(Received for publication, November 18, 1994; and in revised form, January 27, 1995)
From the
E-selectin is a member of the selectin family of proteins that
recognize carbohydrate ligands in a Ca-dependent
manner. In order to better understand the role of Ca
in E-selectin-ligand interactions, we examined the E-selectin
structure by limited proteolysis. Apo-Lec-EGF-CR6, a
Ca
-free form of soluble E-selectin containing the
entire extracellular domain, was sensitive to limited proteolysis by
Glu-C endoproteinase. Amino-terminal sequencing analysis of the
proteolytic fragments revealed that the major cleavage site is at
Glu
which is in the loop (residues 94-103) adjacent
to the Ca
binding region of the lectin domain. Upon
Ca
binding, Lec-EGF-CR6 was protected from
proteolysis. This Ca
-dependent protection was further
augmented upon sialyl Lewis x (sLe
) ligand binding. These
results implied that Ca
binding to E-selectin induces
a conformational change and perhaps facilitates ligand binding. The
sLe
-bound complex in turn stabilizes Ca
binding. Lec-EGF-CR6 contains only one high-affinity
Ca
site (K
=
3.5 µM) as determined by equilibrium dialysis. In
addition, we found that Ba
was a potent antagonist in
blocking Lec-EGF-CR6-mediated HL-60 cell adhesion. By competitive
equilibrium dialysis and proteolysis analysis, we demonstrated that
Ba
bound to apo-Lec-EGF-CR6 5-fold tighter than
Ca
and abolished ligand binding activity.
Sr
also bound to apo-Lec-EGF-CR6 tighter than
Ca
. However, Sr
-regenerated
Lec-EGF-CR6 showed 50% ligand binding activity. Mg
bound to apo-Lec-EGF-CR6 with much weaker affinity than
Ca
and did not show any activity. Thus, E-selectin
function can be modulated by different metal ions.
E-selectin is expressed on endothelial cells upon cytokine
(interleukin-1 and tumor necrosis factor) stimulation and plays a
pivotal role in the initial rolling step of neutrophil adherence to
endothelial cells (Lasky, 1992; Butcher, 1991; Bevilacqua et
al., 1987, 1989). It is a member of the selectin family of
proteins, which also include P- and L-selectin (for reviews, see McEver
(1992) and Yednock and Rosen(1989)). They all consist of an
amino-terminal lectin (Lec) ()domain, an epidermal growth
factor (EGF)-like domain, followed by several consensus repeats (CR)
homologous to those of complement regulatory proteins, a
membrane-spanning region, and a short cytoplasmic carboxyl-terminal
tail (Bevilacqua et al., 1989; Lasky et al., 1989;
Siegelman et al., 1989; Johnston et al., 1989). By
engineering a series of domain-deletion E-selectin constructs, we (Li et al., 1994) and others (Walz et al., 1990; Pigott et al., 1991) have demonstrated that Lec-EGF, a construct
containing only the lectin and EGF domains, is capable of mediating
neutrophil or HL-60 cell adhesion. However, Lec-EGF-CR6 (a construct
containing the lectin, EGF, and 6 CR domains) blocks neutrophil
adherence to cytokine-stimulated HUVEC significantly better than other
shorter constructs (Li et al., 1994). These results suggest
that although the lectin and EGF domains are necessary for mediating
cell adhesion, the presence of the additional CR domains enhances
ligand binding.
The selectin family of proteins are members of the
C-type animal lectins, which bind to carbohydrate ligands in a
Ca-dependent manner (Drickamer, 1988). The crystal
structure of the lectin domain of one of the C-type lectins, the rat
mannose binding protein (MBP), reveals that it contains two
Ca
binding sites (Weis et al., 1991a). More
recently, the crystal structure of Lec-EGF has also been determined
(Graves et al., 1994). Although the lectin domains of these
two proteins share only
30% sequence homology, their overall
structures are very similar. However, a major difference between these
two proteins is that E-selectin contains only one Ca
binding site. The amino acids coordinating with Ca
in E-selectin are conserved with one of the sites in the MBP.
This conserved site is involved in ligand binding in the MBP as
revealed by the co-crystallization of this protein with its
carbohydrate ligand (Weis et al., 1992).
In order to
further understand the mechanism responsible for the
Ca-dependent modulation of E-selectin function, we
studied the effects of Ca
and ligand binding on
Lec-EGF-CR6 conformation by limited proteolysis. We report here that
apo-Lec-EGF-CR6 was sensitive to Glu-C proteinase digestion, but upon
Ca
and ligand binding, the protein was protected from
proteolysis. From amino-terminal sequencing analysis, we identified
that the cleavage sites are located in the Ca
binding
region of the lectin domain and demonstrated that although the
proteolytic fragments were connected by disulfide bonds, Ca
binding was completely abolished. We also determined the binding
affinity for Ca
as well as other metal ions and
showed that E-selectin function could be modulated by these metal ions.
These results provide further understanding of the effects of metal
ions on E-selectin structure/function.
Figure 1:
Limited proteolysis of Lec-EGF-CR6 by
endoproteinase Glu-C. A, apo-Lec-EGF-CR6 (0.2 mg/ml) was
digested with various amounts of Glu-C for 1 h at 37 °C and
analyzed by SDS-PAGE under reducing conditions (see ``Experimental
Procedures''). Lanes 2-6 (3 µg/lane) contain
samples digested with enzyme:substrate ratios of 1:100, 1:50, 1:25,
1:15, and 1:10 (w/w), respectively. An undigested sample is shown in lane 1. B, apo-Lec-EGF-CR6 was regenerated with
increasing concentrations of Ca (0 µM,
10 µM, 20 µM, 40 µM, 80
µM, 160 µM, and 1 mM; lanes
1-7, respectively), digested with Glu-C (enzyme:substrate
ratio, 1:15) for 1 h at 37 °C, and samples (3.75 µg/lane) were
analyzed by SDS-PAGE under reducing conditions. Protein bands were
visualized by Coomassie Blue staining.
To determine
the sites of cleavage, the Glu-C-digested fragments were separated by
SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and
subjected to amino-terminal sequencing analysis. The 85-kDa protein
band contains a major (75%) amino-terminal sequence
(HN-Lys-Asp-Val-Gly-Met-, corresponding to the sequence
starting at residue 99 in the intact protein), indicating that the
major cleavage site is located at Glu
in the lectin
domain. In addition, two minor sequences
(H
N-Ile-Tyr-Ile-Lys-Arg- and
H
N-Arg-(Cys?)-Ser-Lys-Lys-) were also observed,
suggesting that glutamic residues at positions 92 and 107 were also
sensitive to Glu-C digestion. The 15-kDa fragment showed a sequence
identical with the amino terminus of the intact protein. The
Ca
form of Lec-EGF-CR6 after being treated with Glu-C
was also subjected to amino-terminal sequence analysis. The sequence
corresponding to the amino terminus of the intact protein was found,
indicating that indeed no significant cleavage occurred.
Figure 2:
sLe-protected proteolysis.
Apo-Lec-EGF-CR6 (0.2 mg/ml, 2 µM) was regenerated with
increasing concentrations of Ca
(0.5
µM-2.5 mM) at 4 °C overnight. The
samples were then incubated with various concentrations of sLe
(
, 0 mM;
, 0.2 mM;
, 0.6
mM;
, 1.2 mM) for 1 h at 37 °C, digested
with Glu-C (enzyme:substrate ratio, 1:15), and analyzed by SDS-PAGE as
described under ``Experimental Procedures.'' For
quantitation, the Coomassie Blue-stained gels were scanned by a
densitometer (Molecular Dynamics).
Figure 3:
Ca binding to soluble
E-selectins. Equilibrium dialysis experiments were performed as
described under ``Experimental Procedures.'' A, the
data are plotted as moles of Ca
bound per mol of
Lec-EGF (
) or Lec-EGF-CR6 (
) versus the free
[Ca
] concentration in solution. Data from a
single representative experiment are shown. Three or more experiments
were done for each E-selectin, and the results were similar. B, Scatchard plot of Lec-EGF data. C, Scatchard plot
of Lec-EGF-CR6 data. The plots were generated by best-fit to nonlinear
regression of a two-site binding model.
When Glu-C-digested fragments of
Lec-EGF-CR6 were analyzed by SDS-PAGE under nonreducing conditions,
only one protein band with the same mobility as that of undigested
material was observed, indicating that the fragments were associated
with each other by disulfide bonds. The results were also in agreement
with that predicted from the disulfide bond locations
(Cys-Cys
and
Cys
-Cys
) in the crystal structure of Lec-EGF
(Graves et al., 1994). We therefore examined whether or not
the proteolytic fragments retained Ca
binding
activity by equilibrium dialysis. Only the low affinity Ca
site was observed (data not shown), indicating that proteolytic
cleavage of Lec-EGF-CR6 completely abolished high affinity
Ca
binding. Furthermore, when the proteolyzed protein
was coated directly onto microtiter plates and examined for HL-60 cell
adhesion, no activity was observed. To rule out the possibility that
lack of cell adhesion activity was due to poor coating of proteolyzed
protein on the plates, we measured the amounts of coated protein by
enzyme-linked immunosorbent assay using an anti-E-selectin monoclonal
antibody, 9A1, that recognizes the first CR domain (Erbe et
al., 1992). The results showed that the extent of the antibody
binding to proteolyzed Lec-EGF-CR6 on the plate was about the same as
that of the intact protein, suggesting that both proteins were coated
equally on the plates. We also examined whether or not the proteolyzed
Lec-EGF-CR6 was able to inhibit E-selectin-mediated HL-60 cell adhesion
by the assay procedure as described previously (Li et al.,
1994). The results showed that proteolyzed Lec-EGF-CR6 (up to 6
µM) was not capable of inhibiting E-selectin-mediated cell
adhesion. Under the same conditions, intact Lec-EGF-CR6 was able to
completely block HL-60 cell adhesion. Taken together, these results
demonstrated that the high affinity Ca
site is
responsible for ligand binding.
To examine whether the weak ligand binding activities of
Mg-, Sr
-, and
Ba
-regenerated Lec-EGF-CR6 were due to the inability
of these metal ions to bind to the protein, we studied their
sensitivity to Glu-C proteolysis. Lec-EGF-CR6 was incubated with the
above metal ions at three different concentrations (5 µM,
50 µM, and 500 µM) and subjected to Glu-C
digestion. The results indicated that at all three concentrations, the
Sr
- and Ba
-regenerated Lec-EGF-CR6
were more resistant to proteolysis than the Ca
form (Fig. 4). However, the Mg
form was more
sensitive to proteolysis. Even at 500 µM,
Mg
-regenerated Lec-EGF-CR6 was completely
proteolyzed. In contrast, the Ca
regenerated
Lec-EGF-CR6 was completely protected from proteolysis at this
concentration. Thus, Sr
and Ba
bound to apo-Lec-EGF-CR6 tighter than Ca
, but
Mg
had much weaker affinity. To confirm these
results, we also examined whether Mg
,
Sr
, or Ba
was able to compete for
Ca
binding by equilibrium dialysis. As shown in Fig. 5, both Sr
and Ba
showed concentration-dependent competitive binding, whereas
Mg
did not. The calculated K
values for Sr
and Ba
were
about 0.7 µM, which was 5 times lower than that of
Ca
.
Figure 4:
Limited proteolysis of Lec-EGF-CR6
regenerated with divalent cations. Apo-Lec-EGF-CR6 was regenerated with
Ba, Sr
, Ca
, and
Mg
at 5 µM, 50 µM, and 500
µM concentrations, digested with Glu-C, and analyzed by
SDS-PAGE. Proteins were visualized by Coomassie Blue
staining.
Figure 5:
Divalent cation competition with
Ca binding to Lec-EGF-CR6. Equilibrium dialysis
experiments measuring the amounts of Ca
bound to
Lec-EGF-CR6 in the presence of competing divalent cations were carried
out as described in ``Experimental Procedures.''
Ba
(
), Sr
(
), and
Mg
(
) at various concentrations (1
µM-1 mM) were used to compete with
Ca
(10 µM) binding. Data are the mean
± S.E. of triplicate samples.
Since Ba and Sr
showed higher binding affinities to apo-Lec-EGF-CR6 than
Ca
, we then examined whether they were able to
compete with Ca
and inhibit E-selectin function.
Immobilized Lec-EGF-CR6 (the Ca
form) was
preincubated with various concentrations of Ba
or
Sr
at 4 °C overnight in a buffer containing 0.5
mM Ca
. The mixtures were then assayed for
E-selectin function by HL-60 cell adhesion or CEA binding. In both
cases, the results showed that Ba
is a potent
antagonist (IC
= 0.4 mM, Fig. 6, A and B). Sr
also showed weak
inhibitory activity. At high concentrations, Sr
should quantitatively replace Ca
, and the
activity observed was probably due to Sr
-regenerated
Lec-EGF-CR6. As expected, Mg
did not show any
inhibition. The results confirmed that both Ba
and
Sr
could replace Ca
and inactivate
E-selectin function.
Figure 6:
Inhibition of E-selectin function by
divalent cations in the HL-60 cell adhesion assay (A) and CEA
binding assay (B). The assays were carried out as described
under ``Experimental Procedures.'' In both assays, all wash
and incubation buffers contained 0.5 mM CaCl plus
competing cations (
, Ba
;
,
Sr
;
, Mg
) at various
concentrations (50 µM-4 mM). Data are the
mean ± S.E. of triplicate samples.
Although previous studies demonstrated that Ca is required for E-selectin function, its specific role is not
known. In the present studies, we examined whether binding of
Ca
to E-selectin induces a conformational change and
promotes ligand recognition. From fluorescence spectroscopic studies,
Geng et al.(1991) proposed a similar mechanism for the role of
Ca
in P-selectin function. We initially attempted to
study E-selectin conformation by the same techniques as those reported
by Geng et al.(1991). Although we observed a small difference
in fluorescence intensity between apo- and Ca
-bound
Lec-EGF-CR6, we did not detect any significant emission wavelength
shift. In contrast, Geng et al.(1991) reported that both the
fluorescence intensity and emission wavelength were affected upon
Ca
binding to P-selectin. We therefore examined
whether Ca
induces protein conformational changes by
comparing the sensitivity to proteolysis of the apo- and Ca
forms of Lec-EGF-CR6. Limited proteolysis studies have previously
been employed to show Ca
-dependent conformational
changes for many proteins. These include members of the C-type lectins, e.g. rat MBP (Weis et al., 1991b) and the
asialoglycoprotein receptor (Loeb and Drickamer, 1988), the Src
homology region 2 (SH2) domains of p85, and Ras GTPase activating
protein (Mahadevan et al., 1994), erythrocyte spectrin (Wallis et al., 1993), and Nereis sarcoplasmic
calcium-binding protein (Durussel et al., 1993). Our results
showed that while the apo-Lec-EGF-CR6 was sensitive to Glu-C digestion,
the Ca
-bound form was not, suggesting that
Ca
binding induced a conformational change to protect
the protein from proteolysis. In view of our results from fluorescence
spectrum studies, we speculate that such a conformational change may be
subtle.
The crystal structure of Lec-EGF reveals that Ca binding to E-selectin is through coordination of the side chain
carbonyl groups of Glu
, Asn
,
Asn
, and Asp
, as well as the main chain
carbonyl of Asp
and two water molecules, to form a
pentagonal bipyramid sphere (Fig. 7A). Interestingly,
one of the water molecules interacts with the side chain carbonyl group
of Asn
through hydrogen bonding, enhancing Ca
binding. From amino-terminal sequencing analysis of the digested
fragments, we determined that the major Glu-C cleavage site is at
residue 98 which is located in the loop (residues 94-103)
connecting
4 (residues 90-93) and
5 (residues
104-107) strands (Fig. 7A, Graves et
al., 1994). The crystal structure also reveals that Glu
is 18 Å away from the bound Ca
atom (Fig. 7B). Although residues in this loop do not
directly interact with Ca
, they adopt an interesting
conformation such that the loop is tilted toward Ca
.
We speculate that removal of Ca
from E-selectin
allows Asn
and Asp
to adopt a more flexible
conformation and consequently influence the adjacent loop conformation
such that it is susceptible to proteolysis. Data from site-specific
mutagenesis studies (Graves et al., 1994; Erbe et
al., 1992) revealed that Tyr
and Arg
in
this loop are critical in mediating cell adhesion. Thus, it is possible
that the unusual conformation of this loop in the Ca
form of the protein facilitates its interaction with carbohydrate
ligand. Our results that the proteolytic fragments, even though they
were associated with each other through disulfide bonds, did not retain
Ca
or ligand binding activity may also be explained
by the fact that breaking a peptide bond in this loop disrupts its
conformation and consequently its functional role. In addition to the
major cleavage site at Glu
, we also observed two minor
cleavage sites at Glu
and Glu
. Although
these two residues are not directly coordinated with
Ca
, the crystal structure reveals that they are close
to each other and adjacent to the site (
6 Å). Furthermore,
Glu
forms a hydrogen bond with the amide group of
Asn
, which is a Ca
ligand. It is
therefore possible that removal of Ca
from E-selectin
also induces a conformational change in this region.
Figure 7:
A, stick drawing of the E-selectin
structure (residues 78-109) highlighting the Ca binding region. The peptide backbone is highlighted in green, and the side chains of Glu-92, 98, and 107 are
illustrated in red. The magenta sphere represents the
Ca
atom while the corresponding coordinating bonds
are drawn in gold (two water molecules are not shown). B, space-filling model of the Ca
binding
region of E-selectin. The dotted sphere indicates the calcium
ion. Residues Glu
, Glu
, and Glu
are shown in cyan.
By equilibrium
dialysis, we determined that Lec-EGF and Lec-EGF-CR6 contain only one
high affinity Ca binding site with K
values of 2.5 and 3.5 µM, respectively. In addition,
we also observed that at very high Ca
concentrations
more than one Ca
was able to bind to Lec-EGF and
Lec-EGF-CR6. The Scatchard plot analyses indicated that the affinities
for the second sites in both E-selectins were much lower than those of
the high affinity sites. These results were in agreement with those
observed from the crystal structure of Lec-EGF. When Lec-EGF was
crystallized under high concentrations of CaCl
, three sites
were observed (Graves et al., 1994). However, only one showed
high affinity coordination, and the other two were adventitious
resulting from the crystallization conditions. When crystals were grown
under low Ca
concentrations, only one Ca
was observed. Geng et al.(1991) recently reported that
P-selectin contains two indistinguishable high affinity Ca
sites (K
= 22 µM by
equilibrium dialysis and 4.8 µM by fluorescence emission
intensity). These results suggest that although the structures of E-
and P-selectin are highly homologous, P-selectin contains an additional
high affinity Ca
site, whose location yet remains to
be determined. Thus, the selectin family of proteins as well as other
members of the C-type lectins contain a highly homologous
Ca
binding site which is involved in ligand binding.
Some members of this family of proteins contain additional high
affinity Ca
sites, whose roles remain to be
determined.
Our data from divalent cation reconstitution experiments
indicated that the Sr-reconstituted Lec-EGF-CR6
exhibited partial (50%) ligand binding activity whereas the
Ba
or Mg
reconstituted forms did
not show significant activity. To examine whether the lack of ligand
binding activity was due to the inability of these metal ions to bind
to Lec-EGF-CR6, we performed competitive equilibrium dialysis and
limited proteolysis experiments. We concluded that lack of ligand
binding activity by Mg
was attributed to the weak
binding to Lec-EGF-CR6. However, Ba
and
Sr
bound to Lec-EGF-CR6 tighter than
Ca
. Furthermore, from metal ion competitive
functional assays, we demonstrated that both Ba
and
Sr
were antagonists. These results were of interest
because they demonstrated that E-selectin function could be modulated
by divalent metal ions. One possible explanation for the diverse
effects exhibited by these metal ions is that they have different ionic
radii (1.34 Å, 1.12 Å, 0.99 Å, and 0.66 Å for
Ba
, Sr
, Ca
, and
Mg
, respectively). Because of that, they may induce
different conformational changes upon binding to E-selectin. For
example, because Mg
has a smaller radius and
different coordination chemistry from Ca
, it at best
binds to E-selectin weakly and does not induce proper conformational
changes. Ba
, although it can bind to E-selectin
tightly, induces a conformation that is not favorable for carbohydrate
ligand binding due to its larger radius than Ca
. On
the other hand, Sr
, with a similar radius as
Ca
, binds to E-selectin tightly and induces a
conformation retaining partial ligand binding activity.
In summary,
our results reported here suggest that Ca binding to
E-selectin induces a minor, yet critical, conformational change.
Perturbations in the conformation of the Ca
binding
region by either limited proteolysis or substitutions with other metal
ions completely abolished E-selectin function. Understanding these
metal ion-induced conformational changes may help us to design specific
antagonists to block E-selectin-mediated cell adhesion events.