Edition of Heparan sulfate by human endosulfatases monitored by several analytical techniques - Archive ouverte HAL Accéder directement au contenu
Poster De Conférence Année : 2023

Edition of Heparan sulfate by human endosulfatases monitored by several analytical techniques

Résumé

Glycosaminoglycans (GAGs) are anionic polysaccharides of remarkable molecular complexity involved in various biological and physio-pathological processes. The determination of structure-function relationships among these molecules is of great interest; however, the complex structure of GAGs, of which heparan sulfate (HS) is the most challenging representative, and the lack of tools for deciphering complex GAG sequences has restricted advances in the GAGs field. In fact, at the molecular level, HS constitutive disaccharide units can be modified by acetylation, epimerization, and sulfation at multiple positions by highly regulated biosynthetic machinery. These modifications are completed by a post-synthetic editing process involving endosulfatases that finely tune the sulfate code along the HS chain. In humans, HSulf-1 and HSulf-2 are extracellular sulfatases that regioselectively remove the 6-O-sulfate groups from HS. HSulfs action alters HS ligand binding properties and modulates multiple signaling pathways. To gain new insights into the functional properties of HSulf enzymes, we set up a robust and resolving analytical method based on hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry (MS). This method allowed the structural determination of the enzyme products from various sulfated oligosaccharide substrates and the monitoring of the 6-O-sulfate hydrolysis of natural sulfated substrates by HSulf enzymes. HILIC-MS methods are developed in our laboratory to allow the separation of GAG-sulfated oligosaccharides by size and sulfate patern. A specific methodology was developed to monitor the progress of the enzyme reaction catalyzed by the endosulfatase HSulfs on various heparin (Hp)-derived oligosaccharides and characterize both the structure and the kinetics of the formation of the enzyme products. We followed the desulfation reaction on various heparin-oligosaccharide substrates over time. The reaction conditions of the heparin oligosaccharide substrates with the enzymes were optimized by adjusting buffer parameters to allow monitoring of the reaction by HILIC-MS. We observed the sequential hydrolysis of 6-O-sulfate groups within the sulfated oligosaccharides, confirming that HSulfs act processively while desulfating their substrates. In addition, we compared a variety of HP oligosaccharides to determine the influence of the sulfated motifs on the HSulf enzyme activity, taking us a step further in deciphering the catalytic mechanism of these specific editing enzymes
Fichier non déposé

Dates et versions

hal-04429176 , version 1 (31-01-2024)

Identifiants

  • HAL Id : hal-04429176 , version 1

Citer

Nesrine El Omrani, Salomé Poyer, Cédric Przybylski, Yoshiko Takeda-Uchimura, Kenji Uchimura, et al.. Edition of Heparan sulfate by human endosulfatases monitored by several analytical techniques. IMMS 2023, Jul 2023, Paris, France. ⟨hal-04429176⟩
12 Consultations
0 Téléchargements

Partager

Gmail Facebook X LinkedIn More