JCJC SIMI 7 - JCJC - SIMI 7 - Chimie moléculaire, organique, de coordination, catalyse et chimie biologique

Deciphering the sorting mechanism of GAGs by chemical synthesis of effectors of glycosaminyltransferases – GAG-Sorting

Synthesis of new molecules for therapeutic use : study of the enzymes involved in the biosynthesis of proteoglycans

Proteoglycans are a family of complex macromolecules involved in a plethora of natural biological processes but also in the pathogenesis of several diseases including arthropathies, Alzheimer’s disease and cancer. <br />We propose to synthesize new molecules in order to better understand the enzymes and particularly the glycosaminyltransferases involved in the biosynthesis of proteoglycans.

Synthesis of a large library of oligosaccharides as potential substrates of the two enzymes EXTL3 and CSGalNAcT-1

Despite the fact that most of the GTs involved in the biosynthesis of GAGs have been identified, their molecular mechanism of action remains obscure. We will focuse our study on two human glycosaminyltransferases involved in the biosynthesis : EXTL3 and CSGalNAcT-1. To this end, we propose to devise new and efficient synthetic routes for the preparation of new sulfated and unsulfatedoligosaccharides as potential substrates of the targetted enzymes. In addition, a large library of variably modified oligosaccharides will be synthesized as potential specific inhibitors of EXTL3 or CSGalNAcT-1. These new compounds will be tested on the enzymes by our partner. A better understanding of the mechanism of the enzymes will not only provide insight into a fundamental biological process but will also constitute an important step forwards towards the development of new oligosaccharide-based therapeutics.

The first part of the project is focused on the synthesis of new oligosaccharides sulfated or non-sulfated. Standard chemical processes using protection/deprotection, glycosylation as well as sulfation steps will be applied. New chemical pathways included in particular chemo-enzymatic synthesis using sulfatases will also be developed. Total synthesis of all the possible oligosaccharide sulfoforms will help to better understand the role of sulfation pattern on the substrate specificity of the enzymes. In a second part of the project, we plan to synthesize a library of oligosaccharides modified by fluorine, or methoxy for example as potential inhibitors of the targetted enzymes. Beside the synthetic aspect of the project, new tools and methods will be developed to produce the human recombinant EXTL3 and CSGalNAcT-1 and evaluate their enzymatic activities towards the new synthesized oligosaccharides.

Several tri- and tetrasaccharides sulfated or note have been synthesized and are currenlty under enzymatic tests on the CSGalNAcT-1 which has been expressed by our biologist partners. Synthesis of disaccharides is also under investigations in order to study the influence of the chain size on the enzymatic activity.
We also initiated the synthesis of modified oligosaccharides and isolated a 6-deoxy disaccharide.

We envisage to complete the library of sulfated or modified oligosaccharides in order to study the influence of the substitution patterns on the enzymes. Introduction of fluorine atom or of a methoxy group is actually under investigations. Development of new synthetic pathways using in particularyl sulfatases that would allow the obtention of sulfated moiety in regioselective and effective manner.

Communications on the project :
1. Invited conference : Lopin-Bon, C. ; Fournel-Gigleux, S. 25ième journée Journées du Groupe Français des Glucides, Mai 2014 – Paris
2. Invited conference : Lopin-Bon, C. Novembre 2014 - Amiens
3. Oral

Proteoglycans (PGs) are a family of complex macromolecules characterized by the presence of glycosaminoglycan (GAG) chains covalently linked to a core protein. GAGs play important roles in a plethora of biological processes, such as cell growth and proliferation, embryonic development, and the coagulation cascade. They are also involved in the pathogenesis of several diseases including arthropathies, Alzheimer’s disease and cancer. The biosynthesis of PG-GAG chains involves the sequential action of glycosyltransferases (GTs) responsible for the formation of a tetrasaccharide sequence GlcA-ß-1,3-Gal-ß-1,3-Gal-ß-1,4-Xyl-ß-O-attached to a L-serine residue of a core protein. This GAG-linkage region serves as a primer for polymerization of two types of GAG chains, heparin/heparan sulfates (Hep/HS) and chondroitin sulfates/dermatan sulfate (CS/DS). While the elongation is in progress, the GAG chains are modified by the cooperative action of multiple sulfotransferases and epimerases to yield the final complex GAG structure. It has been reported that the linkage region may be modified by sulfation or phosphorylation but the exact role of these substitutions in not yet fully understood and it has been suggested that the phosphate and/or sulfate groups in the linkage region may represent biosynthetic sorting signals controlling maturation and growing of GAG chains. Despite the fact that most of the GTs involved in the biosynthesis of GAGs have been identified, their molecular mechanism of action remains obscure. The major goal of the present project is to advance our knowledge in the biosynthetic pathways of GAGs and particularly in the catalytic mechanism of the GTs implied. We will focuse our study on two human GTs: EXTL3 and CSGalNAcT-1 which orientate the biosynthesis towards the HS/Hep or CS/DS chains respectively. To this end, we propose to devise new and efficient synthetic routes, including chemo-enzymatic synthesis using sulfotransferases for the preparation of new sulfated and unsulfated trisaccharides of the linkage region as potential substrates of the target GTs. Total synthesis of all the possible oligosaccharide sulfoforms will help to better understand mechanism of GTs involved in the orientation of the GAG chains and to shed light the role of sulfation pattern on the substrate specificity of the GTs. In addition, a large library of variably modified disaccharides of the linkage region will be synthesized as potential specific inhibitors of EXTL3 or CSGalNAcT-1. Beside the synthetic aspect of the project, new tools and methods will be developed to (i) produce the human recombinant EXTL3 and CSGalNAcT-1 and (ii) evaluate their enzymatic activities towards the new synthesized oligosaccharides. A better understanding of the mechanism underlying the bifurcation of the synthesis process towards one or the other type of GAG will not only provide insight into a fundamental biological process but will also constitute an important step forwards towards the development of new oligosaccharide-based therapeutics.

Project coordinator

Madame Chrystel Lopin-Bon (Institut de Chimie Organique et Analytique) – chrystel.lopin-bon@univ-orleans.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

ICOA Institut de Chimie Organique et Analytique

Help of the ANR 174,720 euros
Beginning and duration of the scientific project: December 2013 - 48 Months

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