Crosslinking of N-acetyllactosamine-containing glycoproteins to galectin-1 with an introduced cysteine using a photoactivatable sulfhydryl reagent.
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.
Tamura
Mayumi
M
Laboratory of Molecular Immunology, Faculty of Pharmaceutical Sciences, Josai University, Japan.
Igarashi
Takanori
T
Kasai
Ken-ichi
K
Arata
Yoichiro
Y
D000606:Amino Sugars / Q000737:chemistry*; D000818:Animals; D001212:Asialoglycoproteins / Q000737:chemistry; D001577:Benzophenones / Q000737:chemistry* / Q000528:radiation effects; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D060748:Fetuins; D037483:Galectin 1 / Q000737:chemistry* / Q000235:genetics; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D007797:Laminin / Q000737:chemistry; D037102:Lectins; D008301:Maleimides / Q000737:chemistry* / Q000528:radiation effects; D055668:Photochemical Processes; D011817:Rabbits; D013439:Sulfhydryl Reagents / Q000737:chemistry* / Q000528:radiation effects; D000509:alpha-Fetoproteins / Q000737:chemistry
D000606:Amino Sugars; D001212:Asialoglycoproteins; D001577:Benzophenones; D003432:Cross-Linking Reagents; D060748:Fetuins; D037483:Galectin 1; D006023:Glycoproteins; D007797:Laminin; D037102:Lectins; D008301:Maleimides; D013439:Sulfhydryl Reagents; D000509:alpha-Fetoproteins; C017029:asialofetuin; C000458:N-acetyllactosamine; C046109:benzophenone-4-maleimide; D003545:Cysteine