Acceptor specificity of the human leukocyte 3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

106

Background: During inflammation, the selectins engage glycosylated macromolecules expressed on blood leukocytes under fluid shear conditions. Results: Although all three myeloid ␣1,3-fucosyltransferases FUT9, FUT7, and FUT4 regulate human E-selectin ligand biosynthesis, FUT7 and FUT4 are sufficient to form L/P-selectin ligands. Conclusion: FUT9 plays a significant role during human, but not mouse, leukocyte-endothelial interactions. Significance: This study identifies potential ␣(1,3)FUTs regulating inflammation in humans.

Section: Cellsupporting

confidence: 75%

Silencing α1,3-Fucosyltransferases in Human Leukocytes Reveals a Role for FUT9 Enzyme during E-selectin-mediated Cell Adhesion

Buffone

Mondal

Gupta

et al. 2013

106

“…The synthesis of GSPs with core 2-based O-glycans containing polyfucosylated, polylactosamine sequences may help to address these possibilities. In vitro, FucT-VII can generate a terminal sLe x moiety on a polylactosamine sequence, but cannot add internal fucosyl residues to generate the polyfucosylated, polylactosamine sequence found in the O-glycans of PSGL-1 (50,51). Human and murine leukocytes contain a second ␣1,3-fucosyltransferase, termed FucT-IV, that adds fucosyl residues to internal sequences of polylactosamine (50).…”

Section: Gsp-6 Is a Potent Inhibitor Of Neutrophil Adhesion To Immobimentioning

confidence: 99%

A Novel Glycosulfopeptide Binds to P-selectin and Inhibits Leukocyte Adhesion to P-selectin

Leppänen¹,

Mehta²,

Ouyang³

et al. 1999

201

213

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␣233Gal␤134[Fuc␣133]GlcNAc␤13 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...

“…Whereas synthetic glycosulfopeptides containing the simple monosialylated monofucosylated C2-O-sLe x bind to P-selectin, the role of sialylated PFPL O-glycans in binding to P-selectin has not been studied. Efficient generation of sialylated PFPL in vitro requires the participation of two human ␣1,3-fucosyltransferases expressed in leukocytes, FucT-IV and FucT-VII (14,15). FucT-IV appears to preferentially fucosylate internal GlcNAc residues within the polylactosamine backbone, whereas FucT-VII fucosylates a GlcNAc residue of the nonreducing sialylated N-acetyllactosamine unit to generate the sLe x terminus (14).…”

mentioning

confidence: 99%

Glycosulfopeptides with O-Glycans Containing Sialylated and Polyfucosylated Polylactosamine Bind with Low Affinity to P-selectin

Leppänen

Penttilä

Renkonen

et al. 2002

P-selectin glycoprotein ligand-1 (PSGL-1), a dimeric mucin on leukocytes, is the best characterized ligand for selectins. P-selectin binds stereospecifically to the extreme N terminus of PSGL-1, which contains three clustered tyrosine sulfates (TyrSO 3 ؊ ) adjacent to a Thr residue with a core 2-based O-glycan expressing sialyl Lewis x (C2-O-sLe x ). GSP-6, a synthetic glycosulfopeptide modeled after the N terminus of PSGL-1, containing three TyrSO 3 ؊ residues and a short, monofucosylated C2-OsLe x bound to P-selectin with high affinity (K d ϳ650 nM). However, PSGL-1 from human HL-60 cells contains higher levels of O-glycans that are sialylated and polyfucosylated polylactosamines (PFPL). Furthermore, studies with fucosyltransferase-deficient mice suggest that sialylated PFPL structures contribute to binding to P-selectin. To resolve whether sialylated PFPL O-glycans participate in binding of PSGL-1 to human P-selectin, we synthesized glycosulfopeptides, designated GSP-6 and GSP-6؆, with three TyrSO 3 ؊ residues and either difucosylated polylactosamine (C2-O-Le x -sLe x ) or trifucosylated polylactosamine (C2-O-Le x -Le x -sLe x ). Binding of the GSPs to P-selectin was measured by affinity chromatography, fluorescence solid-phase assays, and equilibrium gel filtration. Unexpectedly, both GSP-6 and GSP-6؆ bound to P-selectin with low affinity (K d ϳ37 M for GSP-6 and K d ϳ50 M for GSP-6؆). Binding of GSP-6 and GSP-6؆ to P-selectin required fucosylation and, to a lesser extent, sialylation as well as the sulfated peptide backbone of GSP-6 and GSP-6؆. These results demonstrate that contrary to expectations, a core 2 O-glycan containing sialylated PFPL does not promote high affinity binding of PSGL-1 to P-selectin.