Human galectins have functionally divergent roles, although most of the members of the galectin family bind weakly to the simple disaccharide lactose (Gal1-4Glc). To assess the specificity of galectin-glycan interactions in more detail, we explored the binding of several important galectins (Gal-1, Gal-2, and Gal-3) using a dose-response approach toward a glycan microarray containing hundreds of structurally diverse glycans, and we compared these results to binding determinants on cells. All three galectins exhibited differences in glycan binding characteristics. On both the microarray and on cells, Gal-2 and Gal-3 exhibited higher binding than Gal-1 to fucose-containing A and B blood group antigens. Gal-2 exhibited significantly reduced binding to all sialylated glycans, whereas Gal-1 bound ␣2-3-but not ␣2-6-sialylated glycans, and Gal-3 bound to some glycans terminating in either ␣2-3-or ␣2-6-sialic acid. The effects of sialylation on Gal-1, Gal-2, and Gal-3 binding to cells also reflected differences in cellular sensitivity to Gal-1-, Gal-2-, and Gal-3-induced phosphatidylserine exposure. Each galectin exhibited higher binding for glycans with poly-N-acetyllactosamine (poly(LacNAc)) sequences (Gal1-4GlcNAc) n when compared with N-acetyllactosamine (LacNAc) glycans (Gal1-4GlcNAc). However, only Gal-3 bound internal LacNAc within poly(LacNAc). These results demonstrate that each of these galectins mechanistically differ in their binding to glycans on the microarrays and that these differences are reflected in the determinants required for cell binding and signaling. The specific glycan recognition by each galectin underscores the basis for differences in their biological activities.
L-selectin requires a threshold shear to enable leukocytes to tether to and roll on vascular surfaces. Transport mechanisms govern flow-enhanced tethering, whereas force governs flow-enhanced rolling by prolonging the lifetimes of L-selectin–ligand complexes (catch bonds). Using selectin crystal structures, molecular dynamics simulations, site-directed mutagenesis, single-molecule force and kinetics experiments, Monte Carlo modeling, and flow chamber adhesion studies, we show that eliminating a hydrogen bond to increase the flexibility of an interdomain hinge in L-selectin reduced the shear threshold for adhesion via two mechanisms. One affects the on-rate by increasing tethering through greater rotational diffusion. The other affects the off-rate by strengthening rolling through augmented catch bonds with longer lifetimes at smaller forces. By forcing open the hinge angle, ligand may slide across its interface with L-selectin to promote rebinding, thereby providing a mechanism for catch bonds. Thus, allosteric changes remote from the ligand-binding interface regulate both bond formation and dissociation.
P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin on leukocytes that binds selectins. The molecular features of PSGL-1 that determine this high affinity binding are unclear. Here we demonstrate the in vitro synthesis of a novel glycosulfopeptide (GSP-6) modeled after the extreme N terminus of PSGL-1, which has been predicted to be important for P-selectin binding. GSP-6 contains three tyrosine sulfate (TyrSO 3 ) residues and a monosialylated, core 2-based O-glycan with a sialyl Lewis x (C2-O-sLe x ) motif at a specific Thr residue. GSP-6 binds tightly to immobilized P-selectin, whereas glycopeptides lacking either TyrSO 3 or C2-O-sLe x do not detectably bind. Remarkably, an isomeric glycosulfopeptide to GSP-6, termed GSP-6, which contains sLe x on an extended core 1-based O-glycan, does not bind immobilized P-selectin. Equilibrium gel filtration analysis revealed that GSP-6 binds to soluble P-selectin with a K d of ϳ350 nM. GSP-6 (<5 M) substantially inhibits neutrophil adhesion to P-selectin in vitro, whereas free sLe x (5 mM) only slightly inhibits adhesion. In contrast to the inherent heterogeneity of post-translational modifications of recombinant proteins, glycosulfopeptides permit the placement of sulfate groups and glycans of precise structure at defined positions on a polypeptide. This approach should expedite the probing of structure-function relationships in sulfated and glycosylated proteins, and may facilitate development of novel drugs to treat inflammatory diseases involving P-selectin-mediated leukocyte adhesion.The interactions between selectins and their carbohydratebased ligands initiate adhesion of leukocytes to the vascular wall during inflammation. Although L-, E-, and P-selectin can bind a simple glycan containing sialyl Lewis x (sLe x ) 1 (NeuAc␣233Gal134[Fuc␣133]GlcNAc13 R) in a Ca 2ϩ -dependent manner, each selectin binds with higher affinity to a limited number of macromolecular ligands expressing sialylated and fucosylated glycans (1-4). P-selectin, which is expressed by activated platelets and endothelial cells, demonstrates the most discriminating ligand specificity of any selectin. It interacts predominantly with a disulfide-bonded dimeric mucin on leukocytes termed P-selectin glycoprotein ligand-1 (PSGL-1) (subunit mass ϳ120 kDa) (5).Each 120-kDa subunit of human PSGL-1 contains numerous sialic acids and approximately 70 extracellular Ser and Thr residues, which are potential sites for O-glycosylation, plus three potential sites for N-glycosylation (6, 7) (Fig. 1). These features suggested that the large amount of carbohydrate on the mucin might promote high avidity binding to P-selectin. However, indirect evidence suggests that the extreme N-terminal extracellular region of mature PSGL-1, which begins at residue 42, is important for high affinity binding to P-selectin (reviewed in Ref. 3). Specifically, tyrosine sulfate residues and O-glycans within that region have been considered essential for binding (Fig. 1). A monoclonal antibody directed to a peptide ep...
The dendritic cell specific C-type lectin dendritic cell specific ICAM-3 grabbing non-integrin (DC-SIGN) binds to ''self'' glycan ligands found on human cells and to ''foreign'' glycans of bacterial or parasitic pathogens. Here, we investigated the binding properties of DC-SIGN to a large array of potential ligands in a glycan array format. Our data indicate that DC-SIGN binds with K d < 2 lM to a neoglycoconjugate in which Galb1-4(Fuca1-3)GlcNAc (Le x ) trisaccharides are expressed multivalently. A lower selective binding was observed to oligomannose-type N-glycans, diantennary N-glycans expressing Le x and GalNAcb1-4(Fuca1-3)GlcNAc (LacdiNAc-fucose), whereas no binding was observed to N-glycans expressing corefucose linked either a1-6 or a1-3 to the Asn-linked GlcNAc of N-glycans. These results demonstrate that DC-SIGN is selective in its recognition of specific types of fucosylated glycans and subsets of oligomannose-and complex-type N-glycans.
x and 6-sulfo-sLe x did not support any Siglec-8 binding at the highest concentration tested (300 pmol/spot). We conclude that Siglec-8 binds preferentially to the sLe x structure bearing an additional sulfate ester on the galactose 6-hydroxyl.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.