Fluorogenic glycopolymers available for determining the affinity of lectins by intermolecular FRET.

A convenient assembly of fluorogenic glycopolymers having various polymer compositions was accomplished from the corresponding glycomonomer and dansyl monomer by means of radical polymerization, and the water-soluble glycopolymers gave typical fluorescence spectroscopic profiles due to the dansyl moieties on the glycopolymer in aqueous media. Biological evaluation of the polymer against wheat germ agglutinin (WGA) was accomplished on the basis of fluorescence changes due to tryptophan residues on WGA, and the affinities between the glycopolymers and WGA were estimated to be 4.7 × 105 to 9.3 × 105 M-1. In order to apply the fluorogenic glycopolymers for further biological measurements, efficient resonance energy transfer from tryptophan moieties on WGA to dansyl moieties on the fluorogenic glycopolymers was examined. FRET profiles of both fluorophores were similar compared to the binding profiles on the basis of fluorescence changes of tryptophan residues. This approach is applicable for the determination of an affinity constant between a carbohydrate and a lectin in which no fluorophore exists near the binding site.

Rapid endocytosis and recycling of wheat germ agglutinin binding sites on CHO cells: evidence for two compartments in a nondegradative pathway.

The internalization and recycling of CHO cell plasma membrane components have been quantified by using iodinated wheat germ agglutinin (WGA) as an adsorptive tracer. Most of these binding sites are thought to be composed of a subpopulation of plasma membrane proteins called high-molecular-weight acidic glycoproteins (HMWAG). Greater than 90% of the WGA bound on the cell surface can be removed by brief treatment with N-acetylglucosamine (GlcNAc). At 37 degrees C, endocytosis of WGA that had been allowed to bind to the surface at 4 degrees C is curvilinear with an initial rapid phase occurring with a t1/2 of 6-8 min. Within 20 min, accumulation has slowed gradually to steady-state with 65% of the cell-associated WGA located intracellularly or resistant to removal by GlcNAc. These portions are unaffected by increasing the extracellular concentration of WGA from 0.003 microM to 2.8 microM. By using pulse-chase experiments, the observed decrease in rate of endocytosis is shown to be due to return of the WGA-HMWAG complexes to the cell surface. More than 60% of the WGA that had been internalized is recycled within 30 min, with a mean t1/2 of 17 min. Recycling involved at least two intracellular populations where a significant fraction (less than 30%) of the WGA-HMWAG complexes are lost gradually from the rapidly recycling pool. Most of the WGA-HMWAG complexes that had internalized are not delivered to the lysosome. These results demonstrate the magnitude of rapid and continuous recycling of WGA binding sites between the cell surface and endosomes in fibroblasts.