From the Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom
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ABSTRACT |
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The mechanism of oligosaccharide binding to the selectin cell adhesion molecules has been analyzed by transferring regions of the carbohydrate-recognition domains of E- and P-selectin into corresponding sites in the homologous rat serum mannose-binding protein. Insertion of two basic regions and an adjacent glutamic acid residue leads to efficient binding of HL-60 cells and sialyl-Lewisx-conjugated serum albumin. Substitution of glycine for a histidine residue known to stabilize mannose in the binding site of wild type mannose-binding protein results in dramatic loss of affinity for mannose without decreasing binding to sialyl-Lewisx. The accumulated effect of these changes is to alter the ligand binding selectivity of the domain so that it resembles E- or P-selectin more closely than it resembles the parental mannose-binding domain. Affinity labeling using sialyl-Lewisx in which the sialic acid has been mildly oxidized has been used to verify this switch in specificity and to show that the sialic acid-containing portion of the ligand interacts near the sequence Lys-Lys-Lys corresponding to residues 111-113 of E-selectin. The binding of sialyl-Lewisx-serum albumin is inhibited dramatically at physiological and higher salt concentrations, consistent with a significant electrostatic component to the binding interaction. The binding characteristics of these gain-of-function chimeras suggest that they contain many of the selectin residues responsible for selective ligand binding.
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INTRODUCTION |
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The selectin cell adhesion molecules mediate initial binding
interactions between moving leukocytes and stationary endothelia (1).
The interaction of E- and P-selectins on endothelial cells with
saccharide ligands on leukocytes represents a well characterized example of a carbohydrate-based cell-cell recognition mechanism. Among
the best counter-receptors for the selectins that have been identified
to date are derivatives of either the Lewisx trisaccharide,
Gal1
4(Fuc
1
3)GlcNAc, or of the Lewisa
trisaccharide, Gal
1
3(Fuc
1
4)GlcNAc, which are sialylated
and/or sulfated on the 3 position of the terminal galactose residue
(2).
The sugar binding activity of each selectin has been mapped to an NH2-terminal C-type carbohydrate-recognition domain (CRD)1 that is homologous to carbohydrate-binding domains in other calcium-dependent animal lectins (3). The structures of this module and the adjacent epidermal growth factor-like domain have been determined by x-ray crystallography (4). No complex with a saccharide ligand has been reported. However, complexes of sugar ligands with CRDs from two other members of the C-type animal lectin family have been deduced by x-ray crystallography (5, 6). These studies of rat serum and liver mannose-binding proteins (MBPs) reveal that terminal mannose, N-acetylglucosamine, or fucose residues interact directly with the protein as well as indirectly through a bound Ca2+.
Modification of a cluster of three lysine residues near the COOH terminus of the E-selectin CRD has been shown to decrease or abolish binding to leukocytic cells as well as to 3'-sialyl-Lewisx (sLex) test ligand (4, 7-10). In previous studies, a gain-of-function mutation in the CRD of MBP was created by incorporation of this cluster of basic residues (11). The resulting chimeric CRD binds to both mannose and to sLex on HL-60 cells and conjugated to BSA. Subsequent structural analysis (12) revealed that the fucose residue of sLex binds to this mutant CRD in much the same way as mannose binds to the wild type domain, forming direct coordination bonds to Ca2+ and hydrogen bonds to amino acid side chains that serve as additional coordination ligands for this Ca2+. The Lewisx portion of the ligand has the same conformation as observed in solution by NMR studies and in crystals of the free oligosaccharide (for review, see Ref. 13), but the Gal and GlcNAc residues make only limited contact with the protein. The appended NeuAc residue is rotated approximately 180° from the orientation observed in solution, as predicted from previous transferred NOE analysis of ligand bound to the native E-selectin CRD (13). This residue does not make direct contact with the protein but is located in the vicinity of the incorporated cluster of lysine residues.
To probe further the mechanism of ligand binding to the selectins, additional gain-of-function mutations of MBPs have been created and analyzed. Binding and affinity labeling experiments demonstrate that a switch of preferred ligand can be achieved so that the mutants are closer to the selectins than to the parental MBPs in ligand binding specificity. The effect of changes in the lysine cluster and the sensitivity of binding to salt concentration suggests that electrostatic attractions may play an important role in initiating or stabilizing the binding interaction.
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EXPERIMENTAL PROCEDURES |
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Mutagenesis-- Mutant cDNAs were generated by substitution of synthetic double-stranded oligonucleotides, purchased from GenoSys or Applied Biosystems, for restriction fragments excised from the wild type cDNA for MBP. The mutations were created in an SacI to BamHI fragment of the cDNA in the vector pGEM-3 (14) and were then transferred into the cDNA in the expression vector pINIIIompA2 containing the wild type CRD (15). All mutations were confirmed by DNA sequence analysis using a Sequenase II kit from Amersham. Other reagents for molecular biology were purchased from New England BioLabs.
Protein Expression and Purification--
Proteins were expressed
in Escherichia coli strain JA221 grown in the presence of
Ca2+ to allow isolation of correctly folded and
disulfide-bonded CRD directly from the periplasm. LB medium (500 ml)
containing 50 µg/ml ampicillin was innoculated with 10 ml of an
overnight culture and grown at 23 °C shaking at 200 rpm until the
A550 nm reached 0.8 (6-8 h). After the
addition of isopropyl--D-thiogalactopyranoside to a
final concentration of 40 µM and CaCl2 to a
concentration of 100 mM, incubation was continued at
23 °C for a further 16 h. Cells were harvested by
centrifugation at 3,600 × g for 10 min in a Beckman
JS-4.3 rotor and resuspended in 40 ml of loading buffer (1.25 M NaCl, 25 mM CaCl2, 25 mM Tris-Cl, pH 7.8). Sonication was performed with the
microprobe of a Branson model 250 sonifier for five bursts of 1 min
interspersed with cooling on ice. The extract was centrifuged for 10 min at 11,000 × g in a Beckman JA-21 rotor and then
1 h at 100,000 × g in a Ti55.2 rotor. The final
supernatant was passed over a 1-ml column of Man-Sepharose or
invertase-Sepharose, which was washed with 5 ml of loading buffer and
eluted with 5 aliquots of 0.5 ml of eluting buffer (1.25 M
NaCl, 2.5 mM EDTA, 25 mM Tris-Cl, pH 7.8).
Man-Sepharose was prepared by the method of Fornstedt and Porath (16),
and invertase-Sepharose was prepared following exactly the same
protocol but substituting a 20% (w/v) solution of yeast invertase,
obtained from Sigma Chemical Company, for the mannose.
Binding Assays--
Protein was adjusted to a concentration of
approximately 0.1 mg/ml in loading buffer before coating into Immulon 4 Removawell strips from Dynex Technologies. Wells were coated with CRD
and blocked with BSA, and HL-60 cell binding assays were conducted as
described previously (11). 125I-sLex-BSA was
prepared by the chloramine-T method (17) using 50 µg of
sLex-BSA (containing 12.6 mol of conjugated
oligosaccharide/mol of BSA; from Oxford GlycoSciences) and 1 mCi of
Na125I from Amersham. For standard binding assays, ligand
was diluted to a concentration of approximately 1 µg/ml in
HEPES-buffered saline (136 mM NaCl, 2.7 mM KCl,
0.5 mM MgCl2, 0.9 mM
CaCl2, 19 mM Na-HEPES, pH 7.5) containing 0.1 mg/ml BSA. Ligand solution (50 µl) was incubated in CRD-coated wells
for 2 h at room temperature and then removed by aspiration
followed by three washes with 200 µl/well HEPES-buffered saline
containing 0.1 mg/ml BSA. Separated wells were counted in a Wallac
Wizard counter. Results reported for both HL-60 cell and
S-Lex-BSA binding assays are the average ± S.D. for
3-28 experiments, each performed in triplicate. Background values
obtained in the presence of 10 mM EDTA were always less
than 1% of values in the absence of EDTA.
Affinity Labeling-- sLex obtained from Oxford GlycoSciences was labeled with [3H]NaBH4 (NEN Life Science Products) and purified by washing after spotting on Whatman 3MM chromatography paper by published procedures (18). An aliquot (60 nmol) of lyophilized oligosaccharide was dissolved in 100 µl of 100 mM sodium acetate, pH 5.6, containing 5 mM NaIO4 and reacted for 10 min on ice (19). The reaction was quenched by the addition of 100 µl of 10 mM ethylene glycol followed by further incubation on ice for 10 min. The oxidized oligosaccharide was separated from reagent by chromatography on a 2.5-ml column (0.8 × 5 cm) of Sephadex G-10 eluted with water. Fractions of 100 µl were collected, and eluted oligosaccharide was detected by scintillation counting of 2-µl aliquots. Aliquots of pooled oligosaccharide in approximately 300 µl were mixed immediately with protein (at a concentration of approximately 50 µg/ml) in the presence of 25 mM Na-HEPES, pH 7.8, containing 10 mM CaCl2. NaCNBH4 was added to a final concentration of 10 mM. After incubation for 10 min at room temperature, the protein was precipitated by the addition of 0.5 volume of 30% (w/v) trichloroacetic acid and incubation on ice for 10 min. The precipitated protein was collected by centrifugation for 5 min at 16,000 × g and washed twice with 100-µl aliquots of 50:50 ethanol:ether with intermediate spins of 2 min at 16,000 × g. After drying under vacuum, the protein was dissolved directly in SDS-polyacrylamide gel sample buffer containing 1% 2-mercaptoethanol, incubated 5 min at 100 °C, and resolved on a 17.5% polyacrylamide gel (20). The gel was stained with Coomassie Blue and then treated by Amplify solution (Amersham) for fluorography. After exposure to Kodak XAR-5 film for 1-8 days, protein on the stained gel and the radioactive bands were quantified using a Bio-Rad model 620 densitometer. Data were imported into the Origin data analysis program (Microcal) for integration.
For hydroxylamine treatment, protein at a concentration of approximately 0.4 mg/ml was labeled using a reagent concentration of 60 µM and precipitated as described above. The protein was dissolved in 0.5 ml of 6 M guanidine hydrochloride and 100 mM Tris-Cl, pH 8.5, containing 100 mM 2-mercaptoethanol and reacted for 30 min at 37 °C. Iodoacetamide was added to a final concentration of 70 mM, and alkylation was allowed to proceed for 30 min. After dialysis against two changes of 400 volumes of water, the alkylated protein was lyophilized. Hydoxylamine reagent was prepared at a concentration of 2 M in the presence of 2 M guanidine hydrochloride and 0.2 M potassium carbonate and adjusted to pH 10 with NaOH. Reagent was added directly to the dried protein and incubated for 6 h at 45 °C (21). Controls were performed by incubation for 6 h at 0 °C and 45 °C in 2 M guanidine hydrochloride and 0.1 M potassium carbonate adjusted to pH 10. After incubation, all reactions were dialyzed against two changes of 1,000 volumes of water and lyophilized. Aliquots were analyzed by SDS-polyacrylamide gel electrophoresis and fluorography as described above. ![]() |
RESULTS |
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Incorporation of Additional Regions of the Selectin CRDs-- The effects of incorporating three different regions of E-selectin into the CRD of MBP have been investigated previously (11). The regions, two of which are shown in Fig. 1, were selected based on earlier mutagenesis studies conducted with E-selectin because changes in these regions lead to loss of binding activity (4, 7, 22). Only region 5, when tested by itself, is able to confer HL-60 cell binding activity onto MBP. In an attempt to improve the binding of MBP to these cells, further chimeras have been constructed in which both regions 4 and 5 are included. Because E- and P-selectin differ substantially in this region, sequences corresponding to both were tested. The results (Table I) indicate that when the three lysine residues of region 5 are present, E-selectin region 4 improves the HL-60 cell binding activity, whereas region 4 of P-selectin actually causes a loss of binding activity.
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Affinity Labeling-- The failure of the mutants containing glycine at position 189 to bind to Man-Sepharose suggested that the combination of this change with the insertion of lysine residues in region 5 results in a change in the preferred ligand for the CRD of MBP from mannose to sLex. The limited quantities of sLex and related ligands available make competition binding assays in the usual format impractical, so an alternative approach using affinity labeling to compare affinities was developed. Previous studies have demonstrated that the interaction between L-selectin and its ligand can be stabilized by mild oxidation of the ligand resulting in removal of C-8 and C-9 of NeuAc and generation an of aldehyde group at C-7 (25, 26). Upon binding, this group forms a Schiff's base with lysine residues in the selectins, which can then be stabilized by reduction with cyanoborohydride. To create an affinity label, this approach was applied to sLex oligosaccharide labeled reductively in the GlcNAc moiety with [3H]NaBH4.
The results of incubating oxidized 3H-sLex with mutant CRD containing Gly189 plus regions 4 and 5 of E-selectin are shown in Fig. 4. Concentration-dependent labeling can be detected after gel electrophoresis and fluorography. Controls in the presence of EDTA at a concentration of 25 mM show no labeling, confirming that the binding is Ca2+-dependent (data not shown).
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Region 5 Interactions-- The importance of the three lysine residues in region 5 was explored further in two ways. First, the roles of individual lysine residues in this region were examined. Mutations with single lysine residues in the absence of region 4 did not display any Ca2+-dependent binding activity (data not shown). Therefore, mutations containing one, two, or three lysine residues in each possible combination were created in the presence of region 4 (Table II). The results indicate a cumulative effect of the lysine residues, with particularly the first and third residues capable of supporting some binding. The very limited effect of the middle residue may correlate with the disposition of this side chain furthest from the sialic acid portion of the bound ligand (Fig. 2). The HL-60 cell binding data suggest that the only combination of two residues which supports maximal binding is the second and third. However, the results using 125I-sLex-BSA as test ligand, which has relatively narrower error margins, suggest that any combination of two lysines supports full binding. The data indicate that no one residue is essential for binding activity and reveal a partial correlation of binding with total positive charge in this region. These findings are consistent with the structural results showing that none of the lysine side chains makes a specific, direct contact with the NeuAc portion of the ligand. Moreover, the effective binding achieved in the absence of Lys211 suggests that the hydrogen bond between this residue and the galactose moiety is not an essential component of the binding interaction.
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Effect of Salt on Binding-- The fact that the NeuAc residue in sLex does not make direct contact with lysine residues in region 5 and the apparent role of overall charge in this region of the CRD surface suggest that there may be a strong electrostatic component to the interaction between the selectin-like binding site and its ligand. To assess this possibility, the effect of salt on binding activity was evaluated. As shown in Fig. 6, the interaction of wild type CRD with Man-BSA is relatively insensitive to salt, whereas the interaction of a selectin-like mutant with sLex-BSA is weakened drastically as the ionic strength is increased. At physiological salt concentration, roughly 40% of the activity in the absence of added NaCl is observed. Previous studies with L-selectin on cell surfaces revealed a very similar inhibition of binding, with roughly 40% of activity retained under physiological conditions (27). Similar effects, over a more limited concentration range, have been observed for the other selectins (28). Thus, in addition to providing evidence for the electrostatic contribution to binding of the selectin-like mutant, these studies also demonstrate further parallels between the activity of the mutant and natural selectins.
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Role of Loop 3-- Studies using the extracellular portion of E-selectin have suggested that residues in loop 3 of the CRD might affect ligand binding activity. Substitution of a lysine residue from this region of MBP into the E-selectin CRD has been proposed to introduce mannose binding activity into the domain (22). Therefore, it was of interest to examine the effect of changing the residues in this loop to resemble selectin sequences to see if they affect the relative affinity for Man-BSA and sLex-BSA. In the case of mutants designed to resemble E-selectin either at the single position tested previously (residue 182 in MBP, corresponding to position 77 in E-selectin) or for all three residues in this loop which differ between MBP and E-selectin (positions 182-184), there was no detectable effect on relative binding to these two ligands (Table IV). It should be noted that the absolute value of the binding ratio has no direct meaning, as the ligands are not used at precisely the same concentration, and, more importantly, the degree of substitution with sugars is substantially different. Nevertheless, the results do not support a role for loop 3 residues in ligand discrimination.
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DISCUSSION |
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Mutational analysis of the selectins has revealed that changes at roughly 15 positions affect their ligand binding activity (4, 7-9, 22, 29). Several of the critical residues are Ca2+ ligands, whereas many of the rest have been included in the current studies, which extend the analysis of loss-of-function mutations in the selectins. The changes introduced into the MBP framework have been analyzed for increased binding to selectin ligands and thus represent gain-of-function mutations. It is often easier to interpret such effects rather than loss of function because they are less likely to result from indirect alterations in CRD structure and stability. Similarly, the use of purified CRDs rather than proteins expressed on cell surfaces or as Fc chimeras, combined with use of sLex-BSA ligand as well as HL-60 cells as target ligands, eliminates possible effects on cell surface expression and other confounding factors.
The fact that all of the mutants analyzed show Ca2+-dependent binding to either Man-Sepharose or invertase-Sepharose argues that they have achieved the correct overall conformation. Differential binding to invertase, sLex-BSA, and HL-60 cells by the various mutants investigated therefore presumably reflects conformational differences near the modified binding sites. The lack of any detectable retardation of the glycine-containing mutants of MBP on Man-Sepharose suggests that the affinity for invertase-Sepharose is not just a result of higher concentrations of mannose residues on these columns. Previous studies indicate that mannose phosphate residues in yeast polysaccharides are ligands for L-selectin (30), so similar structures in the invertase preparation might provide binding sites for the selectin-like mutants of MBP.
The phenotypes of mutant CRDs containing various portions of the E- and P-selectin CRDs suggest specific roles for several of these regions in ligand discrimination and stabilization of ligand-CRD complexes. Affinity for sLex seems to be dependent primarily upon the presence of a sufficient number of lysine residues in region 5. Selectin mutagenesis data in this region are not entirely consistent with each other, but several studies have suggested that neither Lys111 nor Lys113 is essential for ligand binding (4, 7-9, 29). These results are consistent with the findings reported in Table II.
In the crystal structure of the initial MBP mutant containing only the three lysines of region 5, the lysine side chains align closely with the arrangement seen in the natural E-selectin domain (4, 12). Thus, although the addition of a glutamic acid residue that hydrogen bonds to one of these lysine residues results in increased binding activity, its role is likely to be in stabilization of the binding site rather than orientation of the lysine side chains. Mutagenesis studies with E- and P-selectin indicating that alanine substitutions for either of these residues results in at least partial loss of binding activity (4, 9) are consistent with this suggestion.
The effects of region 4 are more subtle because the E-selectin sequence
inserted in this region supports increased binding to HL-60 cells and
sLex-BSA only in the presence of a histidine residue at
position 189. When present, this side chain has a potential role in the
binding site because the imidazole ring makes contact with the
N-acetylglucosamine portion of sLex in the
crystal structure of the region 5 mutant (31). Thus, the effect of the
E-selectin sequence in the presence of His189 may be caused
by a slight reorientation of the ligand resulting from interactions
with either the -carbon or the imidazole ring, or it may reflect
compensating interactions with the ligand which are only evident when
the binding site has an initially weaker affinity.
In addition to providing insight into common aspects of ligand binding to the selectins, these studies provide some initial suggestion of why different selectins show preferential binding to different ligands. In particular, the differing behavior of mutants containing E- and P-selectin sequences in region 4 implicates this segment of the CRD in differential ligand binding. In the most selectin-like constructs, the effect can be viewed as a relative loss of affinity for the simple test ligand, sLex-BSA, in the presence of the P-selectin rather than the E-selectin sequence (Table III). High affinity P-selectin ligands contain a tyrosine-sulfated region in addition to a specific set of sugar structures (32-34). Additional interactions with such a region, either through the CRD or the adjacent EGF-like domain, might compensate for lower affinity for sLex reflected in the behavior of the mutants analyzed here.
Mutagenesis studies with E-selectin identified one additional portion of the protein, region 3, which might be involved in sLex-BSA binding, although the phenotype resulting from changes in this segment were less severe than changes in regions 4 and 5 (4, 7, 22). Previous studies showed that incorporation of region 3 alone does not confer sLex-BSA binding activity on MBP (11). Further studies in which regions 3, 4, and 5 of E-selectin were all incorporated into MBP failed to show any enhancement of sLex-BSA binding compared with regions 4 and 5 alone.2 These results and the relatively large distance between region 5 and sLex in the crystal structure of the selectin-like mutant of MBP (Fig. 2) suggest that loss of binding caused by changes in region 3 of E-selectin represents an indirect effect on stability or on the arrangement of residues that are directly in the binding site.
The current results do not support a role for loop 3 of the CRD (region 6) in ligand binding selectivity suggested by previous studies with E-selectin (22). The earlier conclusions were based in part on the effect of mutations in this region on binding to yeast and to yeast invertase. The results reported here indicate that binding activity for commercial preparations of invertase is actually correlated with sLex binding activity, a result that probably confounds the previous use of such material as a test for mannose binding activity.
The oligomeric state of the selectin molecules has not been documented
clearly. However, many previous studies have utilized chimeric
constructs containing immunoglobulin Fc domain fused to the COOH
terminus of CRD and epidermal growth factor domains of the selectins.
Such chimeras form either dimers, when the Fc from IgG is employed, or
higher oligomers in the case of the Fc domain of IgM. All mutants
employed in the present studies contain the neck region from MBP and
are thus almost certainly trimers stabilized by the formation of a
coiled coil of -helices as observed in the crystal structures of
both wild type MBP and the initial region 5 selectin-like mutant (12,
35). Neither the Fc chimeras nor the MBP mutants are likely to reflect
the relative arrangement of multiple CRDs in intact selectins at the
cell surface. Therefore, the fact that the binding activities of Fc
fusions and MBP chimeras closely parallel the activity of the
membrane-bound molecules argues against the need for a specific
geometry of interaction between an array of ligands and the multiple
binding sites in oligomeric selectins. However, the way CRDs are
presented may have more influence on transient interactions with
ligands observed in cell rolling assays in contrast to static,
equilibrium assays employed in the present studies.
Several modified versions of the CRD from MBP have been crystallized successfully in forms that are isomorphous with the wild type CRD, and the spacing in these crystals accommodates introduction of ligands by soaking (12, 36). Thus, structural analysis of some of the mutants described here may prove feasible. The evidence presented showing that the binding activity of some of these mutants closely parallels the activity of the natural selectins suggests that such studies may provide a viable route to a detailed molecular understanding of ligand binding to the selectins.
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ACKNOWLEDGEMENTS |
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We thank Maureen Taylor, Bill Weis, and Ken Ng for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by a grant from the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Dept. of Chemistry, University of Edinburgh, West
Mains Rd., Edinburgh EH9 3IJ, U. K.
§ Wellcome Principal Research Fellow. To whom correspondence should be addressed: Dept. of Biochemistry, University of Oxford, South Parks Rd., Oxford OX1 3QU, U. K. Tel.: 44-1865-275-727; Fax: 44-1865-275-339; E-mail: kd{at}glycob.ox.ac.uk.
1 The abbreviations used are: CRD, carbohydrate-recognition domain; MBP, mannose-binding protein; sLex, 3'-sialyl-Lewisx tetrasaccharide; BSA, bovine serum albumin.
2 K. Drickamer, unpublished observations.
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REFERENCES |
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