High-performance liquid chromatographic/mass spectrometric studies on the susceptibility of heparin species to cleavage by heparanase.
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.
Bisio
Antonella
A
Institute for Chemical and Biochemical Research G. Ronzoni, Via G. Colombo 81, 20133 Milan, Italy. bisio@ronzoni.it
Mantegazza
Alessandra
A
Urso
Elena
E
Naggi
Annamaria
A
Torri
Giangiacomo
G
Viskov
Christian
C
Casu
Benito
B
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
D000991:Antithrombins; D005966:Glucuronidase; D006493:Heparin; D006497:Heparan Sulfate; D009844:Oligosaccharides; D000099175:Heparanase