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

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.

SUBMITTER: Tang TMS

PROVIDER: S-EPMC7441500 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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

Tang T M Simon TMS Cardella Davide D Lander Alexander J AJ Li Xuefei X Escudero Jorge S JS Tsai Yu-Hsuan YH Luk Louis Y P LYP

Chemical science 20200512 23

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 reactio ...[more]

PMID: 32874509

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Project description:Background: Protein theranostics integrate both diagnostic and treatment functions on a single disease-targeting protein. However, the preparation of these multimodal agents remains a major challenge. Ideally, conventional recombinant proteins should be used as starting materials for modification with the desired detection and therapeutic functionalities, but simple chemical strategies that allow the introduction of two different modifications into a protein in a site-specific manner are not currently available. We recently discovered two highly efficient peptide ligases, namely butelase-1 and VyPAL2. Although both ligate at asparaginyl peptide bonds, these two enzymes are bio-orthogonal with distinguishable substrate specificities, which can be exploited to introduce distinct modifications onto a protein. Methods: We quantified substrate specificity differences between butelase-1 and VyPAL2, which provide orthogonality for a tandem ligation method for protein dual modifications. Recombinant proteins or synthetic peptides engineered with the preferred recognition motifs of butelase-1 and VyPAL2 at their respective C- and N-terminal ends could be modified consecutively by the action of the two ligases. Results: Using this method, we modified an EGFR-targeting affibody with a fluorescein tag and a mitochondrion-lytic peptide at its respective N- and C-terminal ends. The dual-labeled protein was found to be a selective bioimaging and cytotoxic agent for EGFR-positive A431 cancer cells. In addition, the method was used to prepare a cyclic form of the affibody conjugated with doxorubicin. Both modified affibodies showed increased cytotoxicity to A431 cells by 10- and 100-fold compared to unconjugated doxorubicin and the free peptide, respectively. Conclusion: Bio-orthogonal tandem ligation using two asparaginyl peptide ligases with differential substrate specificities is a straightforward approach for the preparation of multifunctional protein biologics as potential theranostics.

Project description:Creating covalent protein conjugates is an active area of research due to the wide range of uses for protein conjugates spanning everything from biological studies to protein therapeutics. Protein Farnesyltransferase (PFTase) has been used for the creation of site-specific protein conjugates, and a number of PFTase substrates have been developed to facilitate that work. PFTase is an effective catalyst for protein modification because it transfers Farnesyl diphosphate (FPP) analogues to protein substrates on a cysteine four residues from the C-terminus. While much work has been done to synthesize various FPP analogues, there are few reports investigating how mutations in PFTase alter the kinetics with these unnatural analogues. Herein we examined how different mutations within the PFTase active site alter the kinetics of the PFTase reaction with a series of large FPP analogues. We found that mutating either a single tryptophan or tyrosine residue to alanine results in greatly improved catalytic parameters, particularly in kcat. Mutation of tryptophan 102β to alanine caused a 4-fold increase in kcat and a 10-fold decrease in KM for a benzaldehyde-containing FPP analogue resulting in an overall 40-fold increase in catalytic efficiency. Similarly, mutation of tyrosine 205β to alanine caused a 25-fold increase in kcat and a 10-fold decrease in KM for a coumarin-containing analogue leading to a 300-fold increase in catalytic efficiency. Smaller but significant changes in catalytic parameters were also obtained for cyclo-octene- and NBD-containing FPP analogues. The latter compound was used to create a fluorescently labeled form of Ciliary Neurotrophic Factor (CNTF), a protein of therapeutic importance. Additionally, computational modeling was performed to study how the large non-natural isoprenoid analogues can fit into the active sites enlarged via mutagenesis. Overall, these results demonstrate that PFTase can be improved via mutagenesis in ways that will be useful for protein engineering and the creation of site-specific protein conjugates.

Dataset Information

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

Publications

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

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