From the
E-selectin (ELAM-1) is a member of the selectin family of
cellular adhesion molecules. This family of proteins possesses an
amino-terminal Ca
The selectins are a family of three carbohydrate-binding
proteins (for review see Ref. 4).These proteins are expressed on the
surface of vascular endothelial cells (E- and P-selectin), platelets
(P-selectin), and leukocytes (L-selectin) and function in binding
ligands present on the surface of heterologous cells to promote
intercellular adhesion. E-selectin is thought to be expressed only on
activated endothelial cells and that expression is induced by
cytokines
(5) . The binding of E-selectin to sLe
Disease states in which selectins
have been implicated include reperfusion injury, septic shock,
rheumatoid arthritis, psoriasis, asthma, lupus, diabetes, and cancer
metastasis
(4) . In most cases, it is therapeutically important
to reduce the inflammation associated with these diseases or in the
case of cancer, the metastatic character of some types of tumor cells,
and it seems likely that specific selectin binding inhibitors might
produce such effects. Consequently, it is pharmacologically relevant to
identify such compounds, and the elucidation of the precise molecular
interactions that occur between sLe
The E-selectin
lectin or carbohydrate recognition domain (CRD) is a sequence of
approximately 120 amino acids located at the amino terminus of the
mature protein
(7) . The amino acid sequence of this lectin
domain is similar to a variety of other calcium-dependent
carbohydrate-binding proteins known as C-type lectins
(15) . The
selectins, the asialoglycoprotein receptor, the low-affinity IgE
receptor (CD23), the pulmonary surfactant apoprotein SP-A, together
with the serum and liver mannose-binding proteins are included in this
class of proteins because multiple sequence alignment indicates that
they are evolutionarily related
(16) . Each of these proteins
possesses a relatively high degree of sequence similarity that includes
14 invariant and 17 highly conserved amino acid residues within their
CRDs (15, 16). These conserved amino acid residues are believed to be
essential for proper folding and for the establishment of a hydrophobic
core or scaffold which is a structural characteristic of this protein
class
(15, 17) . The rat mannose-binding protein is the
only member of the C-type lectins for which a ligand-bound
three-dimensional structure has been determined
(3) . As a
consequence, the active sites of most members of this class have not
been defined, and therefore their similarity has not yet been
established.
The three-dimensional structure of the E-selectin CRD
(ligand unbound) was determined by other investigators using x-ray
crystallographic methods
(2) . Additionally, several point
mutations that are known to abolish E-selectin binding to sLe
In order to validate our proposed
model, we have completed a more extensive mutagenic analysis of many of
the amino acid residues previously implicated in E-selectin
binding
(2, 18) . Furthermore, we have sought to
substantiate the speculative E-selectin-sLe
The sLe
During initial
docking attempts, it was found that the conformational lability of the
glucosamine N-acyl group complicated this process due to the
tendency of the acyl carbonyl to hydrogen bond to acidic hydrogen
atoms. Since the deacylated glucosamine analog of sLe
After insertion of the tetrasaccharide, energy minimization was
carried out (using the conjugate gradient method, with a convergence
criterion of 0.2 kcal/mol which was achieved in 192 iterations). No
rigid constraints were imposed. The CAChe implementation of the MM2
parameter set was used
(26) . Solvent effects were not accounted
for either explicitly or implicitly. The resulting torsion angles of
the glycosidic linkages are given in the legend to
Fig. 1C. The purpose of this minimization was to relieve
high energy non-bonded interactions, particularly between Arg
To aid in protein stabilization, detection,
quantitation, and purification, this E-selectin cassette was fused to
the hinge region of the mouse IgG
E-selectin-HL60 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 HL60 cells (10
To elucidate the interactions that occur between selectin and
ligand, a model of sLe
This model is based upon the assumption that the C-2 and C-3
hydroxyls of the
Assuming E-selectin
binds fucose in the manner described above, other amino acids
surrounding the binding pocket must interact with sLe
The critical roles of the
Arg
The E-selectin binding cleft
appears to be similar to that of rMBP with the exception of the
E-selectin five amino acid loop, IKREK (residues 95-99), which is
absent in the rMBP sequence (Fig. 1B). Additionally,
rMBP Lys
HL60 binding to the recombinant Ala
In agreement with the filter assay,
the Ala
To further demonstrate that the binding interactions between the
E-selectin Ala
Several observations can be made from inspection of the
mutant binding data presented in Fig. 2B. The
Tyr
Since the E-selectin crystal structure predicts that
Lys
Two other E-selectin residues that have been
hypothesized to directly interact with the sLe
While the information gained by many substitution mutations is
valuable, it is also speculative and subjective. When a specific
mutation destroys enzymatic activity or binding ability, it is not
possible to know if this is the result of altering an amino acid that
directly interacts with the ligand or if the substitution has had an
indirect effect and altered the configuration of the local backbone or
active site, as described above for the relatively conservative
Lys
In summary, our mutagenesis and binding analyses are consistent
with sLe
We thank Peter Vanderslice, Ed Yeh, and Richard Dixon
for discussions and critical reading of this manuscript and Bill Weis
for provision of mannose/rMBP coordinates before publication. We would
like to dedicate this manuscript to the memory of Rick Gayden who was a
dear friend and colleague. Rick's spirit, determination, and
humor will always be with us.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-dependent lectin or carbohydrate
recognition domain that is essential for ligand binding. A known
E-selectin ligand is the carbohydrate antigen, sialyl Lewis
(sLe
)
(Neu5Ac
2-3Gal
1-4(Fuc
1-3)GlcNAc). We
have developed a model of E-selectin binding to the sLe
tetrasaccharide,
(Neu5Ac
2-3Gal
1-4(Fuc
1-3)GlcNAc), using
the NMR-determined, E-selectin-bound solution conformation of sLe
(Cooke, R. M., Hale, R. S., Lister, S. G., Shah, G., and Weir, M.
P.(1994) Biochemistry 33, 10591-10596) together with the
[Medline]
x-ray crystallographic structures of E-selectin (Graves, B. J.,
Crowther, R. L., Chandran, C., Rumberger, J. B., Li, s., Huang, K.-S.,
Presky, D. H., Familletti, P. C., Wolitzky, B. A., and Burns, D. K.
(1994) Nature 367, 532-538) (ligand unbound) and a
[Medline]
related C-type animal lectin, the mannose-binding protein (Weis, W. I.,
Drickamer, K., and Hendrickson, A.(1992) Nature 360,
127-134) (ligand bound). Analysis of this model indicated that
the alteration of one E-selectin amino acid, alanine 77, to a lysine
residue might shift binding specificity from sLe
to
mannose. The results presented here show that an E-selectin mutant
protein possessing this change displays preferential binding to mannose
containing oligosaccharides and that further mutagenesis of this
mannose-binding selectin confers galactose recognition in a predictable
manner. These mutagenesis data support the presented model of the
detailed interactions between E-selectin and the sLe
oligosaccharide.
(
)
and related epitopes on neutrophils, monocytes, a specific
subset of T lymphocytes, eosinophils, and
basophils
(6, 7, 8, 9, 10, 11, 12, 13, 14) is believed to aid in the recruitment of these cells in
response to inflammatory stimuli.
and E-selectin should
aid in the design of novel selectin inhibitors.
have been proposed by others to identify the carbohydrate binding
pocket
(2, 18) . However, the precise amino
acid-carbohydrate interactions that occur between selectin and ligand
have not yet been elucidated
(2, 18, 19) . To
extend our knowledge of those interactions, we constructed a model of
the E-selectin carbohydrate recognition domain with sLe
bound. This model was generated using the x-ray crystallographic
coordinates of E-selectin
(2) and the recently reported
E-selectin-bound solution conformation of the sLe
tetrasaccharide
(1) . The sLe
tetrasaccharide
in that reported conformation was introduced into the E-selectin
binding pocket by first coordinating the fucose residue with
Ca
in a manner similar to the way in which the rat
mannose-binding protein (rMBP) bindsmannose (3, 20). The resultant
model indicates that E-selectin binds sLe
in a
configuration that is significantly different from that previously
hypothesized to occur between E-selectin and its
ligand
(2, 18) .
binding
interactions by altering ligand specificity. The results presented here
demonstrate that a mutant E-selectin is able to bind oligomannose in a
manner similar to the rat mannose-binding protein which has a
K
of 7.2 mM for free
mannose
(3, 17) . In addition, the mutagenesis data we
present support our proposed fucose coordination (2, 3, 20) as well as
the modeled binding conformation.
Molecular Modeling
Coordinates for E-selectin
and the mannose-binding protein were obtained from the Brookhaven
Protein Data Bank (1ESL and 2MSB,
respectively)
(2, 3, 21, 22, 23) .
Modeling work was conducted using the CAChe WorkSystem and
software or a Silicon Graphics Indy workstation using Biosym commercial
software. The particular force fields and modeling techniques used are
described in greater detail below.
tetrasaccharide model was constructed using the CAChe default MM2
force field and the torsion angles of Cooke et al.(1) that were determined from the NMR transfer nuclear
Overhauser enhancements data generated for the E-selectin-bound
solution conformation of the oligosaccharide. Cooke et al. (1)
reported that the bound sLe
conformation approximated the
previously modeled GESA-C conformer (four low energy structures were
determined and designated GESA-A to GESA-D using the GESA (geometry of
saccharide) algorithm) (24, and references therein). Therefore, the
dihedral angle restraints that were defined for the GESA-C structure
were used to construct the model of sLe
.
had
been shown to have higher affinity for E-selectin (25), the glucosamine
N-acyl function was removed from the tetrasaccharide prior to
its introduction into E-selectin to prevent the formation of bonding
artifacts. Its removal had no significant effect on the torsional
angles of the glycosidic linkages
(25) . This higher affinity may
be a consequence of close proximity of the glucosamine 2-position to
Glu
(Fig. 1, C and D).
Figure 1:
A, fucose and E-selectin
coordination. The proposed interaction of L-fucose with the
E-selectin calcium ion, and its coordinating ligands are illustrated.
The oxygen atoms of Glu, Asn
, and Asn
lie in an approximate plane with the calcium ion, which is
coordinated from below by Asp
(side chain and backbone
carbonyl) and above by the fucose 2- and 3-hydroxyls. The C-3-hydroxyl
oxygen is coordinated to the calcium ion and acts as a hydrogen bond
acceptor from Asn
and a donor to the Glu
carbonyl forming a network of three interactions. The
C-2-hydroxyl oxygen is also coordinated to the calcium ion and is a
hydrogen bond acceptor from the Asn
nitrogen. An
analogous network of coordination and hydrogen bonds are observed with
the 3- and 4-hydroxyls of mannose bound to rMBP (3) with the additional
involvement of Glu
(Glu
in E-selectin). The
relationship between L-fucose and D-mannose
(superimposition of the ring oxygen, 2-, 3- and 4-hydroxyls of fucose
with the ring oxygen, 4-, 3-, and 2-hydroxyls of mannose,
respectively), and its implication to selectin binding was previously
noted (2, 20, 23, 34). B, structural alignment of the
E-selectin lectin domain with the rat mannose-binding protein. The
protein sequence alignment for the human E-selectin and the rat
mannose-binding protein lectin domains as deduced from a comparison of
the respective crystal structures (2). Specific residues discussed in
the text have been noted with an asterisk and vertically
labeled with the appropriate amino acid sequence numbers. C,
stereo view of sLe
bound to E-selectin. The amino acid
backbone is shown in magenta, and the bound calcium is a
yellow sphere. Pertinent amino acid side chains described in
the text are illustrated in color as follows. The side chains,
Arg
and Lys
(vertical) on the five amino acid
insertion loop (residues 95-99) are shown in green. The
Ala
side chain is pictured in gray and white
space-filling spheres; Tyr94 side chain is
turquoise; the Lys
and Lys
are
white, 113 being the residue which extends in front of the
calcium; Glu
side chain (red); Arg
(yellow); sLe
tetrasaccharide (center:
carbon, gray; oxygen, red; nitrogen, blue).
The torsion angles of the glycosidic linkages after minimization as
shown are NeuAc-Gal, -63/-4; Gal-GlcNH
, 32/30; and
Fuc-GlcNH
, 47/37.
Docking
of the oligosaccharide was achieved by a series of superimpositions
using the mannose-mannose-binding protein interactions as a template.
Specifically, the calcium ion, the calcium coordinated mannose residue
(via C-3 and C-4 hydroxyl oxygens), and five calcium coordinating
oxygens from rMBP (Glu, Asn
,
Asn
, and Asp
(two oxygens, one from the
acid and the main chain carbonyl)) were superimposed on the equivalent
calcium ion and metal ligands in E-selectin (Glu
,
Asn
, Asn
, and Asp
(two
oxygens, one from the acid and the main chain carbonyl)). The
root-mean-square deviation for the five oxygen atoms and the calcium
ion was 0.134 Å. The mannose residue was then used as a template
for superimposing the fucose residue of sLe
using the ring
oxygen, C-2, and C-3 hydroxyls of fucose to superimpose the ring
oxygen, C-4, and C-3 hydroxyls of mannose, respectively. The
root-mean-square deviation for this superimposition was 0.060 Å.
Finally, the 2MSB (rMBP) derived template was removed. The hydrogen
bond and coordination network was then constructed with weak binding
interactions (the force constant of the hydrogen bond interaction was
set to one-fifth the force constant of a C-C single bond, which is the
default setting in the CAChe implementation of the MM2 force field).
and the sialyl carboxylate, and Tyr
and the
C-6-hydroxyl of galactose, while maintaining an overall similarity with
the NMR structure previously described. The energy minimization
procedure changed the distances between the non-bonded
tetrasaccharide-protein atom pairs. However, it did not change the
identities of those pairs.
Materials
Tissue culture media, dialyzed fetal
calf serum, phosphate-buffered saline (PBS), and antibiotics were
obtained from Life Technologies, Inc. and fetal calf serum from
Hyclone. The E-selectin cDNA and anti-ELAM-1 antibodies, BBA1 and BBA8,
were purchased from R & D Research. Magnetic beads conjugated to
goat anti-mouse (GAM) IgG, and magnetic separators were obtained from
Dynal Inc. (Great Neck, NY). Unless specifically stated, other
immunochemicals were purchased from Calbiochem. Flexible 96-well assay
plates and Probind 96-well ELISA plates were purchased from Falcon.
Synthetic oligonucleotides were purchased from Oligo's Etc.
Restriction enzymes and T4 DNA ligase were from Life Technologies, Inc.
and New England Biolabs, and Taq DNA polymerase was obtained
from Perkin Elmer.
Cell Culture and Transfection
COS-1 cells were
grown at 37 °C with 5% CO in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum. HL60 cells
were grown at 37 °C with 5% CO
in RPMI supplemented
with 10% fetal calf serum. Prior to electroporation, cells were
trypsinized, harvested, washed twice with PBS, and 10
cells
(0.8 µl) were mixed with 10 µg (1 µg/µl) of plasmid DNA
in a 0.4-cm electrode gap cuvette. Transfections were performed using a
Bio-Rad electroporator following the manufacturer's
recommendations and using 0.22 mV, 960 µF. After transfection, the
cells were grown in 100-mm dishes for 72 h, at which time the culture
media was collected, buffered to a final concentration of 10
mM Tris-HCl, pH 7.5, and centrifuged for 5 min at 600
g to remove cell debris.
Construction and Expression of E-selectin
Mutants
Recombinant DNA techniques and mutagenesis were
performed as described ((27, and references therein). Plasmid DNA for
transfection or sequencing was purified as per the manufacturer's
recommendation using Qiagen reagents. All DNA amplified by polymerase
chain reaction (PCR) or subjected to site-directed mutagenesis
procedures were sequenced by the Sequenase method using reagents
supplied by United States Biochemical Corp.. An E-selectin expression
cassette was made by the following method. To place an EcoRI
site 5` (immediately preceding the ATG initiation codon) and a
kpnI site 3` (at the end of the second complement receptor
repeat), the E-selectin cDNA was amplified by PCR with the
5`-oligonucleotide TCTTGAATTCATGATTGC and the 3`-oligonucleotide
CATGTGGTACCTTTACACGTTGGCT. The PCR-generated 920-base pair
fragment was ligated into M13. Using site-directed mutagenesis, a
BamHI site was then introduced at nucleotide 60 of the coding
sequence, and a HindIII site was removed by mutating
nucleotide 780. The BamHI site changed the alanine codon at
the -1 position of the E-selectin signal cleavage site to a
serine codon while the mutation at nucleotide 780 did not alter the
encoded amino acid.
heavy chain coding
sequence to create a soluble, secreted protein expressed from a
construct similar to that previously described
(28) . 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. Bob
Bjercke, using oligonucleotides GAAAGGTACCAGAGGGCCCACAATC and
GAGCAAGCTTACCCGGAGTCCG. The E-selectin-mIgG fusion gene was constructed
in the mammalian expression vector pJB20, the pCMV plasmid described
(30) with several restriction sites removed. This construct
allowed specific mutations in the E-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 a 350-base pair BamHI/HindIII or
BamHI/BsgI fragment if the mutation altered the
HindIII site.
Protein Purification and Binding
Assays
Recombinant protein levels in the cell supernatants were
monitored by ELISA using alkaline phosphatase-conjugated GAM IgG
antibody (Calbiochem). To affinity purify the recombinant proteins, 10
µl (4 10
beads/ml) of GAM-coated Dynabeads
(Dynal Inc.) were added to 10 ml of culture media harvested from
transfected cells and incubated overnight with rocking at 4 °C. The
beads were concentrated by centrifugation at 600
g for
5 min, and the culture media was removed after the incubation tubes
were placed on a magnetic separator. The beads were resuspended in 1 ml
of PBS (4
10
beads/ml final concentration) and
stored at either 0 or 4 °C. The amount of recombinant protein
recovered on the beads was monitored by ELISA using BBA8, a
biotinylated version of BBA1 (R & D Research). All of the mutant
proteins discussed in this work were readily detectable using this
mouse monoclonal antibody. Furthermore, the ELISA results (data not
shown) indicate that the proteins are 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.
cells/ml RPMI
1640, 10% fetal calf serum) that had been fluorescently labeled with
Calcein AMC-3099 (Molecular Probes) were added to each well followed by
10 µl of beads (4
10
beads/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. The plate was then placed on a
magnetic separator and incubated for 2 more min. While the assay plate
remained on the separator, excess, unbound HL60 cells were removed, and
the wells were washed twice with PBS to remove any remaining unbound
cells. The HL60 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 fluorimetrically using a
Millipore Cytofluor 2350 fluorimeter.
Invertase Filter Binding Assay
Invertase was
coated onto a nitrocellulose filter, and the binding of E-selectin IgG
proteins conjugated to beads was assessed using a slot-blot apparatus
without vacuum using the methods noted by others
(17) with the
following exceptions. E-selectin protein bound to Dynabeads were incubated in the wells of the slot-blot apparatus (Hoefer).
Beads (10
) were incubated in each well in 50 µl of
buffer. It was not necessary to use an enzyme-linked second antibody to
detect the bound protein so this step was omitted. The dark brown areas
shown in the figure are the actual protein-bound beads retained on the
filter after washing. E-selectin mutants were expressed and purified as
described (Fig. 2A).
Figure 2:
A, recombinant human E-selectin IgG cell
binding specificity. E-selectin binding assays were performed in
duplicate wells of 96-well assay plates. Fluorescently labeled HL60
(express sLe), Ramos (no sLe
), or Chinese
hamster ovary (CHO) (no sLe
) cells were mixed with
recombinant human E-selectin IgG-conjugated to immunobeads (Dynal).
Binding was quantitated fluorimetrically using a Millipore Cytofluor
2350 fluorimeter. B, assessment of E-selectin mutant binding
to HL60 cells. E-selectin mutant proteins were immunoprecipitated using
GAM-conjugated beads. After ELISA quantitation, 4
10
E-selectin IgG-adsorbed beads were mixed with 10
fluorescently labeled HL60 cells and incubated together at room
temperature for 10 min. A magnetic separator was used to separate the
bead-bound HL60 cells. Unbound cells were removed by sequential PBS
washes. The number of cells bound by the wild-type E-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.
Assay of E-selectin Mutant Oligo-mannose
Binding
Stock solutions containing 2.5 mg/ml (lyophilized
weight/volume) of yeast invertase, Saccharomyces cerevisiae (yeast), glycated BSA, and bovine fetuin in 25 mM Tris,
pH 7.5, 1 mM 2-mercaptoethanol, 0.5% SDS, and 5% glycerol were
boiled for 10 min, cleared by brief centrifugation, chilled to 0
°C, diluted to final concentrations of 25 µg/ml in PBS, and
coated onto 96 well flexible assay plates. Plates were subsequently
blocked with 3% BSA in PBS. Beads conjugated with various E-selectin
IgG proteins (2 10
beads) were added to each well
in a 55-µl volume of PBS supplemented with 0.5 mM
MgCl
and 2.5 mM CaCl
and incubated for
1 h. Wells were then washed sequentially with PBS until unbound beads
were removed. The number of beads retained in the wells was determined
by ELISA using an alkaline phosphatase-conjugated rabbit anti-goat
antibody. The ELISA absorbance 415 readings were curve fit to serially
diluted mock and E-selectin IgG-coated bead standards, both of which
had essentially identical amounts of goat IgG. The binding of the
E-selectin Ala
Lys mutants to invertase in the presence of
increasing concentrations of free mannose or galactose was performed as
described above except that serial dilutions of free mannose or
galactose were included during the 1-h incubation.
bound by the E-selectin CRD was
developed (see ``Experimental Procedures'' for details). To
construct the model, the calcium ion, the calcium coordinated terminal
mannose, and five calcium coordinating oxygens from the rat
mannose-binding protein (Brookhaven protein coordinate data base entry
code 2MSB)
(3, 21, 22) , rMBP residues
Glu
, Asn
, Asn
, and
Asp
(two oxygens, one from the acid and one from the main
chain carbonyl), were superimposed on the equivalent calcium ion and
metal coordinating atoms in E-selectin. This included E-selectin
residues Glu
, Asn
, Asn
, and
Asp
(two oxygens, one from the acid and the main chain
carbonyl) (Brookhaven protein coordinate data base entry code
1ESL)
(2) . The sLe
fucose ring oxygen together with
the fucose C-2 and C-3 hydroxyls were then superimposed over the bound
terminal mannose ring oxygen and the terminal mannose C-4 and C-3
hydroxyls, respectively. The rMBP-derived template was then removed.
1-3-linked fucose of the sLe
tetrasaccharide coordinate in a calcium-dependent manner with
E-selectin residues Glu
, Asn
,
Asn
, and Asp
in a similar way to the
contacts that are made between the C-4 and C-3 hydroxyls of the
terminal mannose and the mannose-binding protein (rMBP)
(3) with
the exception that E-selectin Glu
(Glu
in
rMBP) is not a calcium ligand (2) (Fig. 1A). Although
the absence of a sixth calcium coordination was a concern, the low
root-mean-square deviation (see ``Experimental Procedures'')
which resulted from superimposing the remaining five oxygen atoms and
the calcium ion indicate that this alteration does not significantly
affect the carbohydrate recognition as modeled and in all probability
merely reflects the amino acid sequence and structural divergence which
has previously been noted for the two proteins
(2) . For
comparison purposes, a structural alignment of E-selectin with rMBP
(2) is shown in Fig. 1B.
and
be responsible for sLe
binding specificity. Although other
modes of docking were investigated and included the use of
calcium-bound water molecules in E-selectin as anchor points for the
fucose 2- and 3-hydroxyls along with rotation around the pseudo C-2
axis of fucose (interchange of the 2- and 3-positions), these
orientations failed to provide rational models for the sialic acid
interaction with the protein, which is a known requirement for binding
activity
(31) . The most satisfactory positioning was found to be
that illustrated in Fig. 1C which delineates the
E-selectin binding cleft as being bordered on one side by the
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)) with the
sLe
sialic acid carboxylate possibly forming a
charge-paired coordination with the Arg
side chain rather
than extending toward the Lys
residue as previously
proposed by others
(2) . It should be noted that the model
postulated by Graves et al.
(2) was prepared using the
sLe
solution conformation rather than the E-selectin-bound
solution conformation which has been used for the modeling detailed
here. In the configuration described here (Fig. 1C), the
tetrasaccharide maintains the structural constraints of the
NMR-prescribed bound conformation
(1) with the torsional angles
of the glycosidic linkages after minimization determined to be
NeuAc-Gal, -63/-4; Gal-GlcNH
, 32/30; and
Fuc-GlcNH
, 47/37 as compared to those projected for the
GESA-C conformer
(24) of NeuAc-Gal, -79/7;
Gal-GlcNH
, 55/7; and Fuc-GlcNH
, 48/25. From
these data one may conclude that there is no significant discrepancy
between the NMR-derived bound conformer and the modeled
sLe
/E-selectin structure.
and Lys
residues in ligand recognition
were previously noted
(2, 18) . Further analysis of the
model presented here indicated that additional interactions between the
galactose 6-hydroxyl and Tyr
and between the glucosamine
and Glu
(Fig. 1C) were also possible,
which was again consistent with known amino acid substitution
data
(2, 18) . To better assess the precise involvement
of these residues in sLe
binding, an E-selectin assay for
sLe
-dependent binding to the human HL60 cell line was
devised (Fig. 2A) and many of these residues were
extensively mutated to determine the effect of further amino acid
substitution on the putative binding pocket (Fig. 2B).
Several of the mutagenesis results shown in Fig. 2B are
consistent with those previously reported by others (2, 18) using
different assay methods. However, the Lys
results are of
particular interest. As described, this residue was previously
implicated in sLe
binding since conservative alteration to
alanine or arginine destroys E-selectin binding
(2, 18) .
The substitution analysis reported here shows that changing Lys
to glutamine or glutamic acid does not affect binding. This
result indicates that the length and/or polarity of the side chain at
this position is perhaps critical for local secondary structure but
also implies that Lys
does not participate in a
charge-paired interaction with the sialyl carboxylate. Similarly, and
in complete agreement with a different type of mutational analysis
reported by others
(2) , one may conclude from the results in
Fig. 2B that Glu
probably does not make
critical contact with the ligand.
extends into the rMBP binding pocket and
interacts with the 4-hydroxyl of mannose-6 on the
(Man9)
1-2(Man6)
1-3(Man4) branch of the
N-linked oligosaccharide chain
(3) . It is notable that
this interaction does not occur during rMBP binding to the second
branch of the N-linked chain
(Man8)
1-3((Man7)
1-6)(Man4) because there is no
similarly linked mannose residue available for binding to the
Lys
residue. The E-selectin amino acid that is analogous
to rMBP Lys
is Ala
, and it is located on a
loop at one end of the predicted sLe
binding cleft
(Fig. 1C). Since the two proteins appeared to share very
similar binding pockets, it seemed possible that replacing E-selectin
Ala
with lysine might provide the required contact
necessary for oligomannose binding and thus confirm the identification
of the proposed ligand interactions. To test this hypothesis,
E-selectin Ala
was changed to lysine and to maximize the
homology between the two proteins, E-selectin Pro
was also
changed to lysine (rMBP Lys
; Fig. 1B).
Lys mutants was
assessed and found to be negligible. Furthermore, the presence of the
Arg
Ala substitution which clearly enhances binding of
sLe
to native E-selectin, was not able to restore the
ability of the Ala
Lys mutants to bind HL60 cells
(Fig. 2B). To further characterize binding specificity,
appropriate amounts of the same preparations of both wild-type and
mutant E-selectin bead-conjugated proteins were assayed for their
ability to bind oligomannose as determined by their adsorption to
highly mannosylated yeast invertase coated onto nitrocellulose (17). As
shown in Fig. 3A, the Ala
Lys mutant clearly
binds to the filter whereas the unmutagenized E-selectin IgG fusion
protein does not. Incubation of the filter in EDTA released the bound
protein, indicating that invertase binding is
Ca
-dependent. Additionally, increasing concentrations
of mannose resulted in specific competitive binding inhibition since
all of the bound E-selectin protein is released from the membrane at a
15 mM concentration of saccharide monomer.
Figure 3:
A, invertase filter binding assay. The
E-selectin IgG wild-type (unsubstituted) and various E-selectin mutants
containing the AlaLys mutation bound to Dynabeads
were assessed for their ability to bind to an invertase-coated
nitrocellulose filter. The dark areas shown in the figure are the
actual protein-bound beads retained on the filter after washing. It is
apparent that each of the Ala
Lys mutants bind the
oligomannose present on yeast invertase and are retained on the filter
whereas the unmutagenized E-selectin wild-type and mock IgG beads are
not. B, assay of E-selectin mutant binding activity. The
results shown are the average of two independent experiments run in
duplicate. Stock solutions containing 2.5 mg/ml (lyophilized
weight/volume) of: 1, yeast invertase; 2, S.
cerevisiae (yeast); 3, glycated BSA; and 4,
bovine fetuin 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 E-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. C, effect of free mannose on mutant invertase
binding. The binding of the E-selectin Ala
Lys mutant to
invertase in the presence of increasing concentrations of free mannose
was determined. This assay was performed as described for B except that serial dilutions of free mannose were included during
the 1-h E-selectin incubation. The concentration of free mannose at
which half-maximal binding was observed is 10 mM. The results
shown in this figure are the averages of two independent experiments
that were completed in duplicate.
To further
validate the mannose binding results, boiled and denatured invertase,
yeast cell lysate, glycated BSA, and bovine fetuin were coated onto
96-well polystyrene plates and E-selectin binding to the coated plates
was determined (Fig. 3B). Only the AlaLys
mutant or various double or triple mutant variants possessing this
change were able to bind invertase. Additionally, these same mutants
bound to denatured yeast protein which is known to be highly
mannosylated, yet they displayed little or no detectable binding to
glycated BSA or bovine fetuin.
Lys mutant binding to denatured invertase and to
denatured yeast cell lysate on polystyrene plates could be effectively
inhibited by increasing concentrations of free mannose
(Fig. 3C). Only subtle differences in the mannose
binding behavior of the four Ala
Lys mutants (Fig. 3,
A and B) were observed. The binding of each mutant
was completely inhibited by 15 mM free mannose and did not
seem to be particularly affected by the presence of the
Pro
Lys or the Arg
Ala second site changes.
Lys mutant and oligomannose are specific and
similar to that of the rat MBP, the Ala
Lys mutant was
mutagenized at Glu
and Asn
. Previous analysis
of the analogous residues in the rat MBP by others
(32) had
shown that changing these amino acids (rMBP Glu
Gln and
Asn
Asp) resulted in altered binding activity such that
galactose rather than mannose became the preferred rMBP ligand. The
results in Fig. 4, A and B, illustrate that a
similar change is apparent in the analogous E-selectin mutant
(Ala
Lys, Glu
Gln, Asn
Asp). We
conclude that the side chain present at position 77 (Ala versus Lys) exerts a significant influence over ligand selectivity and
thereby controls sLe
versus mannose recognition.
Figure 4:
A, effect of free mannose on mutant
invertase binding. The binding of the E-selectin AlaLys
(open squares) and Ala
Lys, Glu
Gln,
Asn
Asp (diamonds) 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
E-selectin incubation. The concentrations of free mannose at which
half-maximal binding was observed are 10 mM
(Ala
Lys) and 45 mM (Ala
Lys,
Glu
Gln, Asn
Asp). The results shown in this
figure are the averages 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 E-selectin
(white squares) Ala
Lys and (diamonds)
Ala
Lys, Glu
Gln, Asn
Asp 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 E-selectin incubation. The concentrations of
free galactose at which half-maximal binding was observed are 100
mM (Ala
Lys) and 25 mM
(Ala
Lys, Glu
Gln, Asn
Asp). The
results shown in this figure are the averages 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. C,
E-selectin Ala
Lys mutant mannose coordination. The
predicted interactions between the E-selectin Ala
Lys
mutant and oligomannose are depicted. The penultimate mannose (Man 6)
together with the
(1,2)-linked terminal mannose (Man 9) are
illustrated with the Man 6 C-4-hydroxyl oxygen hydrogen bonded to the
Lys
-amino group. This interaction corresponds to
that described for rMBP Lys
(3). The ultimate mannose
(Man 9) C-3-hydroxyl oxygen is coordinated to the calcium ion and acts
as a hydrogen bond acceptor from Asn
and a donor to the
Glu
carbonyl forming a network of three interactions.
Similarly, the Man 9 C-4-hydroxyl oxygen is also coordinated to the
calcium ion and is a hydrogen bond acceptor from the Asn
nitrogen. An analogous network of coordination and hydrogen
bonding is observed with the 3- and 4-hydroxyls of mannose bound to
rMBP (3) with the additional involvement of Glu
(Glu
in E-selectin). Although the E-selectin
Asn
side chain occupies a position closest to that
occupied by rMBP Glu
, unlike Glu
,
E-selectin Asn
is probably too distant (4.7 Å) for
direct hydrogen bonding but could be involved via a water molecule.
Oligomannose structures probably do not interact directly with
Arg
and Tyr
. However, the Lys
side chain prevents the set of interactions responsible for
sLe
interaction illustrated in D. D,
E-selectin-sLe
interactions. The modeled polar interactions
predicted between the sLe
tetrasaccharide and E-selectin
are illustrated. The fucose-calcium site coordination is detailed in
the legend to Fig. 1A. The galactose C-6-hydroxyl is located
in close proximity to enable hydrogen bonding interaction with
Tyr
(2.6 Å) and also Asn
-NH
(2.8 Å, not shown), while the sialic acid carboxylate can
form a charge paired interaction with Arg
(3.0 Å).
The glucose N-acetyl nitrogen is in close proximity to
Glu
(2.9 Å).
The ability of the E-selectin AlaLys mutants to bind
oligomannose indicates that the conserved calcium site is capable of
recognizing mannose or fucose. Each of these interactions are
illustrated in Fig. 4, C and D. As depicted
(Fig. 4C), the Lys
side chain provides a
critical additional interaction during E-selectin Ala
Lys
binding to oligomannose, and this probably results from the formation
of a hydrogen bond with the C-4 hydroxyl of the second mannose residue.
The observation that the Ala
Lys mutant is incapable of
binding sLe
and yet is able to bind oligomannose suggests
that the Lys
side chain sterically excludes sLe
from the binding site. This hypothesis is supported by
superimposition of sLe
into the E-selectin binding cleft in
the presence of a lysine residue at position 77 (Fig. 5). The
structures illustrated in Fig. 5demonstrate that the distance
between the substituted Ala
Lys
-NH
and
the sLe
galactose C-5 carbon is only 2.2 Å, which is
again consistent with this interpretation of the binding data.
Figure 5:
Stereo view of sialyl
Lewis/mannose, Ala
/Lys
overlap.
The illustration is a similar orientation to Fig. 1C with the
E-selectin backbone in magenta, the side chains of
Ala
, Tyr
, and Arg
in
green, and the calcium ion as a yellow sphere.
sLe
(red) is coordinated as in Fig. 1C,
while the oligomannose structure (yellow) and Lys
(blue) from rMBP (2MSB) have been superimposed together
using the mannose-fucose comparison previously described with the same
root-mean-square deviation. The lysine-mannose interaction pictured is
derived from the rMBP structure (3). The resulting distance from the
rMBP Lys
C-
to the E-selectin-Ala
C-
is 0.5 Å. The distance between the substituted
E-selectin Lys
-NH
and the galactose C-5
carbon in sLe
is only 2.2 Å, commensurate with the
observation that the Ala
Lys mutant does not bind
sLe
.
Ala substitution was originally reported by Erbe et al.
(18) to
have lost the epitopes for 12 different anti-E-selectin monoclonal
antibodies and was not assayed for binding by this group. More
recently, it was reported that a Tyr
Phe substitution could
not bind neutrophils yet had relatively normal antigenic determinants
as detected by a panel of six monoclonal antibodies
(2) . In
light of these results, it is of interest that neither serine,
arginine, or aspartic acid substitutions restore binding. These data
serve to illustrate the unpredictable nature of many amino acid
alterations. This is perhaps even more dramatically exemplified by the
Glu
Asp substitution that obliterates all HL60 binding
activity while the Glu
Lys mutant binding is greatly
increased and the Glu
Asn replacement has no detectable
effect.
may form an ion pair with Glu
, the
increased binding observed by the charge shift substitution is
difficult to rationalize. However, it seems possible that under
ordinary circumstances, the repulsion between the negatively charged
Glu
and the adjacent Asp
side chains may be
ameliorated by the Lys
-Glu
hydrogen bond,
which enables the proper orientation for Asp
to
coordinate with calcium. As a consequence, Lys
is
essential
(18) unless the Glu
negative charge is
also neutralized
(2) . A lysine at residue 107 may simply alter
the torsional constraints produced by the side chain charge
distribution, altering the steric constraints upon the Asp
calcium coordination. This explanation would be consistent with
the inability of the Glu
Asp mutant to bind HL60 cells
since this more conservative substitution would result in a shorter
amino acid side chain, an increased negative repulsion with
Asp
, and a reduced ability to hydrogen bond with
Lys
.
ligand are
Glu
and Lys
(18, 2) . Our
modeling indicates that like Glu
and Lys
,
these residues may also form an ion pair. If this is true,
substitutions at Glu
and Lys
might
indirectly effect ligand binding. In this regard it is notable that the
Lys
Glu and Lys
Gln substitutions completely
reverse or abolish the amino acid side chain charge and yet are able to
bind HL60 cells with wild-type capability. Furthermore, this
Lys
Glu and Lys
Gln substitution information
when analyzed together with the inability of the Ala
Lys
mutant to recognize sLe
are inconsistent with previous
proposals that describe the interactions of E-selectin with
sLe
(2, 18) and yet appear to be in
agreement with the modeled interactions proposed here (Fig. 5).
Ala, Lys
Arg, Glu
Asp, or
Tyr
Phe
(2, 18) substitutions. As a
consequence, we have sought to substantiate the speculative
E-selectin/sLe
binding interactions by altering ligand
specificity. The results presented here demonstrate that a mutant
E-selectin is able to bind oligomannose in a manner similar to the rat
mannose-binding protein (K
of 7.2
mM(3, 17) compared to the E-selectin
Ala
Lys mutant K
of 10
mM). These data, together with the Glu
Gln,
Asn
Asp substitutions effect on competitive inhibition by
free fucose (data not shown), free mannose, and free galactose
(Fig. 4, A and B) support the proposed fucose
coordination (2, 3, 20). Additionally, the information gained by the
loss of HL60 cell binding when Ala
is replaced by lysine
indicates that sLe
coordination is disrupted by this amino
acid change. This result further supports our proposed binding
conformation and molecular interactions by indicating that the
sLe
tetrasaccharide binds in the same shallow pocket that
is occupied by oligomannose (Fig. 4, C and D,
5).
being bound by E-selectin in a manner that is
similar to that determined for oligomannose binding to the rat
mannose-binding protein
(3) with regard to the fucose versus mannose calcium coordination, and the orientation of the
oligosaccharide toward an area defined by Arg
,
Tyr
, and Ala
. A single Ala
Lys
substitution is capable of obliterating sLe
binding and
facilitating oligomannose recognition (Fig. 5). Our data do not
support previously postulated modes of binding in which the sLe
carboxylate is oriented toward Lys
(2, 33) but in contrast support an interaction with Arg
which is also consistent with known structure-activity data. As
one would expect, the amino acids lining the E-selectin binding pocket,
which is defined on one side by the
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)), are not fully conserved. Yet
functionally, E-selectin, like other members of the C-type lectin
family
(32) has retained a conserved carbohydrate binding and
calcium coordination site. This information should prove valuable to
the rational design of selectin inhibitors with potential therapeutic
efficacy for the treatment of acute inflammation and reperfusion
injury.
, sialyl Lewis
; CRD,
carbohydrate recognition domain; MBP, mannose-binding protein; PBS,
phosphate-buffered saline; GAM, goat anti-mouse; ELISA, enzyme-linked
immunosorbent assay; PCR, polymerase chain reaction; BSA, bovine serum
albumin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.