©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Modulation of E-selectin Structure/Function by Metal Ions
STUDIES ON LIMITED PROTEOLYSIS AND METAL ION REGENERATION (*)

(Received for publication, November 18, 1994; and in revised form, January 27, 1995)

Michael Anostario Jr. Kuo-Sen Huang (§)

From the Department of Inflammation/Autoimmune Diseases, Hoffmann-La Roche Inc., Nutley, New Jersey 07110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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^x) ligand binding. These results implied that Ca binding to E-selectin induces a conformational change and perhaps facilitates ligand binding. The sLe^x-bound complex in turn stabilizes Ca binding. Lec-EGF-CR6 contains only one high-affinity Ca site (K = approx3.5 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.


INTRODUCTION

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) (^1)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 approx30% 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.


EXPERIMENTAL PROCEDURES

Materials

Chelex 100 resin was purchased from Bio-Rad. CaCl(2) (99.99+% pure) and diisopropyl fluorophosphate were obtained from Aldrich. I and CaCl(2) (aqueous solution) were from Amersham Corp. CEA was from Zymed. Endoproteinase Glu-C and Lys-C were from Boehringer Mannheim. 6-Carboxyfluorescein diacetate was from Calbiochem. Iscove's modified Dulbecco's medium was from Life Technologies, Inc. Subtilisin (protease Type VIII), EDTA, EGTA, MgCl(2), SrCl(2), and BaCl(2) were from Sigma. sLe^x and 3`-sialyl-N-acetyllactosamine were from Dr. Donna Huryn, Hoffmann-La Roche Inc. Poly[N-(2-hydroxyethyl)acrylamide]-bound sLe^x was from Syntesome GmbH (Munchen, Germany).

Preparation of Apo-E-selectins

Soluble E-selectins (Lec-EGF and Lec-EGF-CR6) were engineered, produced, and purified as described previously (Li et al., 1994). For most experiments, apo-E-selectins were prepared by dialyzing the proteins extensively against Chelex-treated 50 mM Tris-HCl, pH 8.0, plus 0.02% NaN(3). The formation of apo-E-selectins was monitored by Mono Q-FPLC. Apo-E-selectins bound to Mono Q-FPLC tighter than the metal-bound counterparts. For some experiments (see below), apoproteins were prepared by treatment of immobilized E-selectins with EDTA followed by washing with a Chelex-treated buffer.

Protein Determination

The protein concentrations were determined from the ultraviolet absorption spectrum. The molar extinction coefficients at 280 nm (75,000 M cm and 150,000 M cm for Lec-EGF and Lec-EGF-CR6, respectively) were determined by amino acid composition analysis and the Bradford protein assay (Bio-Rad). The results from both methods were consistent.

Limited Glu-C Proteolysis

Apo-Lec-EGF or apo-Lec-EGF-CR6 (0.2 mg/ml) was prepared by dialysis as described above and regenerated with varying concentrations of Ca or other divalent cations at 4 °C overnight. The samples were then digested with endoproteinase Glu-C at a 1:15 (w/w) enzyme:substrate ratio for 1 h at 37 °C. The reactions were stopped by the addition of diisopropyl fluorophosphate to a final concentration of 2 mM along with reducing sample buffer (Laemmli, 1970). The samples were immediately boiled for 5 min and subjected to SDS-PAGE analyses under reducing conditions using 10-20% Tricine gradient gels (Novex pre-cast). For quantitation Coomassie blue-stained gels were scanned using a densitometer (Molecular Dynamics Personal Densitometer).

Equilibrium Dialysis

Determination of CaCl(2) binding to Lec-EGF or Lec-EGF-CR6 by equilibrium dialysis was performed using ISCO sample concentration cups (Instrumentation Specialties Co., Lincoln, NE) placed in 250-ml beakers. Typically, an aliquot (100 µl) of apo-E-selectin (1 mg/ml as determined by A) in Chelex-treated 50 mM Tris-HCl, pH 7.5, 100 mM NaCl (Buffer A) was placed in the small dialysis chamber of the unit. The sample was then dialyzed through a Spectropor membrane (M(r) cutoff = 8,000) against 50 ml of Buffer A containing 0.14 µCi/ml CaCl(2) (10-40 mCi/mg) and an indicated concentration of CaCl(2) (1 µM-250 µM). Experiments were carried out at room temperature for 15 h. Aliquots (40 µl each) from the protein sample inside the cup as well as 100 µl of the buffer outside were then withdrawn, and the radioactivity was measured by liquid scintillation counting (Beckman LS 3800). The Ca bound was determined by the net counts inside the cup after correcting for counts in the buffer.

Competition Binding by Divalent Cations

The competitive binding of Mg, Sr, and Ba to Lec-EGF-CR6 was determined by equilibrium dialysis. An aliquot (100 µl) of apo-Lec-EGF-CR6 (1 mg/ml) in Buffer A was placed in a cup as described above. The sample was then dialyzed at room temperature for 15 h against a buffer containing 0.14 µCi/ml CaCl(2), 10 µM CaCl(2), and an indicated concentration of competing divalent cations (1 µM-1 mM). The radioactivity associated with the protein was determined by the method described above. The K(d) values for competing divalent cations were calculated by the formula of Cheng and Prusoff (Cheng and Prusoff, 1973). Kd = IC/(1 + ([Ca]/K(d))), where K(d) and K(d) are the dissociation constants for competing divalent cations and Ca, respectively. IC is the divalent cation concentration when 50% of the maximal Ca binding is inhibited. [Ca] is the calcium concentration.

Competitive CEA Binding Assay

This is a modification of the CEA binding assay previously described (Anostario et al., 1994). Typically, microtiter plates (Immulon II, Dynatech) were coated with Lec-EGF-CR6 (100 µl, 500 ng/well) in Iscove's modified Dulbecco's medium. The plates were then blocked with phosphate-buffered saline, 1% BSA, 1 mM CaCl(2), 0.02% NaN(3) (200 µl/well), and washed four times with 50 mM Tris-HCl, pH 7.2, 150 mM NaCl (TBS) plus 0.5 mM CaCl(2) (TBS/CaCl(2)). To each well was then added 100 µl of TBS/CaCl(2) containing various concentrations of competing divalent cations followed by I-labeled CEA (25 ng, 4 times 10^4 cpm/ng). The plates were then incubated at 37 °C for 1 h and washed four times with TBS/CaCl(2) plus divalent cations (200 µl/well for each wash). The bound CEA was then eluted with 35 mM Tris-HCl, pH 8.5, 20 mM EGTA at room temperature for 20 min (200 µl/well). The eluted CEA was quantified by monitoring radioactivity.

CEA Assay with Divalent Cation-reconstituted Lec-EGF-CR6

Lec-EGF-CR6 was coated onto microtiter plates and blocked with BSA as described above. The plates were then treated with TBS, 1 mM EDTA (0.1 ml/well) for 20 min at room temperature and rinsed four times with Chelex-treated TBS followed by incubation with varying concentrations of divalent cations in Chelex-treated TBS overnight at 4 °C. I-Labeled CEA (4 µl, 51 ng, 2.5 times 10^4 cpm/ng) was added to each well and incubated at 37 °C for 1 h. The wells were then washed four times with Chelex-treated TBS containing the appropriate concentration of divalent cation. Bound CEA was eluted and counted in a counter as described above.

Competitive HL-60 Cell Adhesion Assay

The assay was a modification of a procedure previously described (Li et al., 1994). Typically, Immulon II plates were coated overnight at 4 °C with Lec-EGF-CR6 (100 ng/well) in Iscove's modified Dulbecco's medium, blocked with BSA, and washed four times with TBS, 0.5 mM CaCl(2) as described above. HL-60 cells were suspended (5 times 10^6 cell/ml) in TBS, 0.5 mM CaCl(2) and fluorescently labeled with 6-carboxyfluorescein (a stock solution of 5 mg/ml in acetone was added to cells to achieve a final concentration of 40 µg/ml) for 45 min at 37 °C in a CO(2) incubator. The cells were washed with TBS, 0.5 mM CaCl(2) and then resuspended at a final concentration of 2 times 10^6 cells/ml in TBS, 0.5 mM CaCl(2) plus various concentrations of competing divalent cations. The cell suspension was then added (100 µl/well) to Lec-EGF-CR6-coated wells. After incubation for 15 min at room temperature, cells were washed 3 times with TBS, 0.5 mM CaCl(2) containing divalent cations. The fluorescence of the bound cells was measured by a Titertek Fluoroskan II fluorescent plate reader (Flow Laboratories) using an excitation wavelength of 485 nm and an emission wavelength of 538 nm.

Amino Acid Sequencing

The Glu-C-digested fragments were separated by SDS-PAGE, transferred to Millipore Immobilon polyvinylidene difluoride membrane, and visualized by Coomassie Blue staining (Matsudaira, 1987). The blotted fragments were then excised from the membrane support for direct amino-terminal sequencing analyses in an Applied Biosystems Model 470A gas-phase sequencer with an on-line phenylthiohydantoin analyzer.


RESULTS

Ca-dependent Limited Proteolysis of Lec-EGF-CR6

Apo-Lec-EGF-CR6 was incubated with increasing concentrations of Glu-C endoproteinase for 1 h at 37 °C, and the products were analyzed by SDS-PAGE under reducing conditions. At the high Glu-C endoproteinase concentrations (lanes 5 and 6, Fig. 1A), two protein bands with M(r) = 85,000 and 15,000 were observed, indicating that apo-Lec-EGF-CR6 was sensitive to limited proteolysis. However, when apo-Lec-EGF-CR6 was regenerated with varying concentrations of Ca and digested with Glu-C endoproteinase, the extent of proteolysis decreased as the Ca concentrations increased (lanes 1-7, Fig. 1B). At 1 mM or higher concentrations of Ca, no significant proteolysis was observed. As a control, BSA was also digested with Glu-C under the same conditions. The digestion patterns were the same regardless of the CaCl(2) concentration used, indicating that Glu-C enzymatic activity was not affected by CaCl(2). Apo-Lec-EGF-CR6 was also sensitive to limited proteolysis by subtilisin and Lys-C endoproteinase (data not shown). Upon Ca binding, the protein was protected from proteolysis, suggesting that this phenomenon was not limited to the proteinase Glu-C. The results also imply that binding of Ca to apo-Lec-EGF-CR6 induces a conformational change such that the protein is protected from proteolysis.


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 (H(2)N-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(2)N-Ile-Tyr-Ile-Lys-Arg- and H(2)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.

Protection of Lec-EGF-CR6 from Proteolysis by sLe^x

Weis et al. (1991b) reported that the lectin domain of MBP was more resistant to proteolysis upon ligand binding. The crystal structure of MBP complexed with a carbohydrate ligand revealed that Ca forms a ternary complex with the protein and the sugar, consistent with the model that Ca binding to MBP is a prerequisite step for the ligand binding. To study whether or not Ca plays a similar role in E-selectin-ligand interactions, we examined Ca-dependent proteolysis of Lec-EGF-CR6 in the presence of sLe^x. As shown in Fig. 2, the K (the Ca concentration when 50% of the material is undigested) was about 35 µM in the absence of sLe^x. When Lec-EGF-CR6 was incubated with a 100-fold excess of sLe^x, the K was 10 µM, which was 3-4-fold lower than that in the absence of sLe^x. The K values were even lower in the presence of a 300- or 600-fold excess of sLe^x. As a control experiment, we also examined the Ca-dependent proteolysis in the presence of 3`-sialyl-N-acetyllactosamine (fucose-less sLe^x), which is known not to bind to E-selectin (see review by Varki(1994)). As expected, the K was not affected by the presence of 600-fold excess of fucose-less sLe^x. These results imply that the Ca binding affinity is enhanced upon sLe^x binding. (^2)This observation also supports the model that Ca is simultaneously coordinated by sLe^x and the protein, as demonstrated in the co-crystal structure of MBP with oligomannose. However, we could not rule out the possibility that sLe^x does not directly interact with Ca, but still could induce a conformational change in E-selectin to enhance the Ca binding affinity.


Figure 2: sLe^x-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^x (circle, 0 mM; , 0.2 mM; up triangle, 0.6 mM; box, 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).



K(d)Values of CaBinding to Soluble E-selectins

Geng et al.(1991) reported that P-selectin contains two indistinguishable high affinity binding sites for Ca as determined by equilibrium dialysis. The crystal structure of Lec-EGF revealed only one high affinity Ca site. To examine the possibility that E-selectin may contain two high affinity Ca sites with the second site located in the CR domains, we determined the number of Ca binding sites in Lec-EGF and Lec-EGF-CR6 by equilibrium dialysis. Fig. 3A shows the binding of Ca to Lec-EGF and Lec-EGF-CR6 at various Ca concentrations. Both E-selectins showed almost identical Ca binding. On Scatchard plot analyses using a two-site affinity model, both Lec-EGF and Lec-EGF-CR6 contained one high affinity Ca site with similar K values (2.5 µM and 3.5 µM, respectively, Fig. 3, B and C). In addition, both E-selectins also contained a very low affinity site with a K of about 100 times higher than the high affinity site. The specificity of these low affinity sites remains to be demonstrated.


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 (bullet) or Lec-EGF-CR6 (circle) 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.

Metal Ion Effect on E-selectin Function

Recently, members of the CEA family of proteins, e.g. NCA-160 and NCA-90, have been implicated as E-selectin native ligands on neutrophils and shown to carry sLe^x as indicated by their reactivities with anti-sLe^x monoclonal antibodies (Kuijper et al., 1992; Stocks and Kerr, 1993). Since these proteins are not readily available, we have utilized commercially available CEA isolated from human liver adenoma cells as a ligand to develop a cell-free assay to study E-selectin-ligand interactions (Anostario et al., 1994). This assay has been shown to be specific for E-selectin and sLe^x as demonstrated by the ability of anti-E-selectin and anti-sLe^x monoclonal antibodies to block E-selectin-CEA interactions. Therefore, this assay provides an opportunity to study the effects of metal ions on E-selectin function. Apo-Lec-EGF-CR6 was prepared by treatment of immobilized Lec-EGF-CR6 with EDTA. After washing with a Chelex-treated buffer, the apo-Lec-EGF-CR6 was then reconstituted with various metal ions (e.g. Mg, Ca, Sr, and Ba) and examined for binding to I-CEA. The results showed that only Ca could quantitatively regenerate Lec-EGF-CR6 activity (more than 90% of the original activity was regained at 1 mM Ca). Sr also regenerated 50% of the activity. However, Mg and Ba did not show significant activities (data not shown). The results were in agreement with those reported by Asa et al.(1992) whereby they coated sLe^x glycolipids on the plates and examined the binding of metal ion-regenerated E-selectin.

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(d) 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 (circle), Sr (up triangle), and Mg (box) 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(2) plus competing cations (bullet, Ba; , Sr; , Mg) at various concentrations (50 µM-4 mM). Data are the mean ± S.E. of triplicate samples.




DISCUSSION

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 beta4 (residues 90-93) and beta5 (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 (approx6 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(d) 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(2), 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(d) = 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 201-235-5970; Fax: 201-235-3805.

(^1)
The abbreviations used are: Lec, lectin; MBP, mannose binding protein; EGF, epidermal growth factor; CR, consensus repeat; CEA, carcinoembryonic antigen; sLe^x, sialyl Lewis x; BSA, bovine serum albumin; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis; PAA, poly[N-(2-hydroxyethyl)acrylamide]; FPLC, fast protein liquid chromatography.

(^2)
To confirm this, we attempted to determine K values for Ca in the presence of sLe^x by equilibrium dialysis. Due to the prohibitive expense of monomeric sLe^x, poly[N-(2-hydroxyethyl)acrylamide]-bound sLe^x (PAA-sLe^x, M(r) = 30,000) was used because it is retained inside during dialysis (M(r) cutoff = 8,000). When a 10-fold molar excess (monomeric sLe^x to Lec-EGF-CR6) of PAA-sLe^x was added, no differences in K values for Ca were observed between samples with and without PAA-sLe^x. The expense of PAA-sLe^x prohibited us from performing experiments at higher concentrations.


ACKNOWLEDGEMENTS

We thank Dr. Bradford Graves for providing figures of the three-dimensional structure of E-selectin and for helpful discussions. In addition, we acknowledge Dr. Yu-Ching Pan and Kurt Hollfelder for sequencing analysis, Dr. Donna Huryn for providing sLe^x, Shirley Li and Wayne Levin for critical reading of the manuscript, and Dr. Barry Wolitzky for his continued support.


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