Structural and functional characterisation of Human Sulfs – SULF

Methods and approaches that will be developed in this project include:
1- the development of a HSulf expression system, based on the use of a recently patented expression vector (Collaboration Philippe Desprès, Institut Pasteur), and mammalian HEK cells adapted to the culture in suspension and serum-free medium (HEK293, Invitrogen). Libraries of structurally defined HS oligosaccharides will also be prepared, which will supply biologically relevant substrates for our studies.
2- the dynamic and structural characterisation of the enzyme/ligand binding process, using a set of biophysical and biochemical approaches. In particular, the binding of HSulf to HS will be analysed by surface plasmon resonance (SPR) to determine the kinetic parameters of the interaction, and using a heparin binding site mapping technique developed in the laboratory to define the amino acids involved in substrate recognition.
3- the structural analysis of HSulf desulfation process on S-domain like HS oligosaccharides, using advanced methods for saccharide structural characterisation.
4- 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.
5- the determination of HSulf structure, alone or in complex with its natural ligand, by X-ray crystallography.
6- the identification of HSulf inhibitors by automated screening of small-molecule chemical libraries.

As scheduled in the project, the first 6 months of our work have been dedicated to the development of a HSulf-2 expression system. The gene encoding the protein was cloned in a vector enabling addition of a N-terminal tag, to facilitate its detection and improve production yields (collaboration Philippe Desprès, Institut Pasteur). Expression was achieved in suspension HEK cells grown in serum-free medium, and results confirmed that the protein was suitably produced and secreted into the culture medium.
A purification protocol has then been developed, which enabled recovery of functional Hsulf-2, with a purity level of ~95%. Quality of protein preparation was checked and validated by mass spectrometry, N-terminal sequencing, electron microscopy and MALS/DLS. Use of the TEV protease to remove protein tags was also confirmed. In addition, cloning and transfection of HEK cells for the expression of HSulf-1 has been achieved, and mutations to generate HSulf catalytic mutants have been obtained by site directed mutagenesis. Finally, the generation and purification of HS oligosaccharides has been undertaken.

Because of their implication in many physiological and pathological processes, the study of the Sulfs is highly relevant, from both academic and applied research perspectives. However, little is known yet about the activity of these enzymes and the underlying mechanisms involved. This project aims at providing new information on these enzymes, by using multidisciplinary and original approaches. As such, the generation of HS oligosaccharide libraries will constitute a valuable tool that will provide a very wide panel of saccharide samples, representative of HS naturally occurring structural diversity. Analysis of dynamic (binding affinity and kinetic parameters of the interaction with various substrates) and structural (saccharide and protein epitopes involved in the binding) features of the HSulf/HS interaction should help to clarify the mechanism of enzyme/substrate recognition and the catalytic process of enzymatic desulfation.
Characterization in vitro of the Sulfs will be then completed by a functional study, using the biologically relevant model of FGF-2. In parallel, we will undertake the challenging project of solving HSulf structure by X-ray crystallography. Although very complex, obtaining structural data on these enzymes would be a highly significant achievement. Finally, we propose to look for HSulf inhibitors using automated screening of chemical compound libraries. Identification of biologically active molecules by this systematic approach should help defining the structural basis for the development of new anti-HSulf drugs and possible therapeutic applications.

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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.