Analysis of Galectin-Binding Receptors on B Cells

Abstract

The reported roles of the β-galactoside-binding lectin family, known as galectins, in disease development have been advancing at a remarkable pace. Galectins and their glycan counter-receptor ligands are now considered key functional determinants in malignant and metastatic progression, tumor immune evasion, autoimmunity, and immune homeostasis. Their influence in these processes is elicited through coordinated expression in tumor, immune and stromal cellular compartments. While analysis of galectin levels in related research efforts is routinely performed through immunoassays and RT-qPCR, detection, and identification of glycan counter-receptor ligands in their native form on the cell surface has lagged. In this report, we present methods to detect and identify glycan counter-receptor ligands to galectin (Gal)-3 and Gal-9-two galectins at the crosshairs of cancer and immunology research. As a model, we will describe (1) isolation of human B-cell subsets from fresh tonsil tissue, (2) assaying of Gal-3/-9-binding activities on human B cells, and (3) identifying Gal-3/-9 ligands on human B-cell surfaces. These methods, of course, can be implemented on any cell type to provide a cellular and molecular context capable of transmitting a galectin-mediated phenotype. Establishing a galectin-binding activity on specific counter-receptor ligand(s) can help unearth potential critical determinants capable of delivering cellular signals required for disease progression. These advances open new avenues of research investigation that result in novel therapeutic targets and approaches.

Keywords: B cells; Galectins; Ligands; Poly-N-acetyllactosamines; Receptors; T cells.

Fig. 1

Isolation of tonsillar mononuclear cell layer from Ficoll density gradient. Minced tonsillar tissues are processed using the cell strainer. The strained cells are passed through 70-μm nylon mesh to get a single cell suspension, which layered over Ficoll/Histopaque and then centrifuged. The mononuclear cell layer appears as a cloudy, thin layer between the Ficoll and wash media. (Cartoon image created using Biorender illustrating tool (Biorender.com))

Fig. 2

Immunoprecipitation of galectin-binding receptors on B cells. Viable B-cell isolates are first biotinylated and then incubated with rhGalectins to bind specific counter-receptor glycoprotein ligands. Cells are lysed and incubated with anti-galectin antibodies, forming an antibody–galectin–ligand complex. These complexes are then pulled down using Protein A or G coupled beads. (Cartoon image created using Biorender illustrating tool (Biorender.com))