Orthogonal Acyl Acceptor Substrate Pairs and hQC Improve Sequential Ligation by Peptide Asparaginyl Ligases

As highly versatile enzymatic tools for bioconjugation, peptide asparaginyl ligases (PALs) catalyze ligation reactions at Asn/Asp residues through transpeptidation. While Asn-ligation is favored over Asp-ligation, their different pH requirements make it possible to use Asn- and Asp-ligations sequentially for conjugating multiple payloads onto a protein. However, because the protein substrate for sequential ligation contains both an acyl donor at the C-terminus and an acceptor at the N-terminus, it can undergo self-ligation, which leads to cyclized or dimerized side products. Furthermore, the significantly slower Asp-ligation step severely limits the efficiency of the sequential ligation scheme. Here, we report the design of orthogonal acyl acceptor substrates to eliminate cyclization or dimer formation of the protein substrate and the incorporation of human glutaminyl cyclase (hQC) to improve the efficiency of Asp-ligation by rendering it irreversible. As PAL-mediated Asp-ligations and hQC catalysis may require different operating conditions, we performed a pH scan experiment using short model peptide substrates and identified optimal Asp-ligation conditions with OaAEP1b-C247A and hQC at pH 7 that maximize ligation while minimizing unwanted hydrolysis. We further identified orthogonal dipeptide acceptor substrates differing in their ability to be processed by the PAL enzyme. Using the optimal conditions and orthogonal acceptor pair, we then demonstrated C-to-N sequential ligation by first attaching a fluorescein tag to anti-HER2 DARPin, followed by conjugation with anti-EGFR affibody without intermediatory protection/deprotection steps. This produced a fluorescent-labeled anti-EGFR and anti-HER2 bispecific protein that was functional when assessed in representative EGFR- and HER2-positive model cell lines (A431 and BT474). Our work highlights the versatility of PALs in bioconjugation with a wide range of protein sizes and presents a promising approach to producing multicomponent biologics for pharmacological applications.