Binding assay between murine Dectin-1 and β-glucan/DNA complex with quartz-crystal microbalance

Area of Science:

• Biochemistry
• Immunology
• Materials Science

Background:

• Schizophyllan (SPG), a β-glucan, forms complexes with polynucleotides.
• These SPG/oligonucleotide complexes are investigated as Dectin-1 targeted delivery systems for immunocytes.
• Dectin-1 is a membrane receptor recognizing β-glucans, but direct binding of SPG/oligonucleotide complexes to Dectin-1 requires clarification.

Purpose of the Study:

• To investigate the binding affinity between SPG/poly(dA) complexes and the extracellular domain of murine Dectin-1.
• To elucidate the binding mechanism and identify potential binding sites.
• To assess the impact of modifying the oligonucleotide backbone on binding characteristics.

Main Methods:

• Utilized quartz-crystal microbalance (QCM) to measure binding affinity.
• Examined complexes of SPG with poly(dA) featuring phosphodiester and phosphorothioate linkages.
• Used a constructed protein representing the carbohydrate-recognition domain of murine Dectin-1.

Main Results:

• SPG/poly(dA) with phosphodiester linkage showed recognition by Dectin-1, but with lower affinity (larger Kd) than SPG alone.
• SPG/poly(dA) with phosphorothioate linkage (dA60(S)) exhibited significantly enhanced QCM frequency decrease, indicating stronger binding.
• Multiple binding sites were suggested for dA60(S), including the SPG binding site and an additional site involving phosphate anion electrostatic interactions.

Conclusions:

• SPG/oligonucleotide complexes can bind to Dectin-1, with binding affinity modulated by the oligonucleotide backbone.
• Phosphorothioate modification introduces additional binding interactions, potentially increasing the efficacy of Dectin-1 targeted delivery.
• The findings support the potential of SPG/oligonucleotide complexes as targeted delivery vehicles for immunomodulatory therapeutics.