Synthesis and characterization of biologically functional biotinylated RANTES

Area of Science:

• Immunology
• Biochemistry
• Chemical Biology

Background:

• Chemokines mediate immune responses and are implicated in various diseases.
• Labeled chemokines are valuable tools for studying their biological functions.
• RANTES (regulated upon activation normal T cell expressed and secreted) is a key chemokine in inflammatory processes.

Purpose of the Study:

• To synthesize and characterize biotinylated RANTES derivatives.
• To evaluate the binding affinity and biological activity of these derivatives.
• To identify a labeled RANTES variant suitable for studying RANTES-chemokine interactions in physiological and pathological conditions.

Main Methods:

• Chemical synthesis of RANTES derivatives biotinylated at specific residues (1, 25, 33, 45, 67).
• Gel filtration and ultracentrifugation to assess aggregation properties.
• Competition binding assays using CCR5-expressing cells.
• Flow cytometry to analyze binding to primary macrophages.
• Enzymatic treatment with GAG-specific enzymes to assess GAG-dependent binding.
• Functional assays including CCR5 expression modulation and monocyte chemotaxis.

Main Results:

• Biotinylation at positions 45 or 67 reduced RANTES aggregation.
• RANTES derivatives biotinylated at positions 1, 25, and 67 exhibited similar binding affinity to CCR5 as native RANTES.
• RANTES biotinylated at residue 67 (B67-RANTES) showed enhanced binding to primary macrophages.
• Binding of B67-RANTES to cells was dependent on glycosaminoglycans (GAGs).
• B67-RANTES functioned as a CCR5 agonist, modulating CCR5 expression and eliciting monocyte chemotaxis.

Conclusions:

• Biotinylated RANTES derivatives were successfully synthesized and characterized.
• B67-RANTES is a functional CCR5 agonist with enhanced binding to macrophages.
• B67-RANTES is a valuable tool for investigating RANTES function in inflammatory disorders and HIV infection.