Edition of heparan sulfate by endosulfatases monitored by HILIC chromatography coupled to mass spectrometry
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.