Revealing the Biological Effect of the <i>N</i> -glycosylation of High-Mobility Group Box 1 (HMGB1) Facilitated by Chemical Protein Synthesis

High Resolution Image Download MS PowerPoint Slide N -glycosylation is one of the most ubiquitous protein post-translational modifications with both high structural diversity and critical functions in broad molecular biological processes. The effect of N -glycans on high-mobility group box 1 (HMGB1) has not been clearly elucidated. Chemical protein synthesis, in combination with enzymatic transglycosylation, provides a robust approach to obtain homogeneous N -glycoproteins with well-defined glycan structures on designated sites, therefore allowing the investigation of the biological effects of N -glycosylation on site- and structure-specific levels. In this work, the synthetic route toward full-length fully reduced HMBG1 was established, which was adopted to the syntheses of N -glycosylated HMBG1 variants bearing sialylated biantennary N -glycans. The synthetic N -glycoproteins were employed to investigate the effects of N -glycosylation on the interactions of HMGB1 with receptors RAGE, as well as the HMGB1-stimulated cell migration and CXCL12 secretion. The potential effect of N -glycans on the HMGB1/RAGE interaction was investigated via molecular dynamics simulations.