Carbohydrate-based probes for detection of cellular lectins

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Cellular lectins, such as E-selectin, play crucial roles in cell adhesion and recognition.
• Developing specific probes is essential for studying lectin-carbohydrate interactions in biological systems.

Purpose of the Study:

• To synthesize and evaluate novel carbohydrate (Glyc) probes with various tags for studying cellular lectins.
• To assess the specificity and utility of these probes in detecting E-selectin expression on cells and in tumor tissues.

Main Methods:

• Synthesis of Glyc-polyacrylamide (PAA) probes, including radiolabeled (3H), biotinylated, fluorescein (flu), and digoxigenin-labeled variants.
• Binding assays using Chinese hamster ovary (CHO) cells transfected with E-selectin cDNA or mock-transfected.
• Flow cytometry and cell enzyme-linked assays to assess probe specificity and binding.
• In vivo studies using tumor sections from nude mice xenografted with CHO cells.

Main Results:

• Sialyl Lewis X (SiaLeX)-PAA-3H probes showed high binding to E-selectin-transfected cells, confirming minimal contribution from the PAA carrier and spacer.
• Biotinylated probes exhibited high non-specific binding, while digoxigenin-labeled probes performed better.
• Fluorescein-labeled SiaLeX-PAA-flu probes demonstrated specific binding to E-selectin-expressing cells, comparable to anti-E-selectin antibodies and inhibitable by them.
• The fluorescent probe successfully bound to tumor sections from E-selectin-positive CHO cells but not control cells.

Conclusions:

• The developed Glyc-PAA probes, particularly the fluorescein-labeled variant, are effective tools for specifically detecting carbohydrate-binding sites.
• These probes can accurately identify E-selectin expression on cells in culture and in tissue sections, including tumors.
• The findings validate the use of these probes for studying lectin-mediated cellular interactions and diagnostics.