Chemically synthesized solid phase oligosaccharide probes

for carbohydrate-binding receptors

Interactions of the E-, L- and P-selectins with sialyl-Lex and

O-sulphated forms linked to biotin or to polyacrylamide

Davor Pavlovic a, Christine Leteux a, Tatyana Ovchinnikova b, Yury Tsvetkov c,Nikolay Nifant’ev c, Ten Feizi a,*

aThe Glycosciences Laboratory, Faculty of Medicine, Imperial College of Science, Technology and Medicine,

Northwick Park Institute of Medical Research, Watford Road, Harrow, Middlesex HA1 3UJ, UKbLaboratory of Carbohydrate Chemistry, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,

ul Miklukho-Maklaya 16/10, 117871, GSP-7, V-437 Moscow, Russian FederationcSyntesome GmbH, Heimdall Str. 4, D-81739 Munich, Germany

Received 8 November 2001; received in revised form 6 February 2002; accepted 27 February 2002

Abstract

There is a growing interest in chemically defined oligosaccharide reagents for identifying proteins that bind carbohydrates

and determining the specificities of carbohydrate-binding proteins. Here, we compare three sets of chemically synthesized

commercially available oligosaccharide conjugates as immobilized probes, for the binding signals that they elicit with known

carbohydrate-binding receptors of the immune system, the E-, P- and L-selectins. The first set of conjugates is of oligosaccharides

linked to biotin via a nine-carbon spacer. The second and third sets are multivalent derivatives in which the oligosaccharides are

linked, via a three-carbon spacer to poly[N-(2-hydroxyethyl)acrylamide] (PAA) or to biotinylated PAAwith an average of 20%

substitution of the hydroxyethyl-amide groups by carbohydrate. The conjugates were immobilized on streptavidin-coated

microwells if biotinylated, otherwise by drying in uncoated wells. The most robust binding curves, overall, were with the

biotinylated PAA derivatives of the ligands immobilized on streptavidin wells. These reagents have permitted a reevaluation of

selectin binding signals elicited by sialyl-Lewisx (SLex) analogues having sulphate at position 6 of the galactose (6VSuSLex) or ofthe N-acetylglucosamine (6SuSLex). The results clarify the role of 6SuSLex, rather then 6VSuSLex, as a ligand for the selectins.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Selectins; Carbohydrate ligands; E-selectin; P-selectin; L-selectin; Oligosaccharide reagents

0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0022 -1759 (02 )00079 -0

Abbreviations: ABTS, 2,2V-azinobis(3-ethylbenzthiazoline-sulfonic acid); BACH, 6-(biotinyl)-aminocaproyl-hydrazide; CHO, Chinese

hamster ovary; HEV, high endothelial venules; Ig, immunoglobulin; PAA, polyacrylamide; PAA-biotin, biotinylated-polyacrylamide; SLex, 3V-sialyl-Lex; Sp-biotin, spacer-biotin; 6SuSLex, 6-sulpho-3V-sialyl-Lex; 6VSuSLex, 6V-sulpho-3V-sialyl-Lex.

* Corresponding author. Tel.: +44-20-8869-3460/3461; fax: +44-20-8869-3455.

E-mail address: t.feizi@ic.ac.uk (T. Feizi).

www.elsevier.com/locate/jim

Journal of Immunological Methods 264 (2002) 53–58

1. Introduction

There is an increasing awareness of the existence of

lectin-type proteins in the immune system (Crocker,

2001), and thus an increasing need for screening assays

to identify those proteins that bind carbohydrates and

to determine their specificities. As the naturally occur-

ring carbohydrate ligands for such proteins can be

isolated only in relatively small amounts, chemically

synthesized oligosaccharides have played a major part

in consolidating assignments of their carbohydrate-

binding specificities (Auge et al., 1997; Sears and

Wong, 1999) and even in providing clues to the

existence of unsuspected ligands and processing path-

ways for the carbohydrate ligands (Feizi and Galustian,

1999; Galustian et al., 1997a; Komba et al., 1999).

Here we have evaluated as reagents for ELISA-type

carbohydrate-binding assays, chemically synthesized,

commercially available, Lewisx-related oligosacchar-

ides based on a disaccharide backbone, Galh1–4GlcNAc, and presented in three different modes: (a)

as monomers linked via a spacer to biotin, (b) in

oligomeric form linked to polyacrylamide and (c) in

oligomeric form linked to biotinylated-polyacryla-

mide. These have been examined for binding by

soluble forms of the lectin-type proteins of the immune

system, the E-, P- and L-selectins.

2. Materials and methods

2.1. Saccharides

Chemical structures and designations of the three

carbohydrate sequences investigated are as follows:

These oligosaccharides are chemically synthesized,

and are in three forms (provided by Syntesome,

Munich, Germany). The first set of conjugates are of

oligosaccharides linked to biotin via a nine-carbon (11

atoms) spacer (sp-biotin); the second and third are

multivalent conjugates in which oligosaccharides are

linked to poly[N-(2-hydroxyethyl)acrylamide] (PAA)

or biotinylated-polyacrylamide (PAA-biotin) (Bovin,

1998; Game et al., 1998). On average, 20% of

hydroxyethyl-amide groups in PAA or PAA-biotin

are substituted by carbohydrate. In PAA-biotin, 5%

of 2-hydroxyethyl-amide groups are substituted with

biotin. Quantitation of carbohydrates in the aqueous

solutions of the conjugates was performed by the

phenol-sulphuric acid assay (Dubois et al., 1956) in

96-well microwells (Falcon 3872 Primaria). Galactose

was used as a standard; the following response factors

were used to calculate the molar concentrations of the

oligosaccharides: galactose, 1; fucose, 0.6; N-acetyl-

glucosamine and N-acetylneuraminic acid, 0.

2.2. Selectins

Recombinant soluble rat L-selectin in the form of

an IgG Fc chimera (Fcg) (Tamatani et al., 1993) was

from Dr. M. Miyasaka and culture fluids containing

murine forms of E- and P-selectins IgM Fc chimeras

(FcA) (Maly et al., 1996) were from Dr. J.B. Lowe.

2.3. Carbohydrate-binding assays

Selectin binding assays were performed as des-

cribed previously (Leteux et al., 1999) using TBS

(10 mM Tris–HCl, 2 mM CaCl2, 150 mM NaCl, pH

8.0) as a buffer. In brief, dilutions of oligosaccharide

conjugates in phosphate buffered saline (10 mM phos-

phate buffer, 2.7 mM potassium chloride and 137 mM

sodium chloride, pH 7.4, PBS, Sigma) were added to

microwells for immobilization by two methods. For

immobilization via a biotin tag (sp-biotin and PAA-

biotin conjugates), high capacity streptavidin-coated

microtiter wells (Boehringer-Mannheim) were used;

the reaction volumes were 200 Al, and the incubation

time was 16 h at 4 jC. For immobilization by passive

coating (PAA conjugates, and where indicated, the

PAA-biotin conjugates), plastic microwells (Immulon

4, Dynex Technologies, Billingshurst, UK) were used;

the reaction volumes were 50 Al (unsealed), and the

D. Pavlovic et al. / Journal of Immunological Methods 264 (2002) 53–5854

Fig. 1. Binding of the E-, P- and L-selectins to chemically synthesized oligosaccharides of the Lex series. Oligosaccharides linked via a spacer to

biotin (sp-biotin derivatives) [panels A, D and G], or to polyacrylamide tagged with biotin (PAA-biotin) [panels B, E and H], or to non-

biotinylated polyacrylamide (PAA) [panels C, F and I] were immobilized in microwells and the selectin binding signals were assayed as

described under Section 2. Symbols for the oligosaccharides: n, SLex;z, 6VSuSLex; x, 6SuSLex. Results are expressed as means in duplicate

wells with the range indicated by error bars.

D. Pavlovic et al. / Journal of Immunological Methods 264 (2002) 53–58 55

incubation time was 16 h at 37 jC. Thereafter, thewells were incubated with 3% (w/v) bovine serum

albumin in TBS, and the binding of L-selectin Fcg (10

Ag/ml), E-selectin FcA (1/50 dilution of culture super-

natant) and P-selectin FcA (1/3 dilution of culture

supernatant) was assayed using rabbit antihuman IgG

(L-selectin) and antihuman IgM (E- and P-selectins)

followed by protein-A-peroxidase and the substrate,

2,2V- azinobis(3-ethylbenzthiazoline-sulfonic acid), as

described previously (Leteux et al., 1999). Binding

experiments using TBS, containing 2 mM EDTA

instead of 2 mM Ca2 + as diluent, indicated that the

selectin binding to all three species is calcium depend-

ent (results not shown).

3. Results

All three types of oligosaccharide conjugates, sp-

biotin, PAA-biotin and PAA were evaluated for bind-

ing by the three soluble selectins. The binding curves

were steeper with the E- and P-selectins than the L-

selectin (Fig. 1). This is probably due to the recombi-

nant E- and P-selectins being higher oligomers [in the

form of IgM chimeras (Maly et al., 1996)], than the L-

selectin [a spontaneously aggregated IgG chimera

(Galustian et al., 1997b)].

3.1. Binding to SLex conjugates

With the E- and P-selectins, all three conjugates of

SLex elicited robust binding curves (Fig. 1A–F),

whereas with the L-selectin preparation, this was the

case only with the PAA-biotin derivative presented on

the high capacity streptavidin wells (cf. Fig. 1G–I). In

a separate experiment (not shown), where the PAA-

biotin derivative was immobilized by drying in

uncoated microwells, the L-selectin binding curve

was shallow and similar to that in Fig. 1I for the

non-biotinylated PAA derivative. Quantitation of the

relative amounts of the conjugates adsorbed onto the

plates cannot be readily determined, but the results

show that the PAA-biotin conjugate complexed to

streptavidin is an effective means of presenting the

ligand to all three selectin preparations. Thus, in the

experiments shown in Fig. 1A–F and H, comparisons

can be made of the binding of the three selectins to the

SLex, an undisputed ligand for the selectins (Bevilac-

qua andNelson, 1993; Feizi, 1993; Rosen andBertozzi,

1996) and the 6-O-sulphated analogues (sulphated at

GlcNAc or Gal) on which there are conflicting data in

the literature (see Discussion).

3.2. Binding to 6SuSLex and 6VSuSLex conjugates

With E-selectin, 6SuSLex binding signals were

comparable with those of the SLex. With the P- and

L-selectins in contrast, the binding to the 6SuSLex in

all cases was greater. However, there was no detect-

able binding to the 6VSuSLex conjugates in any of the

experiments performed.

4. Discussion

We conclude that the SLex type oligosaccharides

based on a disaccharide backbone, when presented as

PAA or PAA-biotin and sp-biotin derivatives, can

elicit binding signals with the selectins. We have

observed that, overall, the PAA-biotin derivatives

immobilized on streptavidin wells give the most

robust binding curves.

It has been noted previously that when biotinylated

oligosaccharides are used in binding experiments with

antibodies or selectins, the length of both the oligo-

saccharide backbone and of the spacer on the biotiny-

lated tag may be critical factors in the binding signal

(Leteux et al., 1999). The oligosaccharides used in the

present investigation are short structures, based on a

disaccharide backbone. The binding signals that they

elicit contrast with the lack of binding by the three

selectins, reported in a previous investigation (Leteux

et al., 1999) when the same sequences were presented

as biotinylated derivatives with a relatively short

spacer (six carbons or less). The presence of a partic-

ularly long spacer (nine carbons) in the biotinylated

derivatives used in the present investigation most likely

accounts for the binding observed with the selectins.

Among the different probes examined in the

present investigation, the PAA-biotin derivatives

immobilized onto streptavidin-coated plates gave the

strongest, whereas the PAA derivatives gave the weak-

est binding signals. The amount of immobilization in

plastic microwells of the PAA derivatives has been

estimated at around 1% (N.V. Bovin, unpublished

results). With the biotin conjugates, however, the

D. Pavlovic et al. / Journal of Immunological Methods 264 (2002) 53–5856

coating efficiency using the high capacity streptavidin

wells is excellent (Leteux et al., 1999) on account of

the extremely strong avidity of streptavidin for biotin.

Moreover, the display of the oligosaccharides on the

PAA backbone offers multivalence. This clustering is

further enhanced by the binding of four biotin mole-

cules to one streptavidin. PAA-biotin derivatives are

therefore in a multi-clustered state, which makes them

the most appropriate derivatives to use for screening

proteins for carbohydrate binding.

The carbohydrate reagents used in this study have

permitted a reevaluation of the binding signals that the

6SuSLex and 6VSuSLex sequences elicit with the selec-tins, a subject of some controversy (Kannagi and

Kanamori, 1999; Tsuboi et al., 1996). Our results with

these compounds, which have been synthesized inde-

pendently of those synthesized previously (Komba et

al., 1996; Tsuboi et al., 1996; Yoshino et al., 1997),

show clearly that all three selectins give binding

signals with the 6SuSLex but not with the 6VSuSLex

analogue. These results corroborate the earlier obser-

vations on L-selectin binding by Galustian et al.

(1997a) who used the tetrasaccharide-based analogues

linked to lipid. Our findings are also in accord with the

results of L-selectin binding experiments with chemi-

cally synthesized 6,3VSuSLex and 6V,3VSuSLex in whichthe former was strongly and the latter weakly bound by

L-selectin (Galustian et al., 1999).

Data which at first sight contrast with the above

were presented in a previous study by Tsuboi et al.

(1996), and led these authors to conclude that

6VSuSLex, rather than 6SuSLex, is the preferred L-

selectin ligand. These investigators had used a fuco-

syltransferase to transfer en block, and fix onto the

surface of Chinese hamster ovary (CHO) cells, the

6SuSLex or 6VSuSLex oligosaccharides via a chemi-

cally attached fucose residue. This means of attach-

ment would have resulted in the joining of this fucose

residue to a sub-terminal N-acetylglucosamine residue

on oligosaccharides at the CHO cell surface. A possi-

bility we have considered for these conflicting data is

that this artificial method of cell surface attachment

may have physically masked the L-selectin recognition

motif on 6SuSLex, whereas on the 6VSuSLex, it may

have resulted in the presentation of a motif that is

analogous to the short, sulpho motifs that are bound by

L-selectin in a cation-independent manner (Green et

al., 1995; Galustian et al., 2002). An alternative

explanation (M. Fukuda, personal communication),

which is consistent with the foregoing suggestion, is

that this artificial means of oligosaccharide attachment

to cells resulted in marked conformational alterations

and unnatural presentations of the two oligosacchar-

ides. Thus, the present investigation has served to

exclude conclusively a role 6VSuSLex as a ligand for

L-selectin and also for the E- and P-selectins.

Acknowledgements

We are grateful to Drs. M. Miyasaka and J.B. Lowe

for providing the recombinant selectins. This work

was supported by a programme grant (G 9601454)

from the Medical Research Council.

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