High-Performance Liquid Chromatographic/Mass Spectrometric Studies on the Susceptibility of Heparin Species to Cleavage by Heparanase

Antonella Bisio; Alessandra Mantegazza; Elena Urso; Annamaria Naggi; Giangiacomo Torri; Christian Viskov; Benito Casu

doi:10.1055/s-2007-982079

Seminars in Thrombosis and Hemostasis, Table of Contents

Semin Thromb Hemost 2007; 33(5): 488-495
DOI: 10.1055/s-2007-982079

High-Performance Liquid Chromatographic/Mass Spectrometric Studies on the Susceptibility of Heparin Species to Cleavage by Heparanase

Antonella Bisio¹ , Alessandra Mantegazza¹ , Elena Urso¹ , Annamaria Naggi¹ , Giangiacomo Torri¹ , Christian Viskov² , Benito Casu¹

¹Institute for Chemical and Biochemical Research “G. Ronzoni”, Milan, Italy
²Sanofi-Aventis, Vitry sur Seine, France

Abstract

ABSTRACT

Heparanase is an endo-β-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.

KEYWORDS

Heparanase - heparin oligosaccharides - cleavability by heparanase - mass spectrometry.

Full Text

References

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Dr. Antonella Bisio

Institute for Chemical and Biochemical Research “G. Ronzoni”, Via G. Colombo 81

20133 Milan, Italy

Email: bisio@ronzoni.it