(Received for publication, January 17, 1996; and in revised form, January 30, 1996)
From the
The structures of the ligand-binding C-type
carbohydrate-recognition domains of selectin cell adhesion molecules
and of mannose-binding proteins (MBPs) are similar to each other even
though these proteins bind very different carbohydrate ligands. Our
current understanding of ligand binding by E-selectin is based on
structural studies of unliganded E-selectin and of MBP-carbohydrate
complexes, combined with results from mutagenesis of E-selectin. Five
regions of E-selectin that differ in sequence from the corresponding
regions of MBP have been introduced into the carbohydrate-recognition
domain of MBP. Four of the changes have little effect on ligand
binding. Insertion of one stretch of positively charged amino acids
alters the sugar binding selectivity of the domain so that it now binds
HL-60 cells and serum albumin derivatized with sialyl-Lewis tetrasaccharide, thus mimicking the properties of E-selectin.
One of the most striking properties of animal proteins that
contain Ca-dependent carbohydrate-recognition domains
(C-type CRDs) (
)is the diversity of sugar ligands that bind
to different members of this family(1) . In broad terms, these
CRDs fall into two classes. CRDs in one class bind ligands containing
galactose or N-acetylgalactosamine with varying degrees of
selectivity and affinity. CRDs in the second class bind to mannose
and/or N-acetylglucosamine. The distinguishing feature of the
ligands for these two classes is the disposition of the 3- and
4-hydroxyl groups. The results of structural and mutagenesis studies
suggest that in all C-type lectins these hydroxyl groups form part of
the coordination sphere of a bound
Ca
(2, 3, 4) . Preferential
binding of sugars with an equatorial-axial or equatorial-equatorial
arrangement at positions 3 and 4 is determined by the arrangement of
side chains from the protein that form both coordination bonds with the
Ca
and hydrogen bonds with the sugar hydroxyl groups. L-Fucose binds to CRDs in the second class through analogous
interactions of the equatorial 2- and 3-hydroxyl groups. Increased
affinity and selectivity for certain sugars within each of the two
classes of C-type CRDs results from other contacts between the protein
and the sugar. For example, selective binding of N-acetylgalactosamine over galactose by the asialoglycoprotein
receptor requires the presence of additional amino acid residues that
are probably positioned near the 2-acetamido group(5) .
High
affinity binding of C-type animal lectins to complex sugar ligands
results from at least two additional effects. Clustering of multiple
CRDs, each with a single sugar-binding site displayed in an appropriate
geometrical arrangement, results in high affinity binding of
multiantennary sugars(6) . In mannose-binding proteins (MBPs),
such clustering results from oligomerization of polypeptides containing
single CRDs(7) , while there are duplicated CRDs in the single
polypeptide of the macrophage mannose receptor(8) .
Alternatively, additional affinity and selectivity can reflect the
presence of secondary or extended binding sites within single CRDs. In
these cases, a single CRD can interact with more than a single sugar in
an oligosaccharide. Liver MBP (MBP-C) but not serum MBP (MBP-A) appears
to contain such a secondary site. ()
Selectin cell
adhesion molecules mediate the interaction of circulating leukocytes
with vascular endothelial cells, initiating a transient rolling that
eventually leads to firmer attachment and extravasation (9, 10) . E- and P-selectins are found at the surface
of epithelial cells and interact with monocytes and neutrophils, while
L-selectin is expressed on circulating leukocytes and selectively
recognizes the surface of high endothelial venules of the peripheral
lymph nodes. Each of the selectins consists of an
NH-terminal C-type CRD adjacent to an epidermal growth
factor-like domain and a series of complement homology
modules(9, 10) . The natural ligands for the selectins
are not known, but fucose-containing oligosaccharides are the most
effective low molecular weight ligands. The tetrasaccharide
sialyl-Lewis
(9) and various sulfated derivatives (11) have been studied most extensively. Recent evidence
suggests that, in addition to clusters of such oligosaccharide ligands,
high affinity glycoprotein ligands for P-selectin bear sulfated
tyrosine residues (12, 13, 14) .
The
structure of a fragment of human E-selectin consisting of the CRD and
epidermal growth factor-like domains has been established by x-ray
crystallography(15) . This structure reveals a single
Ca-binding site, in contrast to the two sites
observed in the MBP-A and MBP-C CRDs(2, 3) . Evidence
from binding studies suggests first order dependence on Ca
concentration, consistent with the presence of a single
Ca
-binding site(16) . In the absence of
structural information about the sugar-binding sites in the selectins,
mutagenesis studies and modeling based on the MBP-A-oligomannose and
MBP-C-monosaccharide structures have been combined to suggest possible
modes of interaction of E-selectin (16, 17, 18) and P-selectin (19, 20, 21, 22) with
sialyl-Lewis
. The fucose moiety of the ligand probably
binds directly to the single Ca
and surrounding amino
acid residues in the E-selectin CRD in a manner analogous to the
interactions of mannose with Ca
site 2 of MBP-A (2) and of fucose and other sugars with Ca
site 2 of MBP-C(3) , while negatively charged portions of
the ligands are believed to interact with one or more regions of
positive potential on adjacent portions of the CRD surface (16, 17, 18, 19, 20, 21, 22) .
In the present studies, segments of E-selectin have been substituted
into the MBP-A CRD. Replacement of a single segment of 3 amino acid
residues confers upon the CRD the ability to bind HL-60 cells and
sialyl-Lewis-BSA. While many previous experiments have
relied on negative phenotypes of mutants in the E-selectin CRD to
identify functionally important residues, these experiments provide
positive evidence for the importance of a second portion of the CRD
surface in addition to the region directly surrounding Ca
site 2.
Five segments of E-selectin were incorporated into the MBP-A
framework. Two of the regions were hypothesized to be involved in
Ca ligation, while there is evidence that the other
three may be directly associated with oligosaccharide binding.
Figure 1:
Location of mutated regions of MBP-A on
the three-dimensional structure of the CRD. The structure of wild type
MBP-A CRD is shown as a ribbon diagram. Spheres labeled 1 and 2 represent Ca sites 1 and 2, respectively. Regions 1-5, which have been
substituted by sequences from E-selectin, are highlighted by dark
shading and are labeled R1-R5. Regions 1 and 2
contribute side chain ligands to Ca
site 1, while
point mutations in the portions of the E-selectin CRD corresponding to
regions 3-5 affect binding to HL-60 cells. The figure was
prepared using MOLSCRIPT (28) .
Figure 2:
Amino acid sequences of the COOH-terminal
portions of wild type MBP-A and E-selectin and mutants of MBP-A
containing E-selectin sequences. The positions of amino acids that
ligate Ca sites 1 and 2 are designated below the
sequence of MBP-A. The positions of mutations that have previously been
found to affect the binding activity of E-selectin are indicated above
the sequence of E-selectin. Phenotypes are denoted as - for
complete loss of binding activity and ± for partial loss of
activity. Data are summarized from (16, 17, 18) .
In order to
probe which of the amino acid substitutions might be responsible for
the absence of Ca site 1 in E-selectin, two segments
of polypeptide that contribute ligands for this Ca
in
MBP-A were replaced with the corresponding regions of E-selectin. These
segments are designated regions 1 and 2 in Fig. 1. The mutant
CRDs containing changes in regions 1 and 2 were prepared in a bacterial
expression system previously used for analysis of wild type MBP-A CRD.
Interaction of the mutant CRDs with Ca
was analyzed
by measuring the Ca
dependence of ligand binding in a
solid phase assay (Fig. 3). As indicated by the shape of the
curves, both mutant CRDs retain second order dependence on
Ca
, indicating that two Ca
-binding
sites are still present. The measured K
for the
region 1 mutant (2.8 ± 0.3 mM) is substantially weaker
than for the region 2 mutant (1.4 ± 0.1 mM) and wild
type (1.2 ± 0.1 mM(26) ).
Figure 3:
Ca dependence of ligand
binding to mutant MBP-A CRDs. Ca
was diluted in
1.5-fold increments. Curves were fitted to second-order Ca
binding equations(26) .
The second order
dependence on Ca was confirmed for the region 2
mutant using limited proteolysis with subtilisin to detect a
Ca
-dependent conformational change similar to that
observed for the wild type CRD (26) (data not shown). This
assay could not be utilized in the case of the region 1 mutant, because
this CRD proved resistant to proteolysis even in the absence of
Ca
, probably because the initial protease-sensitive
site detected in these assays lies within the loop that has been
substituted.
As shown in Fig. 2, substitution of region 2 of
E-selectin into MBP-A results in a change of one Ca site 1 coordination ligand from aspartic acid to asparagine,
while the substitution of region 1 results in more substantial changes,
as a stretch of 8 amino acid residues including one aspartic acid and
one glutamic acid ligand is replaced with a shorter sequence of 6
residues containing lysine and asparagine residues at the corresponding
positions. These results indicate that Ca
-binding
site 1 is not significantly affected by substitution of region 2, while
it is preserved but weakened by substitution of region 1. The fact that
the Ca
dependence of sugar binding to the region 1
mutant remains second order indicates that both sites 1 and 2 must be
occupied in order to create a sugar-binding site. The absolute affinity
of both region 1 and region 2 mutants for Man-BSA is unchanged from
wild type (data not shown). Thus, although substitution of residues in
region 1 reduces the affinity of this site for Ca
and
thus increases the overall K
, the ultimate
conformation achieved is not distinguishable from that of the wild type
CRD.
Like the region 1 and region 2 mutants, a double mutant
containing both region 1 and region 2 substitutions was produced in the
bacterial expression system in yields comparable with wild type.
However, unlike the single mutants, the double mutant could not be
purified by affinity chromatography on Man-Sepharose. It is likely that
the presence of both regions 1 and 2 in the MBP-A background reduces
the affinity of site 1 for Ca much more than does
either region alone, so a conformation like the doubly
Ca
-ligated form of MBP-A is not stable even at 25
mM Ca
. It is possible that the presence of
regions 1 and 2 from E-selectin is not sufficient to induce the
alternative stable conformation seen in E-selectin in the absence of
Ca
site 1, and thus the double mutant does not fold
correctly. However, since E-selectin itself does not bind to
Man-Sepharose, if the conformation of MBP-A containing both regions 1
and 2 from E-selectin does fold similarly to E-selectin, it might still
not bind to Man-Sepharose. Unfortunately, since it was not possible to
purify the mutant CRD containing both region 1 and 2 substitutions in
soluble form, binding to HL-60 cells or known oligosaccharide ligands
for E-selectin could not be tested.
As an alternative approach to evaluating the
role of regions 3, 4, and 5, each of these segments of E-selectin has
been substituted into the MBP-A background. All three of the modified
MBP-A CRDs can be purified by affinity chromatography on
mannose-Sepharose, suggesting that their ability to bind mannose is not
substantially diminished by the substitutions. The ability to bind
HL-60 cells was utilized to determine if these CRDs have
E-selectin-like binding characteristics. As shown in Fig. 4,
only the region 5 mutant displays Ca-dependent
binding to HL-60 cells. CRDs containing region 5 in combination with
either region 3 or region 4 were purified and found to have binding
characteristics indistinguishable from those of the region 5 mutant
alone (data not shown). These results provide positive evidence that
the cluster of basic residues in region 5 plays a role in binding of
HL-60 surface ligands. Since wild type MBP-A does not bind HL-60 cells,
this binding must reflect a novel activity induced by the presence of
region 5.
Figure 4:
HL-60 binding to wild type MBP-A and
E-selectin and mutants of MBP-A containing E-selectin sequences.
Binding experiments were performed in triplicate, with the results of
five separate experiments averaged, except in the case of region 3 and
4 mutants, which are the average of two experiments. Error bars represent standard deviations. Results are shown for binding in
the presence of 10 mM Ca (+Ca)
or in the presence of 1 mM EDTA (-Ca).
The nature of the binding activity of the region 5 mutant
was investigated further by testing the ability of this mutant to bind
to sialyl-Lewis conjugated to BSA. The iodinated ligand was
bound substantially more effectively to plates coated with the region 5
mutant than to wild type mannose-binding protein (Fig. 5).
Because of the limited quantities of the neoglycoprotein ligand
available, it was not possible to achieve saturation of binding, so an
accurate dissociation constant could not be determined. However, the
results in Fig. 5suggest that incorporation of region 5 results
in a substantial increase in affinity for the sialyl-Lewis
structure. Parallel experiments with
I-Man-BSA show
no change in affinity for this ligand, indicating that the increase in
affinity is specific for the sialylated structures (data not shown).
Figure 5:
Solid-phase binding assays wild type and
mutant MBP-A probed with I-sialyl-Lewis
-BSA.
Experimental data (filled circles) are shown along with
theoretical curves (continuous lines) fitted to the
data.
The fact that binding to sialyl-Lewis-BSA can be
observed in the simple solid phase assay with the region 5 mutant,
while it is difficult to demonstrate with natural E-selectin may
reflect the trimeric nature of the MBP-A derivative(7) . The
relatively close clustering of the three binding sites in the trimer
probably increases the affinity for the polyvalent test ligand. This
result suggests that formation of similar clusters of the E-selectin
CRD may be a useful way to increase the affinity of the natural CRD in
order to simplify measurement of ligand binding.