Peptide ligases: A Novel and potential enzyme toolbox for catalytic cross-linking of protein/peptide-based biomaterial scaffolds for tissue engineering

The fabrication of novel biomaterial scaffolds with improved biological interactions and mechanical properties is an important aspect of tissue engineering. The three-dimensional (3D) protein/peptide-based polymeric scaffolds are promising in vitro biomaterials to replicate the in vivo microenvironment mimicking the extracellular matrix (ECM) for cell differentiation and subsequent tissue formation. Among different strategies in the fabrication of scaffolds, bioorthogonal enzymatic reactions for rapid in situ zero-length cross-linking are advantageous. Peptide ligases as a novel toolbox have the potentiality to enzymatically cross-link natural/synthetic protein/peptide-based polymeric chains for a wide range of biomedical applications. Although natural peptide ligases, such as sortases and butelase 1 are known cysteine proteases with ligase activity, some serine proteases, such as trypsin and subtilisin, are protein engineered to form trypsiligase and subtiligase, respectively, which exhibited efficient ligase activity by linking proteins/peptides with a great variety of molecules. Peptide ligase activity by these engineered proteases is more efficient than the hydrolysis of peptide bonds (peptidase activity). Peptide esters form acyl-enzyme intermediate with serine/cysteine residues of these proteases, with subsequent aminolysis forming covalent peptide bond with N-terminal residue of another polymeric chain. In addition, peptide ligases have the potential to conjugate with cell-adhesive ECM proteins or motifs and growth factors to (bio)polymeric networks to enhance cell attachment, growth, and differentiation. Here, we review the potential and limitations of natural and engineered peptide ligases as an enzyme toolbox with a focus on sortases (classes A-D), butelase 1, trypsiligase, and subtilisin variants, and the mechanisms for their zero-length cross-linking of (bio)polymeric scaffolds for various tissue engineering and regenerative applications.