Structural and molecular glycobiology

The structural and molecular glycobiology team aims to unravel the structure/function relationships of proteins with complex glycans such as glycoconjugates and polysaccharides. Its research is at the chemistry-biology interface with multidisciplinary expertise in bioinformatics, molecular biology, biochemistry and enzymology, X-ray protein crystallography, analysis of protein-carbohydrate interactions and molecular modeling methods. Its research activity is conducted along the following topics:

-The biosynthesis of complex carbohydrates by glycosyltransferases (GTs).

-The degradation of complex polysaccharides by Polysaccharide Utilisation Loci (PULs).

-The recognition of glycoconjugates by lectins.

-The structural analysis of oligo- and polysaccharides.

 

Carbohydrates or sugars or carbohydrates are essential components of any living organism. They represent the most abundant and versatile of the four classes of biomolecules and of Earth’s natural resources. They are the major constituents of any cell surface and form the first layer of information (glycocode) received and sent from a cell. All of the sugars in a cell constitute the glycome, that is a hallmark of cell identity and fate but also a signature of health and disease. Carbohydrates exhibit a wide range of functions: they can serve as structural components, as an energy source or as a key element in various molecular recognition processes. Monosaccharides (> 100) can combine with each other in several types of bonds to form oligo or polysaccharides or with lipids and proteins to form glycoconjugates. Carbohydrates can be branched and / or modified (sulfation, phosphorylation, acetylation). In parallel with their extensive diversity, there is a myriad of enzymes designed to synthesise (glycosyl transferases, GTs) and degrade or modify (glycoside hydrolases, lyases, esterases, LPMOs) carbohydrates as well as proteins to recognize them (lectins). Understanding the mechanism of these enzymes, the lectin recognition process as well as the properties of carbohydrates is of great importance at the fundamental level but also at applied level in particular in the medicinal and industrial fields.

-The biosynthesis of complex carbohydrates by glycosyltransferases (GTs):
C. Breton and O. Lerouxel

Glycosylation is quantitatively the most significant biochemical reaction on Earth and its aberrations are a hallmark of numerous chronic diseases, including inflammation and cancer. Glycosyltransferases are a key factor in these processes and it is important to advance in the knowledge of their regulation and mechanism. In recent years, we have focused our efforts on plant GTs involved in the biogenesis of photosynthetic membranes and the plant cell wall. Major results concern the resolution of the 3D structure of MGD1, the major galactolipid synthase in plants, which reveals critical features of its reaction mechanism and its membrane binding properties. These results allowed us to propose a model for MGD1 membrane association in the chloroplast envelope that may explain the transit of galactolipids to embryonic thylakoids.
Plant cell walls are complex and dynamic networks made mostly of cellulose, hemicelluloses, and pectins. The most abundant hemicellulose in the plant kingdom is xyloglucan, a fucosylated polysaccharide with a cellulose-like backbone. Xyloglucan is a highly valuable target of study to have a comprehensive view on the role of the cell wall remodeling in plant development but even more for biotechnological applications. This renewable polymer has indeed versatile properties and is envisioned to be used in a large variety of applications from health to microelectronics. We tackle the question of the xyloglucan biosynthesis and we were successful in the heterologous expression, purification and resolution of the structure of FUT1, a fucosyltransferase that participates to the synthesis of xyloglucan in Arabidopsis. Our results provide information on the reaction mechanism and selectivity of the enzyme. They also suggest that the functional unit of FUT1 is a dimer, which may explain its preference for large acceptor substrates. This work led to the first structure of a plant cell wall glycosyltransferase. As part of a Glyco@Alps project with SYMMES, we are developing a glycochip to help to characterize the enzymatic activity of GTs and to identify their substrate.

 

-The degradation of complex polysaccharides:
W. Helbert, S. Drouillard & M. Couturier

In this line of research, we are participating in the global effort to assign the function of genes encoding putative polysaccharide degradation enzymes and to understand the degradation of complex polysaccharides. The new discovered enzymes are new tools for the structural characterization of the diversity of polysaccharides. Enzymes also allow producing new oligosaccharides

1-Bioinformatic selection of genes coding for putative polysaccharide degradation enzymes (Data mining). We use two main approaches: i) we select proteins whose sequences are divergent from the predicted or characterized enzymes and ii) we systematically analyze all proteins encoded by the “Polysaccharides utilization loci” (PUL) including predicted and unknown enzymes. We focus on the PULs identified in the genomes of bacteria from the human gut microbiota potentially involved in the degradation of plant polysaccharides and sulphated polysaccharides of marine origin. We mainly study glycoside hydrolases, polysaccharide lyases and carbohydrate sulfatases, but other enzymes such as LPMO and esterases are also of interest to us. This work is carried out in collaboration with the members of the AFMB (Marseilles) responsible for the CAZy database (cazy.org).

2- Enrichment and structural characterization of a unique collection of oligo- and polysaccharides. In order to discover enzymes with new specificities, we maintain and enrich a unique collection of approximately 200 oligo- and polysaccharide substrates. It is regularly supplemented by new substrates prepared in the laboratory or acquired within the framework of collaborations. The most original polysaccharide structures are analyzed in detail using the PCAN service (CERMAV) and the NMR platform (ICMG).

3- Screening and characterization of new enzymes. Since 2016, we have produced around 700 proteins and are planning the synthesis and screening of 600 new targets. To cope with the number of enzymes newly identified in the laboratory and the number of screening experiments, the implementation of a bioinformatics tool "Crazypol" allows us to manage both our collection of substrates and our bank of. protein. We are going to expand our field of investigation with a specific expression system for the study of carbohydrate sulfatases that we have developed in the laboratory. Studies of the modes of action and specificities of the most original enzymes are carried out using modern enzymology approaches including chromatography and spectroscopy (NMR, mass spectrometry) methods.

 

-The recognition of glycoconjugates by lectins:
A. Imberty & A. Varrot

Lectins are protein receptors capable of specifically recognizing carbohydrates, in particular the glycoconjugates which cover the surface of any cell without modifying them. Lectin-glycoconjugate interactions are essential in many cellular processes such as tissue cohesion, immunity, fertilization, but also in pathological processes such as infections. Lectins are able to decipher the glycocode which is one of the main objectives of glycomics, a growing discipline still requiring many tools and technological advances. Lectins have great potential for biotechnological and biomedical applications.

Lectins play an essential role in host-pathogen interactions, either as a first-line defense molecule against foreign organisms, or by promoting microbial adhesion, a crucial step in the initiation of infections. Lectins are now targeted for the development of antiadhesive glycodrugs that could be used as new anti-infective drugs. Many cancerous processes are associated with glycosylation changes that can be recognized specifically by lectins. Lectins are therefore sought-after tools to aid in the diagnosis and prognosis of cancers because they can differentiate healthy cells from cancerous cells, but also for controlling the glycosylation of biotherapeutic products. In collaboration with GLYCODIAG, IBS and DCP, we are developing a recombinant lectin chip for the real-time detection of the main unwanted glycanic structures in biotherapeutic products.

We are interested in the identification of new lectins both in terms of their folding or their specificity, in obtaining the atomic bases governing carbohydrate-protein interactions and in the engineering of lectins to modify their specificity or their multivalency. We also design and produce neo-lectins, based on assembly of different peptide modules, using the tools of synthetic biology.

Since 2005, we have studied about 40 lectins and resolved the crystal structure of 31 lectins of various origins: bacterial, fungal, invertebrates, algae, plants or eukaryotes in apo form or in complex with natural ligands or inhibitors. This led to the deposition of approximately 90 set of coordinates in the Protein Data Bank, the discovery of one new protein fold and several new quaternary arrangements.

 

Data bases

The GBMS team develops tools and databases in glycosciences: Glyco3D  and Unilectin  and participates in the dissemination of knowledge in glycosciences through Glycopedia. Its develops new graphic approaches to visualize (SweeUnityMol), study but also teach glycosciences. A virtual reality platform was set up at CERMAV by S. Perez and is opened to a large public.

 

Fundings

We are partners of the CDP Glyco @ Alps within the framework of the IDEX Université Grenoble Alpes, the Labex Arcane and multidisciplinary networks for innovative training - European Joint Doctoral program: "PhD4GlycoDrug"  and ITN2018 "synBIOcarb"  funded by the European Commission under the Horizon 2020 Marie Skłodowska-Curie action. Several team member participate in several Cost actions: CM1102, CA18132 and CA1803. Our research is also supported by the National Agency for Research : GlycoMime, LectArray, GreenAlgohol, the french cystic fibrosis association “Vaincre la Mucoviscidose”, the Carnot Institute "Polynat" and the CNRS.