title: C5-epimerase and 2-O-sulfotransferase associate in vitro to generate contiguous epimerized and 2-O-sulfated heparan sulfate domains.
abstract: Heparan sulfate (HS), a complex polysaccharide of the cell surface, is endowed with the remarkable ability to bind numerous proteins and, as such, regulates a large variety of biological processes. Protein binding depends on HS structure; however, in the absence of a template driving its biosynthesis, the mechanism by which protein binding sequences are assembled remains poorly known. Here, we developed a chemically defined 13C-labeled substrate and NMR based experiments to simultaneously follow in real time the activity of HS biosynthetic enzymes and characterize the reaction products. Using this new approach, we report that the association of C5-epimerase and 2-O-sulfotransferase, which catalyze the production of iduronic acid and its 2-O-sulfation, respectively, is necessary to processively generate extended sequences of contiguous IdoA2S-containing disaccharides, whereas modifications are randomly introduced when the enzymes are uncoupled. These data shed light on the mechanisms by which HS motifs are generated during biosynthesis. They support the view that HS structure assembly is controlled not only by the availability of the biosynthetic enzymes but also by their physical association, which in the case of the C5-epimerase and 2-O-sulfotransferase was characterized by an affinity of 80 nM as demonstrated by surface plasmon resonance experiments.
authors.affiliation: †Univ. Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38027 Grenoble, France.|‡CNRS, IBS, F-38027 Grenoble, France.|§CEA, DSV, IBS, F-38027 Grenoble, France.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Préchoux
authors.affiliation: †Univ. Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38027 Grenoble, France.|‡CNRS, IBS, F-38027 Grenoble, France.|§CEA, DSV, IBS, F-38027 Grenoble, France.
authors.forename: Célia
authors.initials: C
authors.lastname: Halimi
authors.affiliation: †Univ. Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38027 Grenoble, France.|‡CNRS, IBS, F-38027 Grenoble, France.|§CEA, DSV, IBS, F-38027 Grenoble, France.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Simorre
authors.affiliation: †Univ. Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38027 Grenoble, France.|‡CNRS, IBS, F-38027 Grenoble, France.|§CEA, DSV, IBS, F-38027 Grenoble, France.
authors.forename: Hugues
authors.initials: H
authors.lastname: Lortat-Jacob
authors.affiliation: †Univ. Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38027 Grenoble, France.|‡CNRS, IBS, F-38027 Grenoble, France.|§CEA, DSV, IBS, F-38027 Grenoble, France.
authors.forename: Cédric
authors.initials: C
authors.lastname: Laguri
mesh_terms: D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism*; D002247:Carbon Isotopes; D006497:Heparan Sulfate / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007067:Iduronic Acid / Q000378:metabolism; D047468:Immunoprecipitation / Q000379:methods; D009682:Magnetic Resonance Spectroscopy; D013379:Substrate Specificity; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D002238:Carbohydrate Epimerases; D002247:Carbon Isotopes; D006497:Heparan Sulfate; D007067:Iduronic Acid; D015238:Sulfotransferases; C118596:UST protein, human; C406782:heparan sulfate L-iduronyl 2-O-sulfotransferase; C000607178:glucuronic acid C5-epimerase, human; C019591:heparosan N-sulfate D-glucuronosyl 5-epimerase