title: Investigating cell surface galectin-mediated cross-linking on glycoengineered cells.
abstract: The galectin family of glycan-binding proteins is thought to mediate many cellular processes by oligomerizing cell surface glycoproteins and glycolipids into higher-order aggregates. This hypothesis reflects the known oligomeric states of the galectins themselves and their binding properties with multivalent ligands in vitro, but direct evidence of their ability to cross-link ligands on a cell surface is lacking. A major challenge in fundamental studies of galectin-ligand interactions is that their natural ligands comprise a heterogeneous collection of glycoconjugates that share related glycan structures but disparate underlying scaffolds. Consequently, there is no obvious means to selectively monitor the behaviors of natural galectin ligands on live cell surfaces. Here we describe an approach for probing the galectin-induced multimerization of glycoconjugates on cultured cells. Using RAFT polymerization, we synthesized well-defined glycopolymers (GPs) functionalized with galectin-binding glycans along the backbone, a lipid group on one end and a fluorophore on the other. After insertion into live cell membranes, the GPs' fluorescence lifetime and diffusion time were measured in the presence and absence of galectin-1. We observed direct evidence for galectin-1-mediated extended cross-linking on the engineered cells, a phenomenon that was dependent on glycan structure. This platform offers a new approach to exploring the "galectin lattice" hypothesis and to defining galectin ligand specificity in a physiologically relevant context.
authors.affiliation: Department of Chemistry, and Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California 94720, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Belardi
authors.forename: Geoff P
authors.initials: GP
authors.lastname: O'Donoghue
authors.forename: Adam W
authors.initials: AW
authors.lastname: Smith
authors.forename: Jay T
authors.initials: JT
authors.lastname: Groves
authors.forename: Carolyn R
authors.initials: CR
authors.lastname: Bertozzi
mesh_terms: D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D006224:Cricetinae; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D037483:Galectin 1 / Q000032:analysis / Q000378:metabolism; D037161:Galectins / Q000032:analysis / Q000378:metabolism*; D006001:Glycoconjugates / Q000032:analysis / Q000378:metabolism*; D058105:Polymerization; D011485:Protein Binding
chemical_list: D037483:Galectin 1; D037161:Galectins; D006001:Glycoconjugates