Side Chain Orientation of the Amino Acid Substituted by a Cysteine Residue Is Important for Successful Crosslinking of Galectin to Its Glycoprotein Ligand Using a Photoactivatable Sulfhydryl Reagent

Abstract

We have employed a combination of cysteine mutagenesis and chemical crosslinking using a photoactivatable sulfhydryl reagent, benzophenone-4-maleimide, to obtain a covalent complex between human galectin-1 and a model glycoprotein ligand, asialofetuin. We previously obtained a crosslinked product when Lys 28 of the cysteine-less form of human galectin-1 was mutated to cysteine. To investigate whether substituting either of the two ‰anking amino acid residues in the same b-strand, Ala 27 and Ser 29 , to cysteine could result in crosslinking to the bound asialofetuin, two cysteine-containing mutants were generated. Although both the mutants adsorbed to asialofetuin-agarose and were eluted with 0.1 M lactose, conˆrming their ability to interact with asialofetuin, these mutants did not crosslink to the bound glycoprotein ligand following treatment with benzophenone-4-maleimide. Therefore the orientation of the side chain of the introduced cysteine residue apparently plays an important role in the crosslinking reaction.

Crosslinking of Cys-Mutated Human Galectin-1 to the Model Glycoprotein Ligands Asialofetuin and Laminin by Using a Photoactivatable Bifunctional Reagent

“…10,15,18) Briefly, purified recombinant Cys-less or single-cysteine mutants of hGal-1 were dissolved in PBS containing 1 mM EDTA (pH 7.2) (EDTA-PBS). BPM was dissolved in dimethylformamide, and the BPM solution was added to the recombinant protein in EDTA-PBS (final concentration of BPM, 4 µM).…”

Cross-Link Formation between Mutant Galectins of Caenorhabditis elegans with a Substituted Cysteine Residue and Asialofetuin via a Photoactivatable Bifunctional Reagent

“…11,12) Preparation of Asialofetuin for Cross-Linking Bovine fetuin was treated with sialidase to remove the sialic acid attached to the non-reducing galactose end residues of the sugar chain. The resultant asialofetuin was purified as described previously 14) and used as a model glycoprotein ligand for LEC-1.…”

“…11,12,14) In brief, purified recombinant LEC-1 or the single cysteine-containing mutant of LEC-1 was dialyzed against phosphate-buffered saline (PBS) containing 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 7.2) (EDTA-PBS). BPM was dissolved in dimethylformamide, and the solution was added to the lectin solution (final concentration, 1 mM BPM).…”