Photoaffinity glycoprobes-a new tool for the identification of lectins.
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 Man(9)-glycoprobe (containing Man(9)oligosaccharide) and YEE(ahGalNAc)(3)-glycoprobe (containing a synthetic neoglycopeptide with three terminal N-acetyl-galactosamine 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.
Lauc
G
G
Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovaèiaea 1, 10000 Zagreb, Croatia.
Lee
R T
RT
Dumiae
J
J
Lee
Y C
YC
D000116:Acetylgalactosamine; D000818:Animals; D037263:Asialoglycoprotein Receptor; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002352:Carrier Proteins / Q000097:blood; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry; D037222:Collectins; D004077:Digoxin; D006020:Glycopeptides; D037102:Lectins / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D008099:Liver / Q000737:chemistry; D008358:Mannose; D008969:Molecular Sequence Data; D019920:Photoaffinity Labels; D011134:Polysaccharides; D051381:Rats; D011956:Receptors, Cell Surface / Q000032:analysis; D012712:Serum Globulins / Q000032:analysis / Q000378:metabolism
D037263:Asialoglycoprotein Receptor; D002352:Carrier Proteins; D037222:Collectins; D006020:Glycopeptides; D037102:Lectins; D019920:Photoaffinity Labels; D011134:Polysaccharides; D011956:Receptors, Cell Surface; D012712:Serum Globulins; C024014:conglutinin; C086954:collectin-43 protein, bovine; D004077:Digoxin; D000116:Acetylgalactosamine; D008358:Mannose