Lectin engineering: possibilities and perspectives
Professor Jun Hirabayahsi, National Institute of Advanced Industrial Science and Technology, Japan
Lectins are a wide group of sugar-binding proteins occurring in all kinds of organisms including animals, plants, bacteria, fungi and even viruses. According to a recent report, the number of lectin scaffolds (~Pfam), of which 3D-strcutures are known and sugar-binding functions have been confirmed in literature, exceeds 50, which is far beyond our image in the 20th century. This fact suggests that new lectins will be discovered either by a conventional screening approach or just by chance like in the case of POMGnT1 stem region. It is also expectable that new lectin domains are generated in the future of evolution, although such an attempt has never been done at an experimental level. Based on the current states of the art, various ways of lectin engineering are available, by which lectin specificity and/or stability can be improved with the known lectin scaffold. However, the above observation implies that any other protein scaffold, which has never been described as lectin, is entitled to acquire a sugar-binding function. In this presentation, possible approaches and new items to create sugar-binding properties of synthetic peptides will be described.
Computer-aided engineering of reprogrammed carbohydrate-active enzymes for biotechnological applications
Dr Isabelle André, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés, France
Combined with chemical synthesis, the use of biocatalysts holds great potential to open the way to molecular diversity. Nonetheless, the lack of appropriate enzymatic tools with requisite properties has hampered extensive exploration of chemo-enzymatic routes to complex carbohydrates. To circumvent this limitation, protein engineering has proven to be very efficient to tailor enzymes with novel substrate specificities. However, the outcome of protein engineering strategies strongly depends on our understanding of enzyme catalysis and our comprehension of the inter-relationships between protein structure, activity and dynamics. Herein, Dr André reports the latest work of her laboratory in the development of 'programmed' chemo-enzymatic pathways that take advantage of both knowledge-based and computer-aided enzyme engineering to produce complex microbial cell-surface oligosaccharides entering in the composition of multivalent vaccines to prevent shigellosis.
This lecture will cover and discuss recent developments of Dr André's laboratory.
This work was partially funded by the French National Research Agency (Project GLUCODESIGN ANR-08-PCVI-0002-02; Project CARBUNIVAX ANR-15-CE07-0019-01).
Non-canonical amino acids as building blocks for lectins
Dr Birgit Wiltschi, Austrian Centre of Industrial Biotechnology, Austria
Lectins are carbohydrate binding proteins with a high specificity for their target ligands. They play diverse roles in cellular recognition and signalling processes, as well as in infections and cancer metastasis. Due to their high specificity, lectins find application in biotechnology and medicine for eg blood group typing, purification of glycoproteins or -lipids and as markers, that target cancer cells. In some applications, lectins must be immobilised on a solid support for purification processes or they have to be conjugated with other molecules. However, traditional conjugation reactions are unspecific and in most cases the site of conjugation cannot be pre-defined. Therefore, Dr Wiltschi and her group devised lectins containing non-canonical amino acids with bioorthogonal reactive handles, with which these can be conjugated with other molecules in a pre-defined manner. As a proof of principle, the group conjugated these lectins with small molecules and other lectins. The conjugate lectins might be useful for any process, where lectins shall be conjugated with another module in a pre-defined and site-specific manner.
Lectin-mediated protocell crosslinking and fusion
Professor Winfried Römer, University of Freiburg, Germany
Synthetic membrane systems are extremely useful for the better understanding of complex cellular structures and processes. The engineering of proto-tissues from proto-cells through lectin-glycan interactions represents an important step towards synthetic minimal tissues.
As a first step, Professor Römer's group succeeded in integrating natural and synthetic glyco-modules into giant unilamellar vesicles, which then were specifically recognized by lectins. In a second step, multivalent lectins with opposing carbohydrate binding sites triggered the crosslinking of glycan-functionalised vesicles. The crosslinking process drives the progression from contact puncta into elongated proto-cellular junctions, which form the vesicles into polygonal clusters resembling tissues. Due to their carbohydrate specificity, different lectins can be engaged in parallel with both natural and synthetic glyco-conjugates to generate complex interfaces with distinct lectin domains. In addition, the formation of proto-cellular junctions can be combined with adhesion to a functionalised support by other ligand-receptor interactions to render increased stability against fluid flow.
The group also assembled proto-cells to proto-tissues by employing a chimeric, bispecific lectin, with two rationally oriented and distinct recognition surfaces (from A Imberty). This lectin, coined Janus lectin in allusion to the two-faced roman god, is able to bind independently to both fucosylated and sialylated glyco-conjugates.
Moreover, the group could demonstrate that a synthetic lectin complex (from B Turnbull) consisting of 2-3 cholera toxin B-subunits induces hemifusion and fusion events, and also leakage or rupture of vesicles.