Griffonia simplicifolia I isolectin as a functionally monovalent probe for use in flow cytometry

Abstract

The inherent tendency of lectins to agglutinate cells has limited their use as reagents for the detection of carbohydrate groups on cell surfaces by flow cytometry. In the current study, we demonstrate a method for the use of a fluoresceinated tetrameric isolectin (Griffonia simplicifolia I-A3B, FITC-GS I-A3B) as a functionally monovalent, nonagglutinating probe in flow cytometry. This isolectin contains three A subunits and one B subunit. Both types of subunits bind alpha-D-galactopyranosyl (alpha-D-galp-) end groups with similar affinities; however, the A subunits have a 1,000-fold greater affinity for N-Acetyl-D-galactosamine (GalNAc) than does the B subunit. The addition of low (1-2 mM) concentrations of GalNAc to the FITC-GS I-A3B isolectin results in blockage of the three A subunits without significantly affecting the B subunit; this yields a functionally monovalent probe for the detection of cell surface alpha-D-Galp end groups. This approach has been used to examine two types of cells: Ehrlich ascites tumor cells and rat alveolar macrophages, both of which are known to express cell surface alpha-D-Galp end groups. Lectin binding, as determined by number of positive cells and fluorescence intensity, was dependent upon concentration of the lectin and haptenic sugar. Specificity of the staining was demonstrated by the ability of methyl alpha-D-galactopyranoside (Met alpha-D-Galp) to abolish the binding of the lectin to the cells. Elimination of both GalNAc and Met alpha-D-Galp from the staining solution resulted in agglutination of the cells, indicating that the A subunits were active in the absence of GalNAc.

Preparation of a stable subunit of Japanese elderberry (Sambucus sieboldiana) bark lectin and its application for the study of cell surface carbohydrates by flow cytometry

“…FlTC-labelling of SSA and MSSA, and staining of U937 cells with FlTC-lectins were carried out by the method of McCoy et al [8]. The molar ratio of fluoresceirt/prot¢in of SeA and MSSA was 0.2 for both of them.…”

“…To avoid the agglutination problem in flow eytometry, McCoy et al also used FITC.Griffonia shnpficifolia I-A3B (GS I-A3B) in the presence of GalNAc as the functionally monovalent probe for the detection of cell surface ~-D-Gal residue [8], although this approach can not be generally applied. We describe herein the preparation and characterization of a stable monomeric subunit of Japanese elderberry (Sambucus a'ieboldiana) bark lectin (SSA) [9], which is specific to NeuSAc~2-6Gal/GalNAc sequences, after selective reduction and alkylation with 4-vinylpyridine.…”

The application of lectins to the characterization and isolation of mammalian cell populations

“…One way to overcome these problems was proposed by McCoy and coworkers, who used a tetrameric isolectin of Griffonia simplicifolia [82]. In this investigation a functionally monovalent probe was developed using an intact tetrameric molecule.…”