Deciphering the mechanisms by which functional heparan sulfate motifs are assembled during biosynthesis – GAGOsynth

Obtaining enzymes in native form using various expression systems (insect cells, mammalian cells).

Analysis of interactions by different biophysical approaches (ITC, BLI, MST, SPR, AUC or native mass spectrometry) after obtaining the purified partners.

Oligosaccharide engineering to obtain substrates, specific of each enzyme, using the E Coli K5 strain whose polysaccharide capsule is identical to the HS carbon skeleton (GlcA-GlcNAc).

Structural analysis of the enzymes, alone or in complexes by X-ray crystallography or electron cryomicroscopy.

One of the major challenges in the characterization of EXT1 and EXT2 polysaccharide synthases is their expression and purification. These enzymes perform the HS chain elongation reaction by polymerizing the GlcNAc and GlcA residues. Initial expression tests have shown that co-expression of the two proteins increases yield and stability. NDST, which exists in four isoforms (NDST1-4), is a bi-functional enzyme that catalyzes the N-de-acetylation and N-re-sulfation of GlcNAc residues. We have been able to express and purify isoforms 1 and 2 in sufficient quantities for enzymatic analysis and, by obtaining specific substrates (GlcA-GlcN or GlcA-GlcNAc), we are able to determine their kinetic parameters for both activities, either globally or individually. The biosynthesis enzymes Glce and 2OST have been expressed in HEK293 system, and in order to promote the purification of a Glce-2OST complex, several plasmids have been generated for the co-expression of the catalytic domains of both enzymes in baculovirus and mammalian insect systems (under optimization). We have also cloned human 6OST but the enzyme is not secreted in the culture medium but resides in the cells. In parallel, we generated plasmids allowing the expression of both enzymes in whole form on the surface of extracellular vesicles but biophysical and structural analysis of the purified vesicles did not reveal the presence of the enzymes on their surface.

For the next period, we will continue to optimize the production of enzymes, focus our efforts on the analysis of their interactions and start their structural analysis, alone or in complex. Finally we will characterize the oligosaccharide structure of the products they catalyze.

Annaval T., Wild R., Crétinon Y., Sadir R., VivèsR.R. and Lortat-Jacob H. Heparan sulfate proteoglycans biosynthesis and post synthesis mechanisms combine few enzymes and few core proteins to generate extensive structural and functional diversity. Molecules 25, 4215 (2020)

Heparan sulfate (HS) is a complex polysaccharide expressed on most cell surface. Through its ability to bind hundreds of proteins it regulates a vast array of biological processes. Its extensive functional repertoire is closely related to its structural diversity, enzymatically imprinted onto the molecule backbone during biosynthesis. Because HS biosynthesis is a non-template driven process, how its structure is determined is however poorly known. In that context, the purpose of the project is to decipher how HS biosynthetic enzymes associate to each other to form "nanomachines" committed to specific HS motif assembly. For that purpose we gathered a complementary consortium with the aim to express the HS biosynthetic enzymes, analyze their interactions, investigate the structure of the complexes they form and solve the oligosaccharide sequences they produce. The binding activity of the resulting oligosaccharides against a number of HS binding cytokines will be analyzed to reveal the structure/function relationships of these molecules. The knowledge acquired will be useful for the engineering by enzyme-based approaches of “tailor-made” oligosaccharides with specific targeting activity.