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ANR funded project

Blanc - SVSE 8 - Biochimie, biologie moléculaire et structurale (Blanc SVSE 8) 2012
Projet SULF

Structural and functional characterisation of Human Sulfs

Heparan sulfate (HS) is a sulfated, complex polysaccharide, ubiquitously found at the cell surface and in extra-cellular matrices. It is involved in major cellular processes, as it is able to bind and modulate the activity of a very large array of proteins. These interactions occur through saccharide domains of specific sulfation pattern present within the HS chains. Generation of such domains is orchestrated by a complex biosynthesis machinery and their structure is further regulated at the cell surface by post-synthetic modifying enzymes. Amongst them are the Sulfs, a family of endosulfatases that catalyze the specific 6-O-desulfation of HS within these highly sulfated domains. These modifications have a weak impact on the polysaccharide structure, but great functional consequences, as Sulf alteration of HS 6-O-sulfation patterns dramatically affects its ability to bind and regulate many of its protein ligands.

However, although this mechanism of HS structural tuning is critical for many physiological as well as pathological processes, the study of Sulfs has been hampered by the difficulty to express these enzymes recombinantly, and the complexity of studying their precise effects on HS fine structure. Based on our recent data that highlighted an original, processive and orientated desulfation mechanism for these enzymes, we propose to deliver an extensive study of the structure and activity of the two Human Sulf isoforms HSulf-1 and HSulf-2, using a multidisciplinary approach. The main objectives of this project are: (i) the development of an expression system enabling production in large amounts of pure and functional enzymes, and the preparation of structurally defined HS oligosaccharide libraries, which will supply biologically relevant substrates for our studies ; (ii) the dynamic and structural characterisation of the enzyme/ligand binding process, using a set of biophysical and biochemical approaches ; (iii) the structural analysis of HSulf desulfation process on S-domain like HS oligosaccharides, using advanced methods for saccharide structural characterisation ; (iv) the functional analysis of HSulf activity on the regulation of Fibroblast Growth Factor-2 (FGF-2) and the study of HS 6-O-sulation pattern requirements for the activation of this growth factor ; (v) the determination of HSulf structure, alone or in complex with its natural ligand, by X-ray crystallography and (vi) the identification of HSulf inhibitors by automated screening of small-molecule chemical libraries.

With this project combining both structural and functional studies of Human Sulfs, we expect to make a most significant contribution to the understanding of the fundamental role played by these enzymes. Moreover, our structural data and our chemical library screening approach should provide critical information for the development of biologically active inhibitory compounds and may lead to exciting perspectives for therapeutical applications.

Partners

IBS Institut de Biologie Structurale UMR5075

ANR grant: 199 900 euros
Beginning and duration: février 2013 - 36 mois

 

ANR Programme: Blanc - SVSE 8 - Biochimie, biologie moléculaire et structurale (Blanc SVSE 8) 2012

Project ID: ANR-12-BSV8-0023

Project coordinator:
Monsieur Romain VIVES (Institut de Biologie Structurale UMR5075)
vives@nullibs.fr

 

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The project coordinator is the author of this abstract and is therefore responsible for the content of the summary. The ANR disclaims all responsibility in connection with its content.