title: Expanding the 3-O-Sulfate Proteome--Enhanced Binding of Neuropilin-1 to 3-O-Sulfated Heparan Sulfate Modulates Its Activity.
abstract: Binding of proteins to heparan sulfate is driven predominantly by electrostatic interactions between positively charged amino acid residues in the protein and negatively charged sulfate groups located at various positions along the polysaccharide chain. Although many heparin/heparan-sulfate-binding proteins have been described, few exhibit preferential binding for heparan sulfates containing relatively rare 3-O-sulfated glucosamine residues. To expand the "3-O-sulfate proteome," affinity matrices were created from Chinese hamster ovary (CHO) cell heparan sulfate engineered in vitro with and without 3-O-sulfate groups. Fractionation of different animal sera yielded several proteins that bound specifically to columns containing 3-O-sulfated heparan sulfate modified by two members of the heparan sulfate 3-O-sulfotransferase superfamily, Hs3st1 and Hs3st2. Neuropilin-1 was analyzed in detail because it has been implicated in angiogenesis and axon guidance. We show that 3-O-sulfation enhanced the binding of neuropilin-1 to heparan sulfate immobilized on plastic plates and to heparan sulfate present on cultured cells. Chemoenzymatically synthesized 3-O-sulfated heparan sulfate dodecamers protected neuropilin-1 from thermal denaturation and inhibited neuropilin-1-dependent, semaphorin-3a-induced growth cone collapse of neurons derived from murine dorsal root ganglia. The effect of 3-O-sulfation was cell autonomous and specific to Hs3st2 based on collapse assays of neurons derived from Hs3st1- and Hs3st2-deficient mice. Finally, 3-O-sulfated heparan sulfate enhanced the inhibition of endothelial cell sprouting by exogenous heparan sulfate. These findings demonstrate a reliable method to identify members of the 3-O-sulfate proteome and that 3-O-sulfation of heparan sulfate can modulate axonal growth cone collapse and endothelial cell sprouting.
authors.forename: Bryan E
authors.initials: BE
authors.lastname: Thacker
authors.forename: Emylie
authors.initials: E
authors.lastname: Seamen
authors.forename: Roger
authors.initials: R
authors.lastname: Lawrence
authors.affiliation: Center for Structural Biology, Department of Molecular and Cellular Biochemistry, University of Kentucky , Lexington, Kentucky 40536, United States.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Parker
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina , Chapel Hill, North Carolina 27599, United States.
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina , Chapel Hill, North Carolina 27599, United States.
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
authors.affiliation: Center for Structural Biology, Department of Molecular and Cellular Biochemistry, University of Kentucky , Lexington, Kentucky 40536, United States.
authors.forename: Craig W
authors.initials: CW
authors.lastname: Vander Kooi
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Esko
mesh_terms: D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D051379:Mice; D039942:Neuropilin-1 / Q000378:metabolism*; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D020543:Proteome; D013431:Sulfates / Q000378:metabolism*
chemical_list: D006497:Heparan Sulfate; D039942:Neuropilin-1; D009844:Oligosaccharides; D020543:Proteome; D013431:Sulfates