Global immunostimulatory mechanisms of a high-branched glycogen from Glyptocidaris crenularis eggs in RAW264.7 macrophages via BONCAT-based nascent proteomics.

Marine polysaccharides have emerged as promising immunomodulators. However, conventional analytical approaches have failed to elucidate their activation mechanisms from a global perspective. In this study, we isolated a highly branched glycogen (GCEP2a) from eggs of the sea urchin Glyptocidaris crenularis. Structural characterization revealed that GCEP2a possesses a backbone composed of →4)-α-D-Glcp-(1 → residues, with one C6-branch per five residues, and a molecular weight of 8.06 × 103 kDa. Employing nascent-proteome bioorthogonal non-canonical amino acid tagging (BONCAT) enrichment followed by DIA-FAIMS, we unraveled the global immunostimulatory mechanisms of GCEP2a in RAW264.7 macrophages. This advanced approach enabled dynamic profiling of nascent protein synthesis, uncovering a multi-receptor and multi-pathway regulatory network. Specifically, GCEP2a activates signaling consistent with engagement of the toll-like receptors (TLRs), C-type lectin receptors (CLRs), and RIG-I-like receptors (RLRs), thereby triggering downstream signaling cascades including the NF-κB, MAPK, and PI3K-AKT pathways. Additionally, GCEP2a downregulates the biosynthesis of lacto- and neolacto-series glycosphingolipids, indicating metabolic reprogramming associated with M1 polarization. These findings not only suggest that GCEP2a exhibits potential as an immune adjuvant, but also provide initial insights into the application of BONCAT-based nascent proteomics for investigating the multi-modal immunostimulatory mechanisms of marine polysaccharides.