Chemically synthesized solid phase oligosaccharide probes for carbohydrate-binding receptors. Interactions of the E-, L- and P-selectins with sialyl-Le(x) and O-sulphated forms linked to biotin or to polyacrylamide.
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 PAA with 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-Lewis(x) (SLe(x)) analogues having sulphate at position 6 of the galactose (6'SuSLe(x)) or of the N-acetylglucosamine (6SuSLe(x)). The results clarify the role of 6SuSLe(x), rather then 6'SuSLe(x), as a ligand for the selectins.
Pavlovic
Davor
D
The Glycosciences Laboratory, Faculty of Medicine, Imperial College of Science, Technology and Medicine, Northwick Park Institute of Medical Research, Watford Road, Harrow, Middlesex, UK.
Leteux
Christine
C
Ovchinnikova
Tatyana
T
Tsvetkov
Yury
Y
Nifant'ev
Nikolay
N
Feizi
Ten
T
D000180:Acrylic Resins / Q000378:metabolism*; D000818:Animals; D001710:Biotin / Q000378:metabolism*; D002236:Carbohydrate Conformation; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D019040:E-Selectin / Q000378:metabolism; D019041:L-Selectin / Q000378:metabolism; D007983:Lewis Blood Group Antigens / Q000378:metabolism*; D016256:Lewis X Antigen / Q000031:analogs & derivatives; D008024:Ligands; D015335:Molecular Probes / Q000138:chemical synthesis / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000138:chemical synthesis* / Q000378:metabolism*; D019007:P-Selectin / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D019087:Selectins / Q000378:metabolism*; D000080506:Sialyl Lewis X Antigen / Q000031:analogs & derivatives; D000099166:Polyacrylamides
D000180:Acrylic Resins; D001710:Biotin; D019040:E-Selectin; D019041:L-Selectin; D007983:Lewis Blood Group Antigens; D016256:Lewis X Antigen; D008024:Ligands; D015335:Molecular Probes; D009844:Oligosaccharides; D019007:P-Selectin; D019087:Selectins; D000080506:Sialyl Lewis X Antigen; D000099166:Polyacrylamides; C086949:6'-sulfated sialyl Lewis x