title: High-performance liquid chromatographic/mass spectrometric studies on the susceptibility of heparin species to cleavage by heparanase.
abstract: Heparanase is an endo-beta-D-glucuronidase that cleaves the heparan sulfate chains of heparan sulfate proteoglycans and is implicated in angiogenesis and metastasis. With the aim of establishing a simple and reliable method for studying the susceptibility of heparin/heparan sulfate oligosaccharides to be cleaved by heparanase, an on-line ion pair reversed-phase high-performance liquid chromatographic/electrospray ionization mass spectrometric method was set up. The method works in the micromolar range of concentration and does not require derivatization of the substrate or of the products. It is based on mass identification of oligosaccharide fragments generated by heparanase and their quantification with reference to an internal heparin disaccharide standard. Substrates were (1) the synthetic pentasaccharides GlcN (NS,6S) - GlcA - GlcN (NS,3S,6S) - IdoA (2S) - GlcN (NS,6S) - OMe (AGA*IA (M)) and GlcN (NS,6S) - GlcA - GlcN (NS,6S) - IdoA (2S) - GlcN (NS,6S) - OMe (AGAIA (M)), corresponding to the heparin/heparan sulfate active site for antithrombin, and to the same sequence devoid of the 3- O-sulfate group in the central glucosamine, respectively; and (2) two natural heparin octasaccharides containing the AGA*IA sequence in different locations along the chain. The two pentasaccharides exhibited a higher susceptibility to heparanase cleavage with respect to the octasaccharides. The commercial availability of AGA*IA (M) makes it an ideal substrate to determine the specific activity of heparanase preparations. The present method could also be used for rapid screening of potential heparanase inhibitors.
authors.affiliation: Institute for Chemical and Biochemical Research G. Ronzoni, Via G. Colombo 81, 20133 Milan, Italy. bisio@ronzoni.it
authors.forename: Antonella
authors.initials: A
authors.lastname: Bisio
authors.forename: Alessandra
authors.initials: A
authors.lastname: Mantegazza
authors.forename: Elena
authors.initials: E
authors.lastname: Urso
authors.forename: Annamaria
authors.initials: A
authors.lastname: Naggi
authors.forename: Giangiacomo
authors.initials: G
authors.lastname: Torri
authors.forename: Christian
authors.initials: C
authors.lastname: Viskov
authors.forename: Benito
authors.initials: B
authors.lastname: Casu
mesh_terms: D000991:Antithrombins / Q000737:chemistry*; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D005966:Glucuronidase / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry*; D006497:Heparan Sulfate / Q000737:chemistry*; D009844:Oligosaccharides / Q000737:chemistry*; D012015:Reference Standards; D021241:Spectrometry, Mass, Electrospray Ionization / Q000592:standards; D000099175:Heparanase
chemical_list: D000991:Antithrombins; D005966:Glucuronidase; D006493:Heparin; D006497:Heparan Sulfate; D009844:Oligosaccharides; D000099175:Heparanase