Les héparanes sulfates (HS) sont produits sous la forme d’un précurseur non sulfaté, qui subit ensuite une phase de maturation catalysée par plusieurs sulfotransférases. Une fois synthétisés, ils peuvent subir une dernière modification par les Sulfs, des 6-O-endosulfatases sécrétées. Ces modifications n’ont pas lieu uniformément sur le polymère : il en résulte une large diversité structurale, ce qui va influencer la fixation et les fonctions de nombreux ligands protéiques. Des études récentes ont mis en exergue l’implication des HS dans la régulation de la réponse inflammatoire. Toutefois, les mécanismes qui contrôlent l’expression de leurs enzymes de modification ont été peu étudiés. Dans ce contexte, mon travail de thèse a porté sur l’influence des conditions inflammatoires sur la machinerie de biosynthèse des HS. La première partie a été consacrée à l’étude des variations d’expression des 3-O-sulfotransférases (3-OSTs) dans les monocytes. Nous avons montré que la 3-OST3B est augmentée de façon précoce en réponse à des agonistes des TLRs et au TNF-α, puis son expression est stabilisée par des mécanismes post-transcriptionnels. Dans la deuxième partie de ma thèse, j’ai étudié la régulation de la 6-O-sulfatation des HS. Alors que l’expression des 6-O-sulfotransférases ne varie pas en réponse à des stimuli inflammatoires, Sulf-1 est fortement induite dans des fibroblastes stimulés par le TNF-α. La forte expression s’accompagne d’une diminution des HS 6-O-sulfatés et est corrélée à la désensibilisation des cellules au FGF-1. Ces résultats suggèrent que la 3-OST3 et Sulf-1, en modifiant la structure des HS, participent à la régulation de la réponse inflammatoire. / Heparan sulfates (HS) are produced from a non-sulfated precursor, which is then subjected to the actions of several sulfotransferases. Thereafter, HS can undergo a last modification catalyzed by secreted 6-O-endosulfatases named Sulfs. These modifications do not take place uniformly in the same chain, resulting in a complex organization that influences the functions of many binding proteins. Recent works have highlighted the involvement of HS in the regulation of the inflammatory response. However, little is known about the mechanisms that regulate the expression of HS-modifying enzymes. Thus, my thesis focused on the regulation of the HS biosynthetic machinery in response to inflammatory stimuli. In the first part, I studied the variations of expression of 3-O-sulfotransferases (3-OSTs) in monocytes. This study showed that 3-OST3B was rapidly up-regulated in response to TLR agonists and TNF-α. Thereafter, the high level of expression was stabilized by post-transcriptional mechanisms. The second part of my thesis was dedicated to the regulation of HS 6-O-sulfation. Although the expression of 6-O-sulfotransferases was not modified in response to inflammatory stimuli, the expression of Sulf-1 was strongly induced in fibroblasts exposed to TNF-α. The high expression of Sulf-1 was accompanied by a decrease in the rate of 6-O-sulfated HS and by the desensitization of fibroblasts to FGF-1. Altogether, these results suggest that 3-OST3B and Sulf-1 could participate in the regulation of the inflammatory response by modifying HS structure.

<p>Heparan sulfates (HSs) are linear, negatively charged polysaccharides composed of alternating hexuronic acid (glucuronic acid or iduronic acid) and glucosamine residues that can be substituted to varying degrees with sulfate groups. HS, localized in the extracellular matrix and on the surface of most cells, interacts with a large number of proteins. The actions of HS largely depend on the amount and distribution of its sulfate groups, that provide binding sites for proteins. </p><p>This thesis focuses on the regulation of the structural diversity in HS, in particular the regulation of its 6-<i>O</i>-sulfation patterns that are generated by the combined action of 6-<i>O</i>-sulfotransferases (6OSTs) during biosynthesis, and 6-<i>O</i>-endosulfatases (Sulfs) after completed biosynthesis. In addition, a new model organism is introduced that offers good prospects for investigating the evolutional aspects of HS structural heterogeneity.</p><p>Our studies showed that the three mouse 6OSTs (6OST1-3) exhibit similar substrate specificities <i>in vitro</i>, with minor differences in target preferences. Overexpression of the 6OSTs in cells resulted in increased 6-<i>O</i>-sulfation of both <i>N</i>-sulfated and <i>N</i>-acetylated glucosamine residues. The changes were independent of enzyme isoform but positively correlated to the level of enzyme expressed.</p><p>Quail Sulf1 and Sulf2 enzymes were shown to be cell surface HS 6-<i>O-</i>endosulfatases with preference towards a subset of trisulfated disaccharides within HS chains. The Sulfs contain a “hydrophilic domain” that was shown to be essential for binding of HS, anchorage to the cell surface and endosulfatase activity. QSulf1 was also shown to promote Wnt-Frizzled signaling in cells. </p><p>An HS-like polysaccharide was isolated from the sea anemone <i>Nematostella vectensis</i> and characterized, and all the enzyme families involved in HS biosynthesis and modification in mammalian model systems were also identified. Our results suggest that <i>Nematostella</i> may be a useful tool for understanding the role of evolution in generating HS structural diversity.</p>

Heparan sulfates (HSs) are linear, negatively charged polysaccharides composed of alternating hexuronic acid (glucuronic acid or iduronic acid) and glucosamine residues that can be substituted to varying degrees with sulfate groups. HS, localized in the extracellular matrix and on the surface of most cells, interacts with a large number of proteins. The actions of HS largely depend on the amount and distribution of its sulfate groups, that provide binding sites for proteins. This thesis focuses on the regulation of the structural diversity in HS, in particular the regulation of its 6-O-sulfation patterns that are generated by the combined action of 6-O-sulfotransferases (6OSTs) during biosynthesis, and 6-O-endosulfatases (Sulfs) after completed biosynthesis. In addition, a new model organism is introduced that offers good prospects for investigating the evolutional aspects of HS structural heterogeneity. Our studies showed that the three mouse 6OSTs (6OST1-3) exhibit similar substrate specificities in vitro, with minor differences in target preferences. Overexpression of the 6OSTs in cells resulted in increased 6-O-sulfation of both N-sulfated and N-acetylated glucosamine residues. The changes were independent of enzyme isoform but positively correlated to the level of enzyme expressed. Quail Sulf1 and Sulf2 enzymes were shown to be cell surface HS 6-O-endosulfatases with preference towards a subset of trisulfated disaccharides within HS chains. The Sulfs contain a “hydrophilic domain” that was shown to be essential for binding of HS, anchorage to the cell surface and endosulfatase activity. QSulf1 was also shown to promote Wnt-Frizzled signaling in cells. An HS-like polysaccharide was isolated from the sea anemone Nematostella vectensis and characterized, and all the enzyme families involved in HS biosynthesis and modification in mammalian model systems were also identified. Our results suggest that Nematostella may be a useful tool for understanding the role of evolution in generating HS structural diversity.