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.
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Abstract |
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Key words: carbohydrate recognition/cell adhesion/glycolipid/lectin/ligand binding
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Introduction |
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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, 1997). 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., 1998
). 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., 1992
; Graves et al., 1994
).
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., 1992; Poget et al., 1999
). 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., 1997
; Torgersen et al., 1998
).
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, 1997; Somers et al., 2000
). 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., 2000
).
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.
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Results and discussion |
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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., 1994). 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, 1997
). 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., 1998). 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, 1998). 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., 1992
; Weis and Drickamer, 1994
). 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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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, 1993b; Bajorath et al., 1994
). 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, 1993b
). 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
-helical coiled-coil (Weis and Drickamer, 1994
). 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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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, 1993b; Somers et al., 2000
). 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., 1992
; Graves et al., 1994
; Bajorath et al., 1994
). 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., 1992; Graves et al., 1994
). 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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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., 1992; Poget et al., 1999
).
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, 2001) may suggest the potential for binding anionic glycans through two-site interactions of the type described here.
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Materials and methods |
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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., 1996; Torgersen et al., 1998
). 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., 1998). 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., 1998
).
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 TrisCl, 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 TrisCl, 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 TrisCl, pH 8.0, and 1 mM CaCl2 and eluted with a gradient of 50 mM TrisCl, 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 sulfatepolyacrylamide 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 TrisCl, 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 factorlike 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., 1993).
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., 1963).
Binding assays
Binding assays with 125I-sLex-BSA were performed as described (Blanck et al., 1996; Torgersen et al., 1998
). 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 saltHEPES buffer (1 mM CaCl2, 20 mM Na-HEPES, pH 7.5). Aliquots (100 µl) of electrolyte solutions in low saltHEPES 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 saltHEPES buffer and counted in a Wallac Wizard counter. Ionic strength I was calculated using the equation I = 0.5 ·
(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 Trissaline (150 mM NaCl, 10 mM TrisCl, pH 7.4). Wells were washed with cold Tris-saline, blocked for 2 h at 4°C by 5% BSA in Trissaline, and washed with cold low saltHEPES buffer. Aliquots (100 µl) of E-selectin-immunoglobulin G Fc fusions (about 2 µg/ml) in low saltHEPES 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 saltHEPES 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 (exc = 295 nm with 10 nm band-pass,
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 saltHEPES 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., 1999). 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, 1998
).
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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