Extending Janus lectins architecture: Characterization and application to protocells

Area of Science:

• Synthetic biology
• Biotechnology
• Biomedicine
• Synthetic glycobiology

Background:

• Synthetic biology enables advancements in cell and tissue engineering.
• Engineering glycan-binding proteins is key for tools with precise topology and specificity in synthetic glycobiology.

Purpose of the Study:

• To develop engineered chimeric lectins, termed Janus lectins, with increased valency and specificity.
• To create a novel Janus lectin by fusing domains from *Ralstonia solanacearum* and *Marasmius oreades*.

Main Methods:

• Assembled a fusion protein combining a β-propeller domain (from *R. solanacearum*) and a β-trefoil domain (from *M. oreades*).
• Tested protein activity using glycosylated giant unilamellar vesicles.
• Evaluated binding to H1299 human lung epithelial cancer cells.
• Compared biophysical properties with existing Janus lectins (RSL-CBM40 and RSL-CBM77_Rf).

Main Results:

• The engineered Janus lectin specifically binds fucose and α-galactose simultaneously due to its unique architecture.
• Cross-linking of glycosylated giant unilamellar vesicles by the lectin resulted in proto-tissue-like structures.
• The engineered protein recognizes and binds H1299 human lung epithelial cancer cells.
• Denaturation profiles revealed the significant role of protein fold in stability.

Conclusions:

• Engineered Janus lectins offer enhanced valency and specificity for applications in synthetic glycobiology.
• The novel Janus lectin demonstrates potential for creating proto-tissue structures and targeting cancer cells.
• Protein fold is a critical consideration for stability in protein engineering efforts.