Site-Specific Protein Labeling and Generation of Defined Ubiquitin-Protein Conjugates Using an Asparaginyl Endopeptidase

Abstract

Asparaginyl endopeptidases (AEPs) have recently been widely utilized for peptide and protein modification. Labeling is however restricted to protein termini, severely limiting flexibility and scope in creating diverse conjugates as needed for therapeutic and diagnostic applications. Here, we use genetic code expansion to site-specifically modify target proteins with an isopeptide-linked glycylglycine moiety that serves as an acceptor nucleophile in AEPmediated transpeptidation with various probes containing a tripeptidic recognition motif. Our approach allows simple and flexible labeling of recombinant proteins at any internal site and leaves a minimal, entirely peptidic footprint (NGG) in the conjugation product. We show site-specific labeling of diverse target proteins with various biophysical probes, including dual labeling at an internal site and the N-terminus. Furthermore, we harness AEP-mediated transpeptidation for generation of ubiquitinand ubiquitin-like-modifier conjugates bearing a native isopeptide bond and only one point mutation in the linker region.

One-Step Sortase-Mediated Chemoenzymatic Semisynthesis of Deubiquitinase-Resistant Ub-Peptide Conjugates

“…Ubc9, an E2-SUMO-conjugating enzyme, has also been used for in vitro conjugation of wildtype Ub and ISG15 to recombinant proteins . A couple of combinatorial strategies using genetic-code expansion in combination with either SaSrtA or AEPs were also developed to prepare site-specific Ub and SUMO conjugates in vivo in an inducible fashion. , However, genetic-code expansion is a difficult process and requires optimized systems for the generation of the orthogonal aminoacyl-tRNA synthetase/tRNA pair and incorporation of unnatural amino acids (UAAs) in living cells, whereas in vitro enzymatic generation of site-specific Ub conjugates is less time consuming and relatively easy to perform.…”

“…This method utilizes the intrinsic sequence specificity of Ubc9, an E2-SUMO-conjugating enzyme, for in vitro conjugation of wildtype Ub and ISG15 to recombinant proteins. In a very recent combinatorial approach, AEP in combination with genetic-code expansion was used for the synthesis of site-specific Ub and SUMO conjugates in vivo in an inducible fashion …”

“…This method was modified by replacing the benzyloxycarbonyl (Cbz) with the allyloxycarbonyl (Alloc) group, which can be removed under milder conditions, for the synthesis of more complicated oligo-ubiquitins . Recently, with the discovery and characterization of various ligases, i.e., sortase, asparaginyl endopeptidase (AEP), butelase, and Ubc9, chemoenzymatic synthesis of Ub conjugates has gained attention. ,− Pawale et al used sortase-mediated ligation (SML) for the semisynthesis of SUMO conjugates . Lang and co-workers developed another strategy to ubiquitinate proteins in vivo in an inducible fashion by combining sortase-mediated transpeptidation with genetic-code expansion and biorthogonal Staudinger reduction .…”

“…In a very recent combinatorial approach, AEP in combination with genetic-code expansion was used for the synthesis of sitespecific Ub and SUMO conjugates in vivo in an inducible fashion. 28 Sortases are cysteine transpeptidase enzymes present in Gram-positive bacteria and are responsible for the covalent anchoring of surface proteins on the bacterial cell surface. 29−32 The sortase A from Staphylococcus aureus (SaSrtA) is a prototype sortase and has emerged as a versatile tool for chemical biologists in manipulating proteins and peptides.…”

“…In a very recent combinatorial approach, AEP in combination with genetic-code expansion was used for the synthesis of site-specific Ub and SUMO conjugates in vivo in an inducible fashion. 28 …”