Abstract
Selectins are a family of lectins, that mediate tethering and rolling of leukocytes on endothelium in vascular shear flow. Mild periodate oxidation of the L-selectin ligand CD34, or L-selectin ligands on leukocytes, enhanced resistance to detachment in shear and decreased rolling velocity equivalent to an 8-fold increase in ligand density, yet had little effect on the rate of tethering. Enhanced interactions were also seen with mildly oxidized sialyl Lewisa and sialyl Lewisx glycolipids. Enhancement was completely reversed by borohydride reduction, yielding a strength of interaction equivalent to that with the native ligands. No effect on the strength of P-selectin and E-selectin interactions was seen after mild oxidation of their ligands. Completeness of modification of sialic acid by mild periodate was verified with monoclonal antibody to sialyl Lewisx-related structures and resistance to neuraminidase. The addition of cyanoborohydride to leukocytes rolling through L-selectin on mildly oxidized but not native CD34 caused arrest of rolling cells and formation of EDTA-resistant bonds to the substrate, suggesting that a Schiff base was reduced. Cyanoborohydride reduction of mildly oxidized cells rolling on P-selectin and E-selectin also caused arrest and formation of EDTA-resistant bonds but with slower kinetics. These data suggest that interactions with a sialic acid aldehyde group on mildly oxidized ligands that include interconversion to a Schiff base can occur with three selectins yet only stabilize binding through the selectin with the fastest koff, L-selectin.
Highlights
GlyCAM-1 [6] and CD34 [7]
L-selectin, and Not E-selectin and P-selectin, Shows Enhanced Binding to Ligands Subjected to Mild Periodate Oxidation—The CD34 component of peripheral node addressin (PNAd) immobilized on the lower wall of a flow chamber was subjected to mild (5 mM) periodate treatment, under conditions that are selective for sialic acid and result in cleavage of C-8 and C-9 and oxidation of C-7 of sialic acid to an aldehyde [25, 26]
We have determined the effect of mild periodate oxidation of the carbohydrate ligands of L-selectin, Eselectin, and P-selectin on adhesive interactions in hydrodynamic shear flow
Summary
Monoclonal Antibodies and Cell Lines—Monoclonals 581 (IgG1) and 547 (IgG2a, ) to human CD34 [27, 28] were kindly provided by Dr Gustav Gaudernack (Oslo, Norway). Cells were harvested by a 10-min incubation with H/H medium (0.2% human serum albumin, Hanks’ balanced salt solution, 10 mM HEPES, pH 7.3) containing 5 mM EDTA at 37 °C, washed and resuspended in the same medium at 2 ϫ 107 cells/ml, and kept at room temperature until use. Cells were washed with and suspended in H/H binding medium (Ca2ϩ and Mg2ϩ-free Hanks’ balanced salt solution supplemented with 2 mM Ca2ϩ, 0.2% human serum albumin, 10 mM HEPES, pH 7.3) before flow assays. Perfusion at 0.84 dyn/cm was continued with 5 mM sodium cyanoborohydride (Sigma) in phosphate-buffered saline (pH 7.2) containing 2 mM Ca2ϩ for 5–10 min at room temperature and with Hanks’/HEPES containing 5 mM EDTA to detach noncovalently linked cells
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