is notable that like the Lys-113 mutagenesis results, altering the
charge of each of these residues appeared to have no effect on selectin
binding to HL-60 cells (Table 1). Thus, with the exception of
Tyr-94(20, 23, 24, 40) , we have not
been able to identify other amino acid residues that may directly
contact the bound carbohydrate. This is perhaps not surprising since
the interactions between the selectins and their carbohydrate ligands
are thought to be of relatively low affinity as is evidenced by the
comparably high K
(3 mM) for the
soluble sLe
tetrasaccharide (Table 1). By analogy
with rMBP-oligomannose binding, it seems probable that further contacts
between the selectins and the bound sLe
carbohydrate may be
mediated through water molecules with the peptide backbone and side
chains.
While it is tempting to generalize and simply ascribe the
physical characteristics of one selectin to each of the other members
of this protein family, this is not necessarily true. In this regard,
like the oligomannose binding that we have measured for both E- and
P-selectin A77K mutants, sLe recognition and binding could
be maintained by both proteins without strict conservation of the amino
acid side chains that line the saccharide-binding pocket. Indirect
interactions between the protein and carbohydrate, mediated by water
molecules, might well generate the diversity in ligand recognition that
has already been noted. It seems probable that only ligand-bound
three-dimensional structural determinations will be able to confirm
these possibilities.
In summary, we have attempted to identify the
P-selectin-sialyl Lewis binding interactions by altering
ligand binding specificity. The results reported here are consistent
with those previously reported for E-selectin(23) . However,
they are novel with regard to previously proposed models of
P-selectin-carbohydrate interactions. Additionally, the mutagenesis
data that we have reported for P-selectin Lys-113 indicate that this
residue may not be directly involved in either sLe
or
sulfatide binding. We propose that the P-selectin
sLe
-binding pocket may be partially defined by loop 3
(NWADNE, residues 75-80) on one side, on a second side by loop 5
(IKSPS, residues 95-99), and on a third side by the
5-strand
(WNDE, residues 104-107). It is hoped that this information will
aid in the rational design of pharmacologically active and
therapeutically effective selectin inhibitors.