Quantitative Measurement of Selectin-Ligand Interactions: Assays to Identify a Sweet Pill in a Library of Carbohydrates

Abstract

Soluble oligosaccharides and glycoproteins can inhibit leukocyte adhesion during a range of vascular ailments including inflammation, thrombosis, and cancer metastasis. The design of such molecules in many cases is based on the structure of naturally occurring selectin ligands. In this case, synthetic selectin-ligand mimetics act as competitive inhibitors of cell adhesion. In an alternate approach, cell-permeable, small-molecule oligosaccharides have been shown to alter the metabolic pathways that lead to the biosynthesis of functional selectin-ligands. The addition of such molecules results in glycoproteins that are defective in their ability to bind selectins. Quantitative in vitro testing of the efficacy of the above inhibition strategies ideally requires the application of assays that mimic the in vivo physiological milieu in terms of the valency of selectin and selectin-ligands, the physiological fluid-flow conditions, and the use of blood cells. Assays that are performed in small volumes are preferable when the quantity of available inhibitor is scarce. Finally, the measurements must account for the rapid on- and off-rates of selectin-mediated binding interactions. This chapter addresses these issues by presenting methods to measure selectin function in enzyme-linked immunosorbent assay and flow cytometry-based static assays, cell-adhesion assays performed under shear flow in cone-plate viscometers, and Biacore surface plasmon resonance measurements of molecular-binding kinetics. Examples are presented where such methods are applied to measure the ability of simple oligosaccharides based on sialyl Lewis-X and complex molecules with the core-2 structure to block selectin function. Such methods may be extended to identify potent selectin antagonists in a library of carbohydrates.

Fluorinated per-acetylated GalNAc metabolically alters glycan structures on leukocyte PSGL-1 and reduces cell binding to selectins

“…The specificity of the P-selectin binding assay was confirmed in control studies (supplemental Figure 2). 34,38 Observations similar to those shown in Figure 2E-H were made in runs where cells were not treated with neuraminidase before the addition of acetylated monosaccharide ( Figure 2I-L). Here, too, 50M 4F-GalNAc was more effective compared with 50M 4F-GlcNAc at reducing P-selectin binding (66% vs 22% at 38 hours, Figure 2I), CLA/HECA (82% vs 20% at 38 hours, Figure 2J), and LacNAc (65% vs 22% at 38 hours, Figure 2L) epitope expression.…”

Systems‐level studies of glycosyltransferase gene expression and enzyme activity that are associated with the selectin binding function of human leukocytes

“…Flow cytometry measurements were performed with HL‐60 cells and neutrophils as described previously (4, 20), with minor modifications. Briefly, cells obtained at fixed time points were washed and resuspended in 30 mM HEPES buffer (pH = 7.2) containing 1.5 mM CaCl 2 and 0.1% human serum albumin at 0.5 × 10 6 cells/ml.…”

Biosensors for health applications

“…Non-invasive glucose sensing is the ultimate goal of glucose monitoring and the main approaches being pursued for glucose sensor development are: near infrared spectroscopy, excreted physiological fluid (tears, sweat, urine, saliva) analysis, microcalorimetry, enzyme electrodes, optical sensors, sonophoresis and iontophoresis, both of which extract glucose from the skin (Koschwanez & Reichert, 2007;Beauharnois et al, 2006;Chu et al, 2011). Despite the relative ease of use, speed and minimal risk of infection involved with infrared spectroscopy, this technique is hindered by the low sensitivity, poor selectivity, frequently required calibrations, and difficulties with miniaturization.…”