Minimal requirements for the binding of selectin ligands to a C-type carbohydrate-recognition domain

Samuel Bouyain, Sally Rushton and Kurt Drickamer1

Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK

Received on June 19, 2001; revised on July 20, 2001; accepted on July 23, 2001.


    Abstract
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The C-type carbohydrate-recognition domains of E-selectin and rat serum mannose-binding protein have similar structures. Selectin/mannose-binding protein chimeras created by transfer of key sequences from E-selectin into mannose-binding protein have previously been shown to bind the selectin ligand sialyl-LewisX through a Ca2+-dependent subsite, common to many C-type lectins, and an accessory site containing positively charged amino acid residues. Further characterization of these chimeras as well as analysis of novel constructs containing additional regions of E-selectin demonstrate that selectin-like interaction with sialyl-LewisX can be faithfully reproduced even though structural evidence indicates that the mechanisms of binding to E-selectin and the chimeras are different. Selectin-like binding to the nonfucosylated sulfatide and sulfoglucuronyl glycolipids can also be reproduced with selectin/mannose-binding protein chimeras that contain the two subsites involved in sialyl-LewisX binding. These results indicate that binding of structurally distinct anionic glycans to C-type carbohydrate-recognition domains can be mediated by the Ca2+-dependent subsite in combination with a positively charged region that forms an ionic strength-sensitive subsite.

Key words: carbohydrate recognition/cell adhesion/glycolipid/lectin/ligand binding


    Introduction
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The E- and P-selectin cell adhesion molecules mediate rolling of leukocytes on endothelial cell surfaces prior to firm attachment and extravasation during inflammation. Ca2+-dependent carbohydrate-recognition domains (C-type CRDs) in the selectins interact with sugars linked to glycoproteins and glycolipids on the surface of leukocytes (Figure 1A). The prototype of these ligands is the sialylated, fucosylated tetrasaccharide sialyl-LewisX (sLeX; NeuAc{alpha}2,3Galß1,4[Fuc{alpha}1,3]GlcNAc) (Vestweber and Blanks, 1999Go). E- and P-selectin also bind to 3-sulfated galactose-containing glycolipids (sulfatides) and 3-sulfated glucuronic acid-containing glycolipids (sulfoglucuronyl lipids or SGNLs) (Needham and Schnaar, 1993bGo; Bajorath et al., 1994Go).



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Fig. 1. (A) Structures of selectin ligands. (B) Alignment of E-selectin sequence (top) with MBP sequence (bottom). Regions of E-selectin that have been inserted into MBP to create selectin/MBP chimeras are shaded. Asterisks denote ligands for Ca2+ 1, and plus sign indicates residue 197 of MBP. (C) Sialyl-LewisX-binding site of the region 5 selectin/MBP chimera. Small white, gray, and black spheres represent carbon, nitrogen, and oxygen atoms, respectively. The larger white sphere represents a water molecule, and the large black sphere represents Ca2+. Dashed lines denote Ca2+ coordination bonds and hydrogen bonds. The transparent gray surface indicates the approximate position of region 4 of E-selectin at the front of the structure. This figure has been prepared with MOLSCRIPT using Protein Data Base entry 2 kmb (Kraulis, 1991; Ng and Weis, 1997).

 
In an effort to define the regions of the E-selectin CRD that are necessary and sufficient to obtain binding of selectin ligands to a C-type CRD, the homologous CRD of rat serum mannose-binding protein (MBP) has been modified to display E-selectin–like binding properties (Blanck et al., 1996Go; Torgersen et al., 1998Go). MBP is a C-type lectin that binds D-mannose, N-acetyl-D-glucosamine, and L-fucose but not sLeX. Portions of human E-selectin located near the C-terminus of the CRD have been substituted into the CRD of MBP. Introduction of three lysine residues, designated region 5 of E-selectin (Figure 1B), is enough to induce efficient binding of this selectin/MBP chimera to HL-60 cells and to sLeX-conjugated bovine serum albumin (BSA) (Blanck et al., 1996Go).

The crystal structure of the region 5 chimera complexed with sLeX (Figure 1C) shows that the fucose residue in sLeX binds the chimera at a Ca2+-binding site in much the same way mannose binds MBP, while the carboxylate group of NeuAc is located in the vicinity of the introduced lysine residues (Ng and Weis, 1997Go). Increasingly selectin-like properties were obtained by making three additional changes. Removal of a histidine near the fucose-binding site abolishes mannose binding, whereas introduction of two further portions of E-selectin increases the affinity for sLeX (Figure 1) (Torgersen et al., 1998Go). One of these added segments (region 4) contains a tyrosine residue and an arginine residue known to be important for E-selectin activity, and the other contains a glutamic acid residue that makes a hydrogen bond to one of the lysine residues in region 5 of E-selectin (Erbe et al., 1992Go; Graves et al., 1994Go).

The arrangement of the region 5 chimera suggests that the sLeX-binding site consists of two subsites (Figure 1C). One subsite involves fucose interacting with the Ca2+ in a manner analogous to monosaccharide binding to other C-type CRDs (Weis et al., 1992Go; Poget et al., 1999Go). This Ca2+ site is located in the same position in the structure as the single Ca2+ site in E-selectin. The second ligand-binding subsite appears to involve electrostatic and hydrogen bonding interactions between the sialic acid and galactose portions of sLeX and the lysine side chains in region 5. The electrostatic component of the binding interaction is reflected in the ability of high concentrations of NaCl to inhibit the binding of sLeX to E-selectin and the selectin/MBP chimeras (Koenig et al., 1997Go; Torgersen et al., 1998Go).

The recently described crystal structure of E-selectin complexed with sLeX confirms the extended or two-subsite model, although the details of these interactions are different to those observed in the complex of the region 5 chimera with sLeX (Ng and Weis, 1997Go; Somers et al., 2000Go). In the complex of E-selectin with sLeX, the 3- and 4-hydroxyl (OH) groups of fucose interact with Ca2+, while in the complex with the region 5 chimera, the 2- and 3-OH groups ligate the Ca2+. This change reorients the sialic acid portion of the ligand so that it makes hydrogen bonds and electrostatic interactions with residues in region 4 instead of region 5 (Somers et al., 2000Go).

The differences in the way that sLeX binds to E-selectin and the region 5 E-selectin/MBP chimera suggest that subtle changes in the binding site lead to differences in the preferred orientation of the ligand. The goal of the present work was to attempt to identify which portions of the binding sites in the chimeras are needed to make their biochemical ligand-binding characteristics as much like the selectins as possible. The biochemical binding characteristics of the selectins for both sLeX and sulfated glycolipid ligands can be accurately reproduced in the presence of the Ca2+-dependent subsite with an adjacent ionic strength-sensitive subsite, even though the detailed molecular interactions with the ligand remain different from those seen in the E-selectin crystals. The results indicate that several regions near the primary sugar-binding site of a C-type CRD can participate in binding of complex, anionic glycans.


    Results and discussion
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Effects of ionic strength on sLeX binding
Because the sialic acid moiety of sLeX interacts with two different positively charged regions on the surface of E-selectin and the region 5 E-selectin/MBP chimera, it was of interest to compare the effects of ionic strength on these two binding interactions to see how similar their properties actually are. Previous studies have demonstrated that interaction between the selectins and sLeX is inhibited by salt and that about 50% of the binding activity is retained under physiological salt conditions of roughly 150 mM ionic strength (Stoolman, 1989Go; Koenig et al., 1997Go). For direct comparison with the chimeras, the ionic strength dependence of sLeX interaction with E-selectin was investigated using a mouse E-selectin-immunoglobulin G Fc fusion protein, and 50% inhibition was obtained at an ionic strength of 140 mM. Similar assays showed that binding of the region 5 chimera is more sensitive to NaCl, with 50% inhibition observed at 50 mM NaCl (Table I). The potential role of specific ions in inhibiting interactions between sLeX and the selectin/MBP chimeras was investigated by conducting inhibition assays with several electrolytes. Both Na2SO4 and MgCl2 inhibited binding of 125I-sLeX-BSA to the region 5 chimera with similar characteristics (Figure 2). Thus binding is inhibited by ionic strength rather than by specific ions such as SO42– and probably reflects the strong electrostatic component of the interaction between sLeX and region 5 of the chimera.


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Table I. Inhibition of 125I-sLeX-BSA binding by ionic strength
 


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Fig. 2. Modulation of 125I-sLeX-BSA binding to the region 5 selectin/MBP chimera by ionic strength. Experimental data (dots) are shown along with theoretical curves (lines) fitted to the data (Na2SO4 only). Binding results are normalized to the binding achieved with no added electrolyte.

 
In an effort to analyze the function of individual lysine residues in region 5, the size of region 5 introduced into MBP was reduced to two residues. Although Lys211 makes direct and water-mediated hydrogen bonds to the galactose moiety of sLeX (Figure 1), substituting it with alanine has only a modest effect on sLeX binding (Table II). Removal of Lys213 has a slightly more significant effect, probably reflecting destabilization of the binding site once the hydrogen-bonding partner of Glu197 is lost. Thus it seems that the overall charge in region 5 is the important determinant for sLeX binding, although the exact position of the charged residues in the sequence has some effect. These results are in line with the hypothesis that interactions between sLeX and region 5 involve a substantial electrostatic component.


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Table II. Role of lysine residues in region 5 of selectin/MBP chimeras
 
These experiments were extended to more selectin-like chimeras containing region 4 as well as region 5 of E-selectin. Introduction of region 4 with Gly189 and Glu197 decreases the sensitivity to NaCl so that binding is inhibited by 50% at 90 mM ionic strength. The average inhibition parameters obtained for a variety of electrolytes do not significantly differ from the ones obtained for NaCl only (Table I). These results might suggest that the orientation of the bound ligand in the more selectin-like chimera has changed to resemble the arrangement in E-selectin. However, the crystal structure of this chimera with bound sLeX closely resembles the structure of the region 5 chimera (Feinberg et al., unpublished data). Thus the salt sensitivity of the interaction between sLeX and E-selectin has been reasonably closely mimicked in the chimeras, although the residues involved in these subsites differ.

Role of the accessory Ca2+-binding site in interactions with selectin ligands
A significant difference between the selectin/mannose-binding protein chimeras and E-selectin is the presence of the accessory Ca2+-binding site in MBP, designated Ca2+ site 1, which is not present in E-selectin (Graves et al., 1994Go). The structure of the complex of the region 5 chimera with sLeX suggests that interactions with sLeX are limited to the Ca2+-binding site that is present in both E-selectin and MBP (designated Ca2+ site 2) (Ng and Weis, 1997Go). However, in the light of the differences between the way E-selectin and the region 5 E-selectin/MBP chimera bind sLeX, it was important to demonstrate that Ca2+ site 1 does not play a role in sLeX binding to the chimeras.

To eliminate Ca2+ site 1 from the chimeras, two regions containing three of the four amino acids residues that ligate this Ca2+, designated regions 1 and 2, were replaced by the corresponding sequences from E-selectin (Figure 1). These changes were introduced into the most selectin-like of the previously characterized chimeras, which contains regions 4 and 5 of E-selectin as well as Gly189 and Glu197 (Torgersen et al., 1998Go). The resulting chimera contains regions 1, 2, 4, and 5. Solid phase binding assays demonstrated that removal of Ca2+ site 1 ligands has only a modest effect on binding to 125I-sLeX-BSA. Under conditions where the original protein with Ca2+ site 1 binds 31 ± 3% of input ligand, the new chimera binds 22 ± 3% of input ligand. As expected, this interaction remains Ca2+-dependent due to the critical role of Ca2+ site 2. Removal of Ca2+ site 1 causes only a modest change in sensitivity of ligand binding to ionic strength, rendering it marginally more like E-selectin (Table I).

To confirm that Ca2+ site 1 absent from the new chimera, Ca2+ binding was measured by monitoring changes in intrinsic tryptophan fluorescence (Figure 3A,B). Residues Trp181 and Trp204 are located near the Ca2+-binding sites, and modulation of their intrinsic fluorescence signals reflects local conformational changes resulting from occupancy of the Ca2+-binding sites (Ng and Weis, 1998Go). The KCa determined for the original chimera is 2.3 mM, and the order of the binding reaction is 2.7, which suggests that more than two Ca2+ bind to each CRD. This higher-than-second-order binding might be explained by the presence of a third weak Ca2+-binding site adjacent to Ca2+ site 1 that is observed in crystals of MBP grown at 15 mM Ca2+ (Weis et al., 1992Go; Weis and Drickamer, 1994Go). In contrast to its parent, the chimera containing regions 1, 2, 4, and 5 exhibits first-order binding to Ca2+ (0.8 ± 0.3) with a KCa of 1 mM. These results are consistent with the expected loss of Ca2+ site 1 as well as the adjacent adventitious site. Taken together with the 125I-sLeX-BSA binding data, these results support the notion that Ca2+ 1 does not contribute to the selectin-like properties of the selectin/MBP chimeras. Unfortunately, this chimera has been refractory to crystallization so no direct comparison of the mode of ligand binding can be made (Feinberg et al., unpublished data).



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Fig. 3. Ca2+ binding to selectin/MBP chimeras monitored by changes in tryptophan fluorescence. Experimental data (filled circles) are shown along with theoretical curves (continuous lines) fitted to the data. The KCa and order of reaction are reported as the average ± SD for 2 to 10 experiments.

 
Binding to glycolipid ligands
Because many glycolipid ligands for the selectins lack fucose moieties, it can be imagined that they might bind selectins in a manner very different to the way sLeX binds (Vestweber and Blanks, 1999Go). There is at present no structural information available for selectin-glycolipid complexes, so examination of the behavior of the selectin/MBP chimeras can provide insight into the way glycolipids can be bound by C-type CRDs. The binding to sulfatides was initially investigated in the solid phase binding assay format employed for the ionic strength dependence assays, using a sulfatide head group to inhibit the interaction between 125I-sLeX-BSA and the chimera containing regions 4 and 5. The inhibitory effect of 1-ß-O-azidoethyl-3-O-sulfo-D-galactopyranose (kind gift of Laura Kiessling, University of Wisconsin) was the same as sulfate, which means that the assay reflects inhibition due to ionic strength and not other interactions with the chimeric CRD (data not shown).

In previous studies, selectin binding to sulfatides and other glycolipids was demonstrated using highly multivalent complexes of selectin-immunoglobulin G Fc fusion proteins to bind glycolipid-coated wells (Needham and Schnaar, 1993bGo; Bajorath et al., 1994Go). These results have been reproduced using a mouse E-selectin-immunoglobulin Fc fusion construct and extended to the SGNLs (data not shown). A previous report indicated that the SGNLs do not bind human E-selectin, which may reflect a difference in the behavior of mouse E-selectin used here (Needham and Schnaar, 1993bGo). To test for glycolipid binding by the selectin/MBP chimeras, the chimeras were modified to allow efficient oligomerization. MBP and the chimeras derived from it make up a trimer of CRDs held together by a neck region that forms an {alpha}-helical coiled-coil (Weis and Drickamer, 1994Go). Introduction of a cysteine residue at the junction between the neck and the CRD followed by derivatization with biotin maleimide allows formation of oligomers by binding of multiple trimers to tetravalent 125I-streptavidin (Heise et al., unpublished data).

Binding assays conducted with modified and oligomerized wild-type MBP and with the region 5 chimera revealed that the chimera binds to sulfatide-coated wells in a Ca2+-dependent manner, whereas binding of the wild type CRD is almost undetectable (Figure 4A). Similar results were obtained using plates coated with the more complex SGNL ligand (Figure 4B). Interactions of different chimeras with the glycolipid ligands were compared by quantifying the results of assays like those shown in Figure 4. The amount of each chimera bound was compared to the amount of the region 5 chimera bound at equivalent input concentrations (Table III). The region 5 chimera binds five to eight times better than wild-type MBP, while the binding of the region 4 chimera to glycolipid-coated plates is marginal. These results show that introduction of region 5 but not region 4 of E-selectin into the CRD of MBP is enough to create an efficient glycolipid-binding site.



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Fig. 4. Binding of 125I-streptavidin-biotinylated chimeras to glycolipid-coated plates. Experiments were conducted in the presence of 0.9 mM Ca2+ or 10 mM EDTA. Experimental data for the region 5 chimera (dots), wild-type MBP (WT, squares) and the region 5 chimera in the presence of EDTA (triangles) are shown along with theoretical curves (lines) fitted to the data.

 

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Table III. Binding of selectin/mannose-binding protein chimeras to sulfatides and SGNLs
 
An initial attempt to combine regions 4 and 5 in the presence of the extra cysteine residue used for oligomerization was unsuccessful because this protein is unstable in the bacterial expression system. In contrast, the further modified chimera containing regions 4 and 5 as well as Gly189 and Glu197 was stably produced and could be analyzed. Binding to both sulfatides and SGNLs was significantly enhanced compared to the region 5 chimera (Table III). This increase in binding may originate from several distinct effects. Residues of region 4 may make favorable interactions with glycolipid ligands although they are not able by themselves to sustain efficient binding to the glycolipids. On the other hand, insertion of Glu197, which forms a hydrogen bond with one of the lysine residues of region 5, stabilizes this portion of the binding site and therefore may increase the affinity indirectly. Similarly, removal of His189 may eliminate an unfavorable interaction. Binding of the chimera containing regions 4 and 5 compared to the chimera containing region 5 is enhanced by a factor of 2 for the sulfatides and a factor of 10 for the SGNLs. Because SGNLs bear a pentasaccharide head group, compared to the simple monosaccharide head group of sulfatides, these ligands may make more extensive contacts with portions of the protein surface including region 4 as well as region 5.

Role of the ionic strength-sensitive subsite in glycolipid binding
The role of the ionic strength-sensitive subsite in binding to glycolipids is reflected in the sensitivity of glycolipid binding to increasing NaCl concentrations (Table III). The concentrations of salt that result in 50% inhibition of binding are mostly higher than the concentrations needed to inhibit sLeX binding and are lower for SGNL binding than sulfatide binding. Values obtained with the region 5 chimera and the chimera containing regions 4 and 5 are similar to those obtained for E-selectin. The selectin/MBP chimera containing regions 4 and 5 is a particularly good mimic of E-selectin binding to SGNLs.

The contributions of charge to interactions with sulfatides and SGNLs differ. Introduction of additional E-selectin regions into the region 5 chimera does not significantly change the ionic strength inhibition parameters for sulfatides. However, for SGNLs, inclusion of selectin sequences in addition to region 5 decreases the ionic strength sensitivity of the binding interaction and makes it comparable to the natural E-selectin value. These results are consistent with the suggestion that SGNLs make more ionic strength-independent contacts with the protein surface than sulfatides do.

Role of Ca2+ site 2 in glycolipid binding
E-selectin binds the nonfucosylated sulfatides and SGNLs in a Ca2+-dependent manner, which seems surprising because the Ca2+-binding site mediates critical and specific interactions with the fucose residue in sLeX (Needham and Schnaar, 1993bGo; Somers et al., 2000Go). The importance of the Ca2+-dependent subsite in binding to sLeX and other ligands was investigated by selectively modifying Ca2+ site 2. Previous studies of MBP and E- and P-selectin have demonstrated that changing one of the Ca2+ site 2 ligands from asparagine to aspartate destabilizes binding to sugar ligands, reflecting the importance of this asparagine residue in hydrogen bonding to sugar hydroxyl groups (Figure 1) (Weis et al., 1992Go; Graves et al., 1994Go; Bajorath et al., 1994Go). Therefore, an equivalent mutation was introduced into the region 5 chimera by changing Asn187 to Asp.

The chimera containing the N187D mutation does not bind Man-Sepharose or invertase-Sepharose. Solid phase binding assays demonstrated that this new chimera also exhibits little or no binding to 125I-sLeX-BSA under conditions where its parent binds efficiently (Figure 5). The mutant CRD is resistant to subtilisin digestion in the presence of Ca2+, like the CRDs of E-selectin and MBP, confirming that it is correctly folded and that Ca2+ site 2 is intact (data not shown) (Weis et al., 1992Go; Graves et al., 1994Go). The presence of Ca2+ site 2 was further demonstrated by studies in which Ca2+ binding was monitored using fluorescence (Figure 3C). The KCa of 2.8 mM and an order of binding of 2.5 are comparable to the parameters obtained for binding of Ca2+ to the chimera containing regions 4 and 5, strongly suggesting that this CRD is correctly folded in spite of the N187D mutation and that its Ca2+-binding properties have been conserved. Thus mutation of an asparagine to an aspartate in Ca2+-binding site 2 of the region 5 chimera does not alter its Ca2+-binding properties but abolishes sLeX binding. The phenotype of this mutant reflects the importance of the interactions made by fucose at Ca2+ site 2, confirming that the region 5 chimera resembles E-selectin in this respect.



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Fig. 5. Effect of the mutation N187D on the ligand-binding properties of the region 5 selectin/MBP chimera. Experimental data obtained for 125I-sLeX-BSA (dots) are shown along with theoretical curves (lines) fitted to the data.

 
The effect of the N187D mutation is to reduce the binding to sulfatides and SGNLs by a factor of 2 to 3 (Table III). This decrease suggests that residues in or near Ca2+ site 2 do play a role in glycolipid binding. However, compared to the 15-fold reduction in sLeX binding, the effect on glycolipid binding is modest. It seems unlikely that this decrease in affinity originates from changes in the position of Ca2+ because the Ca2+-binding properties of the modified region 5 chimera are unchanged compared to the chimera containing regions 4 and 5 (Figure 3A,C). Instead, this reduction of binding may indicate that the amido group of Asn187 directly contacts a portion of the sulfatide or SGNL molecule bound to the chimera. Such a contact would be sensitive to a change in the conformation of the asparagine side chain like the one caused when Ca2+ is removed from the binding site (Ng et al., 1998Go). These results would explain the Ca2+ dependence of the glycolipid-binding interaction. Similar mutation of a Ca2+-binding asparagine residue in P-selectin reduces sulfatide binding by a factor of three to four compared to wild type (Bajorath et al., 1994Go). These observations establish a further parallel between the properties of the Ca2+-dependent subsites of E-selectin and the selectin/MBP chimeras in the interaction with glycolipids.

Conclusions
The results presented here suggest that the sLeX-binding site engineered in the CRDs of selectin/MBP chimeras can be dissected into two subsites, which have biochemical properties similar to those of the natural E-selectin. A strong parallel between the binding of sLeX and selectin glycolipid ligands to the chimeric CRDs can be established, which extends the two-subsite model to the interaction with these sulfated, nonfucosylated ligands. Although it is not possible to confirm that the mechanisms of glycolipid binding to the CRDs of E-selectin and the chimeras are identical, the evidence shown here exemplifies how a C-type CRD accommodates structurally diverse ligands using similar binding sites. This study also suggests that the binding of oligosaccharides to C-type CRDs does not always involve the canonical monosaccharide-Ca2+-protein interactions usually described in these domains (Weis et al., 1992Go; Poget et al., 1999Go).

These results provide a basis for identification of potential accessory binding sites in other C-type CRDs. The possibility that either region 4 or region 5 can support ionic strength-sensitive binding to anionic saccharide ligands must be considered when sequences of CRDs derived from genomic sequence are examined. For example, the presence of appropriately spaced basic residues in each of these regions in some Drosophila CRDs (Dodd and Drickamer, 2001Go) may suggest the potential for binding anionic glycans through two-site interactions of the type described here.


    Materials and methods
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
Restriction enzymes were purchased from New England BioLabs. Yeast invertase, biotin maleimide, streptavidin, sulfatides, biotinylated F(ab')2 fragment of goat anti-human immunoglobulin G ({gamma}-chain specific) were obtained from Sigma Chemical Co. sLex12.6-BSA was purchased from Oxford GlycoSciences. Na125I was purchased from Amersham Pharmacia Biotech. Immulon 4 polystyrene wells were from Dynex Technologies. Goat anti-human immunoglobulin G (Fc specific) was from Serotec. Chelex 100 resin was purchased from BioRad Laboratories. Anatop 10 filters (0.1 µm) were obtained from Whatman. SGNLs purified from dog sciatic nerve endoneurium were the kind gift of Ronald Schnaar (Johns Hopkins University School of Medicine) (Needham and Schnaar, 1993aGo).

Mutagenesis
Mutations were introduced into the MBP-A cDNA (CRD and coiled-coil neck region) by substitution of double-stranded synthetic oligonucleotides in place of restriction fragments as previously described (Blanck et al., 1996Go; Torgersen et al., 1998Go). For expression, the modified cDNAs were substituted into the expression vector pINIIIompA2 used for expression of the wild-type CRD. To introduce the mutation K109C to allow subsequent biotinylation at this position (see below), the expression vector was modified so that the sequence of bases 15 to 59 (numbered from the first NcoI site) was CCGGATGTAAGTTCTTTGTGACCAACCATGAAAGGATGCCCTTTT. All mutations were confirmed using a Perkin-Elmer ABI Prism 310 DNA sequencer.

Protein expression and purification
All chimeras were prepared by induction with isopropyl-ß-D-thiogalactopyranoside in the presence of Ca2+ and sonication to release the correctly folded protein from the periplasm as described previously (Torgersen et al., 1998Go). In most cases, purification by affinity chromatography on Man-Sepharose or invertase-Sepharose was followed by reverse-phase chromatography on a C4 column (50 x 4.60 mm, Jupiter series from Phenomenex) (Torgersen et al., 1998Go).

The inability of the region 5 chimera modified by the change N187D to bind to either Man-Sepharose or invertase-Sepharose led to a modification of the final purification steps. Expression and initial purification were the same as for all the other chimeras until the affinity chromatography step. At this stage, 100 ml of cleared bacterial lysate in loading buffer (1.25 M NaCl, 25 mM CaCl2, 25 mM Tris–Cl, pH 7.8) was submitted to ammonium sulfate fractionation. The protein pellet from the 40% to 80% fraction was resuspended in 20 ml of 50 mM Tris–Cl, pH 8.0, and 1 mM CaCl2 and dialyzed extensively against the same buffer to remove traces of ammonium sulfate. The protein was then purified on a MonoQ HR 5/5 anion exchange column under the starting conditions of 50 mM Tris–Cl, pH 8.0, and 1 mM CaCl2 and eluted with a gradient of 50 mM Tris–Cl, pH 8.0, 1 mM CaCl2, and 500 mM NaCl, at a rate of 3.33%/min. Fractions containing the mutant protein were identified by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subjected to further purification by reverse-phase chromatography. The mutant region 5 chimera was loaded under starting conditions of 10% acetonitrile and 0.1% trifluoroacetic acid then eluted with a gradient of 6.25%/min for 4 min and 0.25%/min for 40 min instead of a 1.25%/min gradient over 40 min used for all the other chimeric CRDs. Fractions were dried for 30 min in a Savant Speed-Vac concentrator to remove acetonitrile and lyophilized.

Biotinylation
Chimeras produced in the Cys109-containing vector, freshly eluted from the affinity column, were made 25 mM in CaCl2 and reacted for 60 min at room temperature with biotin maleimide at a final concentration of 0.1 mg/ml. Biotinylated proteins were recovered by affinity chromatography on Man-Sepharose or invertase-Sepharose and further purified by reverse-phase high-performance liquid chromatography (HPLC). The region 5 chimera with the further N187D mutation was biotinylated by addition of biotin maleimide directly into the cleared bacterial lysate to a final concentration of 0.1 mg/ml. After a 60 min reaction at room temperature, the solution was centrifuged in a Ti55.2 rotor at 100,000 x g and dialyzed against loading buffer containing 25 mM Tris–Cl, pH 6.8 instead of pH 7.8. The rest of the purification was carried out by reverse-phase HPLC following the protocol described above for this chimera.

Production of a mouse E-selectin-immunoglobulin G Fc fusion
Chinese hamster ovary cells expressing a mouse E-selectin CRD, the epidermal growth factor–like domain, and the first two complement repeats fused to the hinge region of human IgG1 were the kind gift of Dietmar Vestweber (University of Münster) (Hahne et al., 1993Go).

Radioiodination of proteins
sLex-BSA (50 µg) and streptavidin (0.2 mg) were iodinated by the chloramine T method using 1 mCi of Na125I (Greenwood et al., 1963Go).

Binding assays
Binding assays with 125I-sLex-BSA were performed as described (Blanck et al., 1996Go; Torgersen et al., 1998Go). Results are reported as the average ± SD for three experiments, each performed in duplicate.

Ionic strength inhibition assays
Aliquots (50 µl) of chimeric CRDs in loading buffer (0.1 mg/ml) were pipetted into Immulon 4 wells and incubated overnight at 4°C. Protein solutions were removed and wells were blocked by 5% BSA in loading buffer for 2 h at 4°C. Wells were washed with cold low salt–HEPES buffer (1 mM CaCl2, 20 mM Na-HEPES, pH 7.5). Aliquots (100 µl) of electrolyte solutions in low salt–HEPES buffer containing 1 mg/ml BSA and 125I-sLex-BSA (approximately 0.5 µg/ml) were added to the CRD-coated wells. After 2 h at 4°C, wells were washed three times with cold low salt–HEPES buffer and counted in a Wallac Wizard {gamma} counter. Ionic strength I was calculated using the equation I = 0.5 · {Sigma} (ci · zi2), where ci is the molar concentration and zi is the charge of species i. The salt sensitivity of the interaction between E-selectin and sLeX was measured using a modified version of the assay described above. Immulon 4 wells were coated overnight at 4°C with 50 µl of goat anti-human immunoglobulin G (Fc specific) in Tris–saline (150 mM NaCl, 10 mM Tris–Cl, pH 7.4). Wells were washed with cold Tris-saline, blocked for 2 h at 4°C by 5% BSA in Tris–saline, and washed with cold low salt–HEPES buffer. Aliquots (100 µl) of E-selectin-immunoglobulin G Fc fusions (about 2 µg/ml) in low salt–HEPES buffer supplemented with 1 mg/ml BSA were added to the wells. After 2 h incubation at 4°C, wells were washed with cold low salt–HEPES buffer. At this stage, aliquots of electrolyte solutions containing 125I-sLex-BSA were added to the wells as described, and the remainder of the protocol was identical to that used for the chimeric CRDs.

Ca2+-binding fluorescence assays
Protein at 1 µM in Ca2+-free and Mg2+-free HEPES-buffered saline (136 mM NaCl, 2.7 mM KCl, 19 mM Na-HEPES, pH 7.5, passed through Chelex 100 resin) was filtered through a 0.1-µm Anatop 10 filter. CaCl2 was added and samples were allowed to equilibrate for 1 h at 37°C. Emission spectra were recorded for 30 s on a JASCO FP-777 spectrofluorimeter ({lambda}exc = 295 nm with 10 nm band-pass, {lambda}em = 340 nm with 5 nm band-pass) with a 150 W lamp. The temperature of the cuvette was maintained at 37°C. Measurements were corrected by subtracting the signal obtained with an aliquot of the reaction buffer.

Glycolipid binding assays
Aliquots (50 µl) of sulfatides (1 µM) or SGNLs (0.4 µM) in methanol were pipetted into Immulon 4 wells. Contents of wells were allowed to dry overnight. The wells were rinsed with loading buffer and blocked by 5% BSA in loading buffer for 2 h at room temperature. The plate was rinsed three times with a cold solution of HEPES-buffered saline (136 mM NaCl, 2.7 mM KCl, 0.9 mM CaCl2, 0.5 mM MgCl2, 19 mM Na-HEPES, pH 7.5). Multivalent complexes were formed by mixing 18 µg of biotinylated selectin/MBP chimeras, 4 µg of 125I-streptavidin, and 12 µg of streptavidin in 200 µl of HEPES-buffered saline supplemented with 1% BSA. The mixture was incubated 30 min at 37°C to allow complex formation. In the case of the E-selectin Fc fusion, 2 µg of protein was added to a mixture of 6 µg of a F(ab')2 fragment of a goat anti-human immunoglobulin G biotin conjugate, 1.25 µg of 125I-streptavidin, and 5 µg of streptavidin in 200 µl of HEPES-buffered saline supplemented with 1% BSA. Complex solutions were diluted in HEPES-buffered saline supplemented with 1% BSA, and aliquots (100 µl) added to glycolipid-coated wells. After incubation for 2 h at 4°C, wells were rinsed three times with cold HEPES-buffered saline and counted.

Ionic strength inhibition assays were carried out according to the same protocol using low salt-HEPES buffer instead of HEPES-buffered saline. Aliquots (100 µl) of complex solutions (100 nM input concentration) prepared with increasing concentrations of NaCl were added to glycolipid-coated wells and plates were incubated for 2 h at 4°C. Plates were then washed with low salt–HEPES buffer and counted.

Data analysis
Data were fitted to appropriate equations using the nonlinear least squares fitting program SigmaPlot from Jandel Scientific. Direct binding curves were fitted to an equation describing saturable binding superimposed with a linear increase of non-specific binding (Simpson et al., 1999Go). Results for ionic strength assays were fitted to the equation: Fraction of maximal binding = e–(ln2/I50) • I, where I50 is the ionic strength when binding is 50% of the maximal value and I is the ionic strength. Results reported are the average ± SD for two to five experiments, each performed in duplicate. Data from the fluorescence experiments were fitted as previously described (Ng and Weis, 1998Go).


    Acknowledgments
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We thank Ronald Schnaar for providing the sulfoglucuronyl lipids, Dietmar Vestweber for providing the Chinese hamster ovary cell line expressing the mouse E-selectin-immunoglobulin Fc fusion protein, Laura Kiessling for giving us the sulfatide head group analog, and Stephanie Wragg for help with the fluorescence experiments. We are also grateful to Bill Weis and Maureen Taylor for comments on the manuscript. This work was supported by grant 041845 from the Wellcome Trust and a Wellcome Prize Studentship to Samuel Bouyain.


    Abbreviations
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
BSA, bovine serum albumin; CRD, carbohydrate-recognition domain; HPLC, high-performance liquid chromatography; MBP, mannose-binding protein; sLeX, sialyl-LewisX; SGNL, sulfoglucuronyl lipid.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results and discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Bajorath, J., Hollenbaugh, D., King, G., Harte, W. Jr., Eustice, D.C., Darveau, R.P., and Aruffo, A. (1994) CD62/P-Selectin binding sites for myeloid cells and sulfatides are overlapping. Biochemistry, 33, 1332–1339.[ISI][Medline]

Blanck, O., Iobst, S.T., Gabel, C., and Drickamer, K. (1996) Introduction of selectin-like binding specificity into a homologous mannose-binding protein. J. Biol. Chem., 271, 7289–7292.[Abstract/Free Full Text]

Dodd, R.B. and Drickamer, K. (2001) Lectin-like proteins in model organisms: implications for evolution of carbohydrate-binding activity. Glycobiology, 11, R71–R79.

Erbe, D.V., Wolitzky, B.A., Presta, L.G., Norton, C.R., Ramos, R.J., Burns, D.K., Rumberger, J.M., Rao, B.N.N., Foxall, C., Brandley, B.K., and Lasky, L.A. (1992) Identification of an E-selectin region critical for carbohydrate recognition and cell adhesion. J. Cell Biol., 119, 215–227.[Abstract]

Graves, B.J., Crowther, R.L., Chandran, C., Rumberger, J.M., Li, S., Huang, K.-S., Presky, D.H., Familletti, P.C., Wolitzky, B.A., and Burns, D.K. (1994) Insight into E-selectin/ligand interaction from the crystal structure and mutagenesis of the lec/EGF domains. Nature, 367, 532–538.[ISI][Medline]

Greenwood, F.C., Hunter, W.M., and Glover, J.S. (1963) The preparation of 131I-labelled human growth hormone of high specific radioactivity. Biochem. J., 89, 114–123.[ISI]

Hahne, M., Jager, U., Isenmann, S., Hallmann, R., and Vestweber, D. (1993) Five tumor necrosis factor-inducible cell adhesion mechanisms on the surface of mouse endothelioma cells mediate the binding of leukocytes. J. Cell Biol., 121, 655–664.[Abstract]

Koenig, A., Jain, R., Vig, R., Norgard-Sumnicht, K.E., Matta, K.L., and Varki, A. (1997) Selectin inhibition: synthesis and evaluation of novel sialylated, sulfated and fucosylated oligosaccharides, including the major capping group of GlyCAM-1. Glycobiology, 7, 79–93.[Abstract]

Kraulis, P.J. (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr., 24, 946–950.[ISI]

Needham, L.K. and Schnaar, R.L. (1993a) Carbohydrate recognition in the peripheral nervous system: a calcium-dependent membrane binding site for HNK-1 reactive glycolipids potentially involved in Schwann cell adhesion. J. Cell Biol., 121, 397–408.[Abstract]

Needham, L.K. and Schnaar, R.L. (1993b) The HNK-1 reactive sulfoglucuronyl glycolipids are ligands for L-selectin and P-selectin but not E-selectin. Proc. Natl Acad. Sci. USA, 90, 1359–1363.[Abstract]

Ng, K.K.S. and Weis, W.I. (1997) Structure of a selectin-like mutant of mannose-binding protein complexed with sialylated and sulfated Lewis oligosaccharides. Biochemistry, 36, 979–988.[ISI][Medline]

Ng, K.K.-S. and Weis, W.I. (1998) Coupling of prolyl peptide bond isomerization and Ca2+ binding in a C-type mannose-binding protein. Biochemistry, 37, 17977–17989.[ISI][Medline]

Ng, K.K.-S., Park-Snyder, S., and Weis, W.I. (1998) Ca2+-Dependent structural changes in C-type mannose-binding proteins. Biochemistry, 37, 17965–17976.[ISI][Medline]

Poget, S.F., Legge, G.B., Proctor, M.R., Butler, P.J.G., Bycroft, M., and Williams, R.L. (1999) The structure of a tunicate C-type lectin from Polyandrocarpa misakiensis complexed with D-galactose. J. Mol. Biol., 290, 867–879.[ISI][Medline]

Simpson, D.Z., Hitchen, P.G., Elmhirst, E.L., and Taylor, M.E. (1999) Multiple interactions between pituitary hormones and the mannose-receptor. Biochem. J., 343, 403–411.[ISI][Medline]

Somers, W.S., Tang, J., Shaw, G.D., and Camphausen, R.T. (2000) Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLe(X) and PSGL-1. Cell, 103, 467–479.[ISI][Medline]

Stoolman, L.M. (1989) Adhesion molecules controlling lymphocyte migration. Cell, 56, 907–910.[ISI][Medline]

Torgersen, D., Mullin, N.P., and Drickamer, K. (1998) Mechanism of ligand binding to E- and P-selectin analyzed using selectin/mannose-binding protein chimeras. J. Biol. Chem., 273, 6254–6261.[Abstract/Free Full Text]

Vestweber, D. and Blanks, J.E. (1999) Mechanisms that regulate the function of the selectins and their ligands. Physiol. Rev., 79, 181–213.[Abstract/Free Full Text]

Weis, W.I. and Drickamer, K. (1994) Trimeric structure of a C-type mannose-binding protein. Structure, 2, 1227–1240.[ISI][Medline]

Weis, W.I., Drickamer, K., and Hendrickson, W.A. (1992) Structure of a C-type mannose-binding protein complexed with an oligosaccharide. Nature, 360, 127–134.[ISI][Medline]