Glycobiology, 2000, Vol. 10, No. 4 357-364
© 2000 Oxford University Press
Photoaffinity glycoprobes—a new tool for the identification of lectins
2Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovaèiæa 1, 10000 Zagreb, Croatia and 3Biology Department, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA
One of the proposed functions for the carbohydrate structures on glycoconjugates is the transfer of information through interaction with specific lectin receptors. However, the number of elucidated functional lectin–carbohydrate interactions is still relatively small, largely due to the lack of adequate methods to identify lectin activity in complex biological samples. Aiming to solve this problem, we have developed a method based on the novel group of compounds we named glycoprobes. The glycoprobe consists of three vital parts: (1) glycan, (2) digoxin tag, and (3) photoreactive crosslinker. When incubated in dark, oligosaccharide part of the glycoprobe forms a complex with lectin. After illumination, covalent link between the probe and the lectin is formed resulting in a digoxin-tagged lectin. Using antibodies against digoxin, this complex can easily be identified immuno/cytochemically, or by Western blots. To demonstrate the applicability of glycoprobes we have used Man9-glycoprobe (containing Man9 oligosaccharide) and YEE(ahGalNAc)3-glycoprobe (containing a synthetic neoglycopeptide with three terminal N-acetylgalactosamine residues; Lee and Lee, Glycoconjugate J., 1987, 4, 317) to identify lectins in bovine serum and rat liver membranes. The simplicity of the method enables its application in routine monitoring of changes in lectin activity during various developmental or pathological processes. An example of GalNAc-binding analysis in human serum is shown.