Glycosylation-enabled chemoselective growth factor engineering for biomaterial functionalization

In native tissue environments, growth factors (GFs) are often physically associated with the extracellular matrix (ECM) framework. Despite the enormous potential of chemoselective click chemistry for biomaterial functionalization to recapitulate such GF-ECM association, its application remains limited by constraints in the reliable production of clickable GFs. Here we present a platform technology that leverages intrinsic post-translational protein glycosylation to enable chemoselective engineering of GFs to incorporate click-reactive azido tags for subsequent ECM conjugation. Using Vascular Endothelial Growth Factor as a model, we demonstrated efficient, glycosylation-dependent azido incorporation during its recombinant expression with preserved bioactivity. We further expanded the utility of this strategy to non-glycosylated proteins through engineered N-linked glycosylation via the incorporation of a signal peptide, to direct newly synthesized proteins to the secretory pathway where glycosylation takes place, along with sequons for glycan attachment. The resulting GF with chemoselective azido incorporation can be effectively immobilized within dibenzocyclooctyne-bearing ECM hydrogel via copper-free click chemistry, exhibiting sustained GF retention and delivering augmented angiogenic responses. Our approach thereby offers an opportunity to streamline recombinant protein engineering for biomaterial functionalization in tissue engineering and regenerative medicine applications. STATEMENT OF SIGNIFICANCE: This manuscript describes a novel approach that uses natural sugar modification (glycosylation) of proteins to precisely modify growth factors (GFs) in a site-specific manner. By adding special chemical tags (azido tags) to these proteins, our approach streamlines the use of click chemistry for boosting biomaterial performance. Typically, each GF requires extensive individual optimization; however, our universal method simplifies the process, improving GF retention and functionality within biomaterials. Additionally, the technique can be applied to proteins that don't naturally have these sugar modifications by introducing engineered glycosylation. Overall, this technology offers an easy-to-use platform for researchers in tissue engineering, enhancing their ability to precisely place and deliver therapeutic proteins within biomaterial scaffolds, ultimately benefiting the broader field of regenerative medicine.