Site-Specific Chemoenzymatic Labeling of Aerolysin Enables the Identification of New Aerolysin Receptors

Abstract

Aerolysin is a secreted bacterial toxin that perforates the plasma membrane of a target cell with lethal consequences. Previously explored native and epitope-tagged forms of the toxin do not allow site-specific modification of the mature toxin with a probe of choice. We explore sortase-mediated transpeptidation reactions (sortagging) to install fluorophores and biotin at three distinct sites in aerolysin, without impairing binding of the toxin to the cell membrane and with minimal impact on toxicity. Using a version of aerolysin labeled with different fluorophores at two distinct sites we followed the fate of the C-terminal peptide independently from the N-terminal part of the toxin, and show its loss in the course of intoxication. Making use of the biotinylated version of aerolysin, we identify mesothelin, urokinase plasminogen activator surface receptor (uPAR, CD87), glypican-1, and CD59 glycoprotein as aerolysin receptors, all predicted or known to be modified with a glycosylphosphatidylinositol anchor. The sortase-mediated reactions reported here can be readily extended to other pore forming proteins.

HypE-specific Nanobodies as Tools to Modulate HypE-mediated Target AMPylation

“…Heptamutant Ca 2ϩ -independent sortase A from Staphylococcus aureus (SrtA staph7M ) was purified as described (29). Sortase reaction nucleophiles GGG-TAMRA, GGG-biotin, and GGG-Alexa Fluor 647 were synthesized as described (30,31).…”

“…Ectopic expression of HA-tagged HypE constructs in cells that express GFP-V H H-8, GFP-V H H-1, GFP-V H H-2, and GFP-V H H-100 resulted in robust co-localization of the V H Hs with HA-HypE, whereas the GFP signal remained diffuse in cells expressing GFP-V H H-9. To further test our V H Hs, we engineered V H H-Alexa Fluor 647 fusions by sortagging (29) and used these to stain fixed HypE-overexpressing cells (Fig. 7, A and B).…”

Site‐Specific Protein Labeling via Sortase‐Mediated Transpeptidation

“…Currently, two engineered variants of SrtA staph containing either five (SrtA staph pentamutant) or seven (SrtA staph heptamutant) point mutations have risen to prominence as the preferred enzymes for this process by offering key advantages over the wild-type enzyme. These include enhanced reaction rates (penta-and heptamutant) and lack of the requirement for a Ca 2+ cofactor (heptamutant) in the reaction mixture (Chen, Dorr, & Liu, 2011; Hirakawa, Ishikawa, & Nagamune, 2015; Witte et al, 2015; Wuethrich et al, 2014). Given these features, the authors recommend that initial labeling studies employ either of these engineered SrtA staph derivatives, and complementary labeling protocols for the use of either the penta- or heptamutant are provided below.…”

“…These include the SrtA staph pentamutant, which was identified in a directed evolution screen and exhibits an approximately 120-fold increase in enzyme activity versus wild-type SrtA staph (Chen et al, 2011). The heptamutant builds on this derivative by including two additional point mutations that alleviate Ca 2+ dependency (Hirakawa et al, 2015; Witte et al, 2015; Wuethrich et al, 2014). In terms of reaction reversibility, a handful of strategies have been introduced that drive reaction equilibrium through selective removal or deactivation of transpeptidation products.…”

Site-specific protein modification using immobilized sortase in batch and continuous-flow systems

“…We recently described a sortase A variant, termed heptamutant sortase A (7M) , which combines the pentamutant version with increased catalytic activity with mutations that render sortase calcium independent 18 , resulting in an enzyme with increased activity and no longer requiring Ca 2+ (Fig. 2) 19 . The immobilized variant of this enzyme facilitated straightforward modification of aerolysin and greatly simplified the purification of the modified product.…”