Distinct glycosyltransferases synthesize E-selectin ligands in human vs. mouse leukocytes

The binding of selectins to carbohydrate epitopes expressed on leukocytes is the first step in a multi-step cell adhesion cascade that controls the rate of leukocyte recruitment at sites of inflammation. The glycans that function as selectin-ligands are post-translationally synthesized by the serial action of Golgi resident enzymes called glycosyltransferases (glycoTs). Whereas much of our current knowledge regarding the role of glycoTs in constructing selectin-ligands comes from reconstituted biochemical investigations or murine models, tools to assess the impact of these enzymes on the human ligands are relatively underdeveloped. This is significant since the selectin-ligands, particularly those that bind E-selectin, vary between different leukocyte cell populations and they are also different in humans compared with mice. To address this shortcoming, a recent study by Buffone et al. (2013) outlines a systematic strategy to knockdown upto three glycoTs simultaneously in human leukocytes. The results suggest that the fucosyltransferases (FUTs) regulating selectin-ligand synthesis may be species-specific. In particular, they demonstrate that FUT9 plays a significant role during human, but not mouse, leukocyte-endothelial interactions. Overall, this article discusses the relative roles of the FUTs during human L-, E-, and P-selectin-ligand biosynthesis, and the potential that the knockdown strategy outlined here may assess the role of other glycoTs in human leukocytes also.     

Chemoenzymatic Synthesis of Optically Active Diacylglycerophospholipid Hydroperoxides Derived from Arachidonic Acid

A new derivative of sulfatide, 2-O-α-l-fucopyranosyl sulfatide, was synthesized. The compound inhibited the binding of HL-60 cells, which express sialyl Lewis X, to P- and L-selectin more than the corresponding non fucosylated compound.     

Cell separation mediated by differential rolling adhesion

Recently, we showed a correlation between the maturity of hematopoietic stem and progenitor cells during development and rolling efficiency on selectins. These findings motivated us to explore a novel separation that exploits differences in selectin-mediated rolling adhesion between populations of cells. We extend the use of a previously developed cell-free system to study the separation of populations of sialyl Lewis x (sLe(x))-coated microspheres designed to roll with different average velocities on L-selectin chimeric substrates under well-defined flow. Results show that a separation that exploits differences in average rolling velocities between cell or microsphere populations is attainable. Excellent recovery and purity values for the slower rolling, or more desirable, populations are obtained and can be estimated from rolling velocity measurements. We also assess the feasibility of a selectin-mediated separation of adult bone marrow cell populations using previously obtained rolling velocity and rolling flux data for CD34+ and CD34- adult bone marrow cells on L-selectin substrates. We believe that a cell separation mediated by differential rolling adhesion can be used to enrich populations of hematopoietic stem and progenitor cells from an adult bone marrow cell preparation and that this method possesses several major advantages over existing antibody-mediated cell-affinity chromatography technologies.     

Signal-dependent distribution of cell surface P-selectin in clathrin-coated pits affects leukocyte rolling under flow

Flowing leukocytes roll on P-selectin that is mobilized from secretory granules to the surfaces of endothelial cells after stimulation with histamine or thrombin. Before it is internalized, P-selectin clusters in clathrin-coated pits, which enhances its ability to support leukocyte rolling. We found that thrombin and histamine induced comparable exocytosis of P-selectin on endothelial cells. However, compared with histamine, thrombin decreased the recruitment of P-selectin into clathrin-coated pits, slowed the internalization of P-selectin, and reduced the number and stability of neutrophils rolling on P-selectin. Significantly more RhoA was activated in thrombin- than in histamine-stimulated endothelial cells. Inhibitors of RhoA or its effector, Rho kinase, reversed thrombin's ability to inhibit the internalization and adhesive function of P-selectin in endothelial cells. Experiments with transfected cells confirmed that the inhibitory actions of thrombin and Rho kinase on P-selectin required its cytoplasmic domain. Thus, a signaling event affects both the function and clearance of a protein that enters the constitutive clathrin-mediated endocytic pathway.     

Effect of the sialyl Lewis X (SLe(x)) moiety on splenic accumulation of SLe(x)-carboxymethylpullulan conjugate

Sialyl Lewis X (SLe(x)), an E-selectin ligand, was conjugated with carboxymethylpullulan (CMPul) and the disposition characteristics of this conjugate after intravenous administration were investigated using mice with ear edema. The concentration of 3H-labeled SLe(x)-CMPul in the spleen was significantly high. When CMPul was modified with a saccharide unable to bind to E-selectin, this splenic accumulation was not observed. The uptake of radiolabeled SLe(x)-CMPul by the spleen was completely inhibited by a 100-fold molar of cold SLe(x)-CMPul but not by a sialyl N-acetyllactosamine-CMPul conjugate (SLN-CMPul). Microautoradiography analyses revealed that SLe(x)-CMPul accumulated in the marginal zone of the spleen.     

Dimerization of P-Selectin Glycoprotein Ligand-1 (PSGL-1) Required for Optimal Recognition of P-Selectin

Interactions between P-selectin, expressed on endothelial cells and activated platelets, and its leukocyte ligand, a homodimer termed P-selectin glycoprotein ligand-1 (PSGL-1), mediate the earliest adhesive events during an inflammatory response. To investigate whether dimerization of PSGL-1 is essential for functional interactions with P-selectin, a mutant form of PSGL-1 was generated in which the conserved membrane proximal cysteine was mutated to alanine (designated C320A). Western blotting under both denaturing and native conditions of the C320A PSGL-1 mutant isolated from stably transfected cells revealed expression of only a monomeric form of PSGL-1. In contrast to cells cotransfected with α1-3 fucosyltransferase-VII (FucT-VII) plus PSGL-1, K562 cells expressing FucT-VII plus C320A failed to bind COS cells transfected with P-selectin in a low shear adhesion assay, or to roll on CHO cells transfected with P-selectin under conditions of physiologic flow. In addition, C320A transfectants failed to bind chimeric P-selectin fusion proteins. Both PSGL-1 and C320A were uniformly distributed on the surface of transfected K562 cells. Thus, dimerization of PSGL-1 through the single, conserved, extracellular cysteine is essential for functional recognition of P-selectin.     

P-selectin/ligand unbinding force measured with atomic force microscopy: comparison of two chemical protocols for the tethering of single molecules

Leukocytes, as an indispensable arm of the immune system, need to be recruited from the flowing blood and transferred to the sites of infection. Their extravasation is feasible due to their ability to tether and roll over the activated endothelium, which is much dependent on the association of their selectin molecules with ligands on the activated endothelial cells. In view of the importance of this interaction for the physiological immune functions as well as for autoimmune diseases, specifying the affinity of selectins to their ligands at the single molecule level appears a challenging task to gain insight into the mechanisms that control leukocyte–endothelial avidity. To this end we functionalized substrates with P‐selectin and cantilever probes with its major ligand, the P‐selectin glycoprotein ligand‐1, and used atomic force microscopy to measure their unbinding force. Two different chemical protocols were used for the tethering of the molecules on the substrates, one based on a homobifunctional poly(ethylene glycol) linker and the other on the use of antibody‐specific binding. The unbinding forces measured with the two methods were 312 ± 149 and 230 ± 57 pN, respectively. Measurements on activated endothelials, declaratory of single molecule interactions, gave comparable results. Copyright © 2011 John Wiley & Sons, Ltd.