Carboxylated N-glycans on RAGE promote S100A12 binding and signaling

Area of Science:

• Molecular biology
• Immunology
• Glycobiology

Background:

• The receptor for advanced glycation end products (RAGE) is a key signaling receptor involved in various pathologies.
• RAGE binds diverse ligands, but the structural basis for these interactions remains unclear.
• Previous studies indicated carboxylated glycans on RAGE V-domain facilitate HMGB1 and S100A8/A9 binding.

Purpose of the Study:

• To investigate the role of carboxylated glycans on RAGE in the binding and intracellular signaling of the ligand S100A12.
• To understand how RAGE glycosylation impacts S100A12 interaction and downstream signaling pathways.

Main Methods:

• Analysis of S100A12 binding to RAGE subpopulations with varying glycan enrichment.
• Expression of RAGE in mammalian cells to study glycosylation patterns and S100A12 binding.
• Investigation of RAGE multimerization using deglycosylated RAGE and E. coli-expressed sRAGE.
• Functional assays using an anti-carboxylated glycan antibody (mAbGB3.1) to block S100A12-mediated NF-kappaB signaling.

Main Results:

• S100A12 specifically binds to carboxylated glycans on RAGE.
• A subpopulation of RAGE enriched for carboxylated glycans exhibited >10-fold higher S100A12 binding affinity.
• RAGE glycosylation, particularly at N25IT, is crucial for S100A12 binding and is cell-type dependent.
• Carboxylated glycan-rich RAGE forms higher-order complexes with S100A12, which is impaired by deglycosylation.
• mAbGB3.1 effectively blocked S100A12-induced NF-kappaB signaling in HeLa cells.

Conclusions:

• Carboxylated N-glycans on RAGE are critical for enhancing S100A12 binding affinity.
• These glycans promote RAGE clustering and multimerization, facilitating downstream signaling.
• Targeting these specific glycan modifications on RAGE could offer therapeutic strategies for RAGE-mediated pathologies.