Extending Janus lectins architecture: Characterization and application to protocells.

Synthetic biology is a rapidly growing field with applications in biotechnology and biomedicine. Through various approaches, remarkable achievements, such as cell and tissue engineering, have been already accomplished. In synthetic glycobiology, the engineering of glycan binding proteins is being exploited for producing tools with precise topology and specificity. We developed the concept of engineered chimeric lectins, i.e., Janus lectin, with increased valency, and additional specificity. The novel engineered lectin, assembled as a fusion protein between the ß-propeller domain from Ralstonia solanacearum and the ß-trefoil domain from fungus Marasmius oreades, is specific for fucose and α-galactose and its unique protein architecture allows to bind these ligands simultaneously. The protein activity was tested with glycosylated giant unilamellar vesicles, resulting in the formation of proto-tissue-like structures through cross-linking of such protocells. The engineered protein recognizes and binds H1299 human lung epithelial cancer cells by its two domains. The biophysical properties of this new construct were compared with the two already existing Janus lectins, RSL-CBM40 and RSL-CBM77Rf. Denaturation profiles of the proteins indicate that the fold of each has a significant role in protein stability and should be considered during protein engineering.

Homo- and Heterovalent Neoglycoproteins as Ligands for Bacterial Lectins.

Click chemistry gives access to unlimited set of multivalent glycoconjugates to explore carbohydrate-protein interactions and discover high affinity ligands. In this study, we have created supramolecular systems based on a carrier protein that was grafted by Cu(I)-catalyzed azide-alkyne cycloaddition with tetravalent glycodendrons presenting αGal, ßGal and/or αFuc. Binding studies of the homo- (4 a-c) and heterovalent (5) neoglycoproteins (neoGPs) with the LecA and LecB lectins from P. aeruginosa has first confirmed the interest of the multivalent presentation of glycodendrons by the carrier protein (IC50 up to 2.8 nM). Moreover, these studies have shown that the heterovalent display of glycans (5) allows the interaction with both lectins (IC50 of 10 nM) despite the presence of unspecific moieties, and even with similar efficiency for LecB. These results demonstrate the potential of multivalent and multispecific neoGPs as a promising strategy to fight against resistant pathogens.

Polyvalent Transition-State Analogues of Sialyl Substrates Strongly Inhibit Bacterial Sialidases*.

Bacterial sialidases (SA) are validated drug targets expressed by common human pathogens such as Streptococcus pneumoniae, Vibrio cholerae, or Clostridium perfringens. Noncovalent inhibitors of bacterial SA capable of reaching the submicromolar level are rarely reported. In this work, multi- and polyvalent compounds are developed, based on the transition-state analogue 2-deoxy-2,3-didehydro-N-acetylneuraminic (DANA). Poly-DANA inhibits the catalytic activity of SA from S. pneumoniae (NanA) and the symbiotic microorganism B. thetaiotaomicron (BtSA) at the picomolar and low nanomolar levels (expressed in moles of molecules and of DANA, respectively). Each DANA grafted to the polymer surpasses the inhibitory potential of the monovalent analogue by more than four orders of magnitude, which represents the highest multivalent effect reported so far for an enzyme inhibition. The synergistic interaction is shown to operate exclusively in the catalytic domain, and not in the flanked carbohydrate-binding module (CBM). These results offer interesting perspectives for the multivalent inhibition of other SA families lacking a CBM, such as viral, parasitic, or human SA.