ABSTRACT
Relatively weak interactions between galectins and their potential ligands can hinder identification of physiological lectin ligands using conventional methods such as affinity purification. We have employed a combination of cysteine mutagenesis with chemical crosslinking using a photoactivatable sulfhydryl reagent benzophenone-4-maleimide to obtain a covalent complex between human galectin-1 and the model glycoprotein ligands asialofetuin and laminin which contain an N-acetyllactosamine structure. A crosslinked product was obtained only when galectin-1 with an introduced cysteine interacted with these glycoproteins via their carbohydrate moiety. This procedure should be useful for the detection of important, and as yet unidentified, ligands for galectins which cannot be currently detected because of their relatively weak interaction.
Subject(s)
Amino Sugars/chemistry , Benzophenones/chemistry , Cross-Linking Reagents/chemistry , Cysteine/chemistry , Galectin 1/chemistry , Glycoproteins/chemistry , Maleimides/chemistry , Sulfhydryl Reagents/chemistry , Animals , Asialoglycoproteins/chemistry , Benzophenones/radiation effects , Cattle , Cysteine/genetics , Fetuins , Galectin 1/genetics , Humans , Laminin/chemistry , Lectins , Maleimides/radiation effects , Photochemical Processes , Rabbits , Sulfhydryl Reagents/radiation effects , alpha-Fetoproteins/chemistryABSTRACT
A mixed disulfide reagent for photolabeling is described which reacts stoichiometrically with a cysteine sulfhydryl group of rabbit muscle creatine kinase to form a new mixed disulfide between enzyme and 2-thiobenzyl[14C]diazoacetate. When irradiated at 254 nm for 5 s in a photoreactor, the enzyme-bound diazo group is destroyed, presumably via a carbene intermediate. After photolysis, the enzyme can only be 69+/-2% reactivated by dithiothreitol, and only 67+/-1% of the radiolabel can then be removed from the protein by dialysis in the presence of dithiothreitol. Less than 3% of this permanent labeling occurs if photolysis is carried out in 6 N guanidine hydrochloride, which shows that the native enzyme structure is required for photolabeling. Identification of the tagged products after acid hydrolysis indicates that 30% of the carbene produced on photolysis reacts with the hydroxyl groups of threonine andserine with O-[14C]carboxymethylthreonine as the major product. Photochemical Wolff rearrangement is estimated to occur to less than 30%, and no S-carboxymethylcysteine was detected. The reagent employed and its isomers are proposed as bifunctional photolabeling probes to "scan" the amino acid residues near the active sites of thiol enzymes.
