©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Single Amino Acid Residue Can Determine the Ligand Specificity of E-selectin (*)

Timothy P. Kogan (3) (2)(§), B. Mitch Revelle (1), Stephen Tapp (1), Dee Scott (1), Pamela J. Beck (3) (1)

From the (1) From the Department of Molecular Biology and (2) Department of Medicinal Chemistry, Texas Biotechnology Corporation and the (3) Department of Internal Medicine, The University of Texas Medical School, Houston, Texas 77030

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

E-selectin (ELAM-1) is a member of the selectin family of cellular adhesion molecules. This family of proteins possesses an amino-terminal Ca-dependent lectin or carbohydrate recognition domain that is essential for ligand binding. A known E-selectin ligand is the carbohydrate antigen, sialyl Lewis (sLe) (Neu5Ac2-3Gal1-4(Fuc1-3)GlcNAc). We have developed a model of E-selectin binding to the sLe tetrasaccharide, (Neu5Ac2-3Gal1-4(Fuc1-3)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.


INTRODUCTION

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() 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.

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 and E-selectin should aid in the design of novel selectin inhibitors.

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 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) .

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 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.


EXPERIMENTAL PROCEDURES

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.

The sLe 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.

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 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).

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 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.

To aid in protein stabilization, detection, quantitation, and purification, this E-selectin cassette was fused to the hinge region of the mouse IgG heavy chain coding sequence to create a soluble, secreted protein expressed from a construct similar to that previously described (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.

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 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 AlaLys 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.


RESULTS

To elucidate the interactions that occur between selectin and ligand, a model of sLe 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.

This model is based upon the assumption that the C-2 and C-3 hydroxyls of the 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.

Assuming E-selectin binds fucose in the manner described above, other amino acids surrounding the binding pocket must interact with sLe 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.

The critical roles of the Arg 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.

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 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).

HL60 binding to the recombinant AlaLys mutants was assessed and found to be negligible. Furthermore, the presence of the ArgAla substitution which clearly enhances binding of sLe to native E-selectin, was not able to restore the ability of the AlaLys 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 AlaLys 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 AlaLys 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 AlaLys 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.

In agreement with the filter assay, the AlaLys 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 AlaLys 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 ProLys or the ArgAla second site changes.

To further demonstrate that the binding interactions between the E-selectin AlaLys mutant and oligomannose are specific and similar to that of the rat MBP, the AlaLys 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 GluGln and AsnAsp) 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 (AlaLys, GluGln, AsnAsp). We conclude that the side chain present at position 77 (Ala versus Lys) exerts a significant influence over ligand selectivity and thereby controls sLeversus mannose recognition.


Figure 4: A, effect of free mannose on mutant invertase binding. The binding of the E-selectin AlaLys (open squares) and AlaLys, GluGln, AsnAsp (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 (AlaLys) and 45 mM (AlaLys, GluGln, AsnAsp). 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) AlaLys and (diamonds) AlaLys, GluGln, AsnAsp 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 (AlaLys) and 25 mM (AlaLys, GluGln, AsnAsp). 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 AlaLys mutant mannose coordination. The predicted interactions between the E-selectin AlaLys 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 AlaLys 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 AlaLys 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 AlaLys -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 AlaLys mutant does not bind sLe.




DISCUSSION

Several observations can be made from inspection of the mutant binding data presented in Fig. 2B. The TyrAla 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 TyrPhe 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 GluAsp substitution that obliterates all HL60 binding activity while the GluLys mutant binding is greatly increased and the GluAsn replacement has no detectable effect.

Since the E-selectin crystal structure predicts that Lys 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 GluAsp 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.

Two other E-selectin residues that have been hypothesized to directly interact with the sLe 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 LysGlu and LysGln 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 LysGlu and LysGln substitution information when analyzed together with the inability of the AlaLys 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).

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 LysAla, LysArg, GluAsp, or TyrPhe (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 AlaLys mutant K of 10 mM). These data, together with the GluGln, AsnAsp 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).

In summary, our mutagenesis and binding analyses are consistent with sLe 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 AlaLys 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.


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 should be addressed. Tel.: 713-796-8822; Fax: 713-796-8232.

The abbreviations used are: sLe, 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.


ACKNOWLEDGEMENTS

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.


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