Use of an asparaginyl endopeptidase for chemo-enzymatic peptide and protein labeling.

T M Simon Tang,Davide Cardella,Alexander J Lander,Yu-Hsuan Tsai,Louis Y P Luk,Xuefei Li,Jorge S Escudero

doi:10.1039/d0sc02023k

T M Simon Tang, Davide Cardella + Show 5 more

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https://doi.org/10.1039/d0sc02023k

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Abstract

Asparaginyl endopeptidases (AEPs) are ideal for peptide and protein labeling. However, because of the reaction reversibility, a large excess of labels or backbone modified substrates are needed. In turn, simple and cheap reagents can be used to label N-terminal cysteine, but its availability inherently limits the potential applications. Aiming to address these issues, we have created a chemo-enzymatic labeling system that exploits the substrate promiscuity of AEP with the facile chemical reaction between N-terminal cysteine and 2-formyl phenylboronic acid (FPBA). In this approach, AEP is used to ligate polypeptides with a Asn-Cys-Leu recognition sequence with counterparts possessing an N-terminal Gly-Leu. Instead of being a labeling reagent, the commercially available FPBA serves as a scavenger converting the byproduct Cys-Leu into an inert thiazolidine derivative. This consequently drives the AEP labeling reaction forward to product formation with a lower ratio of label to protein substrate. By carefully screening the reaction conditions for optimal compatibility and minimal hydrolysis, conversion to the ligated product in the model reaction resulted in excellent yields. The versatility of this AEP-ligation/FPBA-coupling system was further demonstrated by site-specifically labeling the N- or C-termini of various proteins.

Highlights

There has been a vast expansion in the toolkit of protein bioconjugation,[1,2,3] drawing on expertise from both biological[4,5,6] and organic[7,8,9,10,11,12] chemistry
These descriptions apply to the bioconjugation reaction mediated by the enzyme asparaginyl endopeptidase (AEP) and the chemical labeling of School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff, CF10 3AT, UK
We propose that the nonnatural secondary amine motif formed between formyl phenylboronic acid (FPBA) and Nterminal cysteine is unlikely to be a reactive substrate for AEP catalysis, and the byproduct of the enzymatic reaction Cys– Leu can be potentially trapped by the addition of FPBA

Summary

Introduction

There has been a vast expansion in the toolkit of protein bioconjugation,[1,2,3] drawing on expertise from both biological[4,5,6] and organic[7,8,9,10,11,12] chemistry. Protein-based approaches offer the ability to function efficiently under mild reaction conditions.[5,6] Transferase,[13,14,15] oxidoreductase,[16,17,18] ligase,[19,20] transpeptidase[2,4,21,22,23,24,25,26,27] and (split-)intein[28,29,30] have been applied for protein bioconjugation. Whereas stability and solubility of intein-fused constructs are extremely case-dependent,[28,29,30] reversible enzymatic reactions need to be suppressed by a large excess of label[24,31] or unstable substrate that has inherent limitation to where bioconjugation takes place.[32,33,34] In contrast, chemical approaches with commercially available reagents are simple to perform and have become standard practice.[4,8,9,10] efficiency and selectivity of these reactions relies on the availability of speci c residues, which varies greatly among proteins.[2,35] These descriptions apply to the bioconjugation reaction mediated by the enzyme asparaginyl endopeptidase (AEP) and the chemical labeling of

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Conclusion