Identification of sortase substrates by specificity profiling

Schmohl, Lena; Bierlmeier, Jan; Kügelgen, Nicolai von; Kurz, Leonie; Reis, Pascal; Barthels, Fabian; Mach, Pia; Schutkowski, Mike; Freund, Christian; Schwarzer, Dirk

doi:10.1016/j.bmc.2017.06.033

Cited by 31 publications

(61 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Certain sortases also tolerated serine (Ser; SrtA staph , SrtA suis , SrtA pneu ) or phenylalanine (Phe; SrtA pneu , SrtA mono , SrtA lactis ). Overall, these results mirrored those of previous studies with SrtA staph and other staphylococcal and streptococcal sortases, indicating that although substrate specificity might be relaxed with respect to position 4, it is still restricted to select substrate variants for class A sortases …”

Section: Resultssupporting

confidence: 85%

“…To identify pairs of sortases and substrates with potential utility for SML, an efficient strategy for substrate screening was required. To date, a variety of screening studies have been reported for sortase substrate identification, which often involve the use of model peptide substrates . Similar to the recent work of Puorger et al., we opted for an initial screening strategy in which select positions within the canonical LPXTG motif were randomized during solid‐phase synthesis .…”

Section: Resultsmentioning

confidence: 99%

“…To avoid complications from undesired disulfide formation and thioether oxidation, cysteine was omitted from the isokinetic coupling mixture, and methionine was replaced with an isosteric norleucine (Nle) residue. In addition, the peptide pool was capped with an N‐terminal 2‐aminobenzoyl (Abz) fluorophore and a C‐terminal 2,4‐dinitrophenyl (Dnp) quencher to allow preliminary monitoring of substrate cleavage by fluorescence . An additional glycine spacer was also included between the five amino acid substrate motif and the C‐terminal Dnp‐labeled lysine residue.…”

Section: Resultsmentioning

confidence: 99%

“…In addition to enzyme engineering, a complementary approach that has seen surprisingly little attention is to exploit differences in reactivity among different homologues within the sortase enzyme family. To date, the only wild‐type sortase other than SrtA staph that has been used extensively in SML is sortase A from Streptococcus pyogenes , and it is only recently that the first study focused on characterizing sortase homologues as tools for SML was reported by Schmohl et al . From these studies, it is evident that sortase homologues offer distinct advantages over SrtA staph .…”

Section: Introductionmentioning

confidence: 99%

“…For example, the ability of S. pyogenes sortase A to process both LPXTG and LPXTA sequences has been leveraged for modifying both termini of a single protein target, layer‐by‐layer surface immobilization of proteins, or labeling different subunits within viral capsids . In the most recent work by Schmohl et al., streptococcal sortases were also found to exhibit a more relaxed substrate specificity and displayed an unexpected preference for LPKLG motifs, which might prove useful for certain SML applications …”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

Expanding the Scope of Sortase‐Mediated Ligations by Using Sortase Homologues

et al. 2017

View full text Add to dashboard Cite

Sortase-catalyzed transacylation reactions are widely used for the construction of non-natural protein derivatives. However, the most commonly used enzyme for these strategies (sortase A from Staphylococcus aureus) is limited by its narrow substrate scope. To expand the range of substrates compatible with sortase-mediated reactions, we characterized the in vitro substrate preferences of eight sortase A homologues. From these studies, we identified sortase A enzymes that recognize multiple substrates that are unreactive toward sortase A from S. aureus. We further exploited the ability of sortase A from Streptococcus pneumoniae to recognize an LPATS substrate to perform a site-specific modification of the N-terminal serine residue in the naturally occurring antimicrobial peptide DCD-1L. Finally, we unexpectedly observed that certain substrates (LPATXG, X=Nle, Leu, Phe, Tyr) were susceptible to transacylation at alternative sites within the substrate motif, and sortase A from S. pneumoniae was capable of forming oligomers. Overall, this work provides a foundation for the further development of sortase enzymes for use in protein modification.

show abstract

Section: Resultssupporting

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Expanding the Scope of Sortase‐Mediated Ligations by Using Sortase Homologues

et al. 2017

View full text Add to dashboard Cite

show abstract

Cell Surface Labeling by Engineered Extracellular Vesicles

et al. 2020

View full text Add to dashboard Cite

Deoxycytidine [3]). Extracellular vesicles (EVs) are increasingly recognized as a new class of multivalent messengers involved in cell-cell communication, both locally and at distance. [4-7] EVs are membranebound particles released by virtually every cell type, with stressed and transformed cells secreting the highest amounts. [8-11] Investigations of EVs' roles in different pathophysiological conditions are thwarted by biases introduced during isolation and reinfusion procedures. In particular, these biases manifest at different levels: (i) certain EV subsets may be excluded, [6] (ii) operator judgment is involved in deciding how many EVs to reinfuse, and (iii) an assumption that EV concentration is highest in blood, whereas several studies have shown that EV biodistribution occurs primarily via lymph fluid. [12-15] To circumvent these issues, the EV-releasing cells of interest can be genetically engineered to express membrane-bound reporters (or other functional biomolecules) in vitro or in vivo. [16] Although this approach is still in its infancy, it avoids the above limitations and to unbiasedly investigate untouched, endogenously released EVs. Although a detailed description of how EVs signal between cells is still missing, two nonexclusive mechanisms have been proposed: (i) membrane fusion between EVs and target cells, thereby releasing EV cargo (e.g., microRNAs and proteins, including oncogenes) directly in the cytoplasm of targeted cells; and (ii) surface binding with subsequent ligand-receptor type signaling. Membrane fusion (also known as horizontal transfer) has been described using several in vitro models (see Pucci and Pittet [17] and references therein), but proved more challenging to demonstrate in vivo. Recently, horizontal transfer of tumor-derived EVs was tested in mice using a sensitive and stringent approach based on the CRE-Lox reporter system. [15,18] Although CRE recombinase has an efficiency of 55%, [19] horizontal transfer was shown to happen mainly between tumor cells in vivo, but not between tumor cells and host cells. [15,18] These results have been further confirmed using other genetic strategies, [20,21] suggesting that surface binding may represent the main mechanism of EV-mediated tumor-host communications in vivo. A major limitation in the study of native EV signaling to host cells in vivo derives from their small size, which restricts the amount of reporter molecules carried by a single EV. As a consequence, only target cells binding the highest levels of EVs [12,14,15] will accumulate enough reporter molecules to be detected and investigated. Nonetheless, many host cell types Extracellular vesicles (EVs) can mediate local and long-range intercellular communication via cell surface signaling. In order to perform in vivo studies of unmanipulated, endogenously released EVs, sensitive but stringent approaches able to detect EV-cell surface interactions are needed. However, isolation and reinfusion of EVs can introduce biases. A rigorous way to study EVs in vivo is by genetically eng...

show abstract

Empowering Site‐Specific Bioconjugations In Vitro and In Vivo: Advances in Sortase Engineering and Sortase‐Mediated Ligation

Zou,

Ji,

Schwaneberg

2023

Angewandte Chemie

View full text Add to dashboard Cite

Sortase‐mediated ligation (SML) has emerged as a powerful and versatile methodology for site‐specific protein conjugation, functionalization/labeling, immobilization, and design of biohybrid molecules and systems. However, the broader application of SML faces several challenges, such as limited activity and stability, dependence on calcium ions, and reversible reactions caused by nucleophilic side‐products. Over the past decade, protein engineering campaigns and particularly directed evolution, have been extensively employed to overcome sortase limitations, thereby expanding the potential application of SML in multiple directions, including therapeutics, biorthogonal chemistry, biomaterials, and biosensors. This review provides an overview of achieved advancements in sortase engineering and highlights recent progress in utilizing SML in combination with other state‐of‐the‐art chemical and biological methodologies. The aim is to encourage scientists to employ sortases in their conjugation experiments.

show abstract

Identification of sortase substrates by specificity profiling

Cited by 31 publications

References 31 publications

Expanding the Scope of Sortase‐Mediated Ligations by Using Sortase Homologues

Expanding the Scope of Sortase‐Mediated Ligations by Using Sortase Homologues

Cell Surface Labeling by Engineered Extracellular Vesicles

Empowering Site‐Specific Bioconjugations In Vitro and In Vivo: Advances in Sortase Engineering and Sortase‐Mediated Ligation

Contact Info

Product

Resources

About