In order to address the specific research objectives, the integration of different underpinning disciplines and methodologies is required. To accomplish its mission, GLYTUNES will be structured into 4 research work packages (WP).
Siglec ligands will be isolated or synthesized for binding and immunological studies. These include microbial glycoconjugates, such as LPS, flagellin, capsular polysaccharides from bacteria (as Campylobacter, Fusobacterium, Acinetobacter, Neisseria strains), whose cell walls mimic the structure of endogenous sialyloglycans. These microbial glycoconjugates will be isolated and characterized in an iterative process (ESR1), and then tested for their activity and binding studies (WP3, ESR2 and ESR4. Advanced sialylated glycan ligand libraries will be developed, using organic and enzymatic synthetic approaches (ESR10, ESR9). Full or fragmented endogenous and exogenous glycan ligands will be recombinantly produced or isolated from mammalian cells or either synthesized, with the aim to deeply investigate the molecular requisites of Siglecs’ interaction with their cognate ligands (by WP2 and WP3, all ESR members).
WP1 is led by Kim Le Mai Hoang of Glycouniverse, Potsdam, Germany.
Recombinant expression of Siglecs will be carried out at bioGUNE, UNIFI, GIOTTO laboratories (ESR3, ESR5, ESR11). The Siglecs V-set domain will be expressed fused to the Fc portion of human IgG1 or GST to increase the solubility, in their unlabeled and 13C, 15N, 2H labeled forms. A central part of the project will be focused on the analysis of the molecular basis of recognition and binding of Siglecs to natural endogenous and exogenous substrates (WP1). To this aim, a multifaceted approach spanning a wide array of structural (i.e. spectroscopic NMR, single-particle electron microscopy and x-ray crystallography), biophysical (i.e. ITC, SPR and biolayer- interferometry (BLI)) and computational techniques will be used (ESR3, ESR5, ESR6, ESR11, ESR12). These methods will be crucial to i) get a detailed molecular picture of the Siglecs-glycans complexes; ii) describe both protein and glycans key portions/residues critical in the recognition and binding events; iii) define the ligand regions in close contact to the receptor (epitope mapping) and describe the bioactive conformation; iv) analyse the energetics and thermodynamics of the molecular interactions; v) fast screen possible binders with respect to a specific target receptor.
WP2 is led by June Ereño-Orbea of CICBIOGUNE, Derio, Spain.
WP3 aims to understand the immune mechanisms and signaling pathways that govern the interaction between Siglecs and both endogenous and exogenous ligands (ESR2 and ESR4). The impact of exogenous and endogenous, synthetic or semi-synthetic, sialylated conjugates will be tested using human antigen-presenting cells, and the analysis of released cytokines, as well as the expression of co-stimulatory surface-markers will indicate the capacity of these molecules to induce/regulate effective T-cells activation. The tumor microenvironment (TME) induces qualitative and quantitative glycosylation changes especially regarding sialic acid expression, and is able to up or down regulate Siglec expression, thus orchestrating the immunological networks that will lead to the series of immunomodulatory events that make the TME “invisible” to our immune system. WP3 will help in understanding this process and how to interfere with these immunomodulatory events, cracking the tumor sialic acid code. Tumors that arise at epithelial barrier surfaces of the body harbor extensive microbiota, and the importance of these microbes in colorectal cancer is now widely acknowledged. We will identify and assess the impact Fusobacterium nucleatum species on Siglec-mediated immune responses.
WP3 is led by Yvette van Kooyk and Fabrizio Chiodo of VUMC, Amsterdam, The Netherlands.
The design of novel glycomimetics optimal in size and shape, finely tuned to the supramolecular architecture of individual Siglecs is required to modulate the activity of Siglecs understudy (WP2). We will take advantage of the pivotal information on the macromolecular Siglec -ligand complexes understudy, manipulation of the chemical information encoded in sugar structures (the sugar code) to control or alter the flow of information read-out by Siglecs upon binding with sialylated glycans (ESR7, ESR8, ESR9). The newly synthesized ligands will be analyzed for their interaction with target Siglecs as above (WP2 and WP3) and the ligands with improved binding affinity will be further developed.
WP4 is led by Thomas Boltje of RAD, Nijmegen, The Netherlands.