Isolation, structural characterization of natural chondroitin sulfate oligosaccharides and their binding study with anti-angiogenic factors

Drugs that inhibit tumor angiogenesis, promote vascular normalization and improve the tumor microenvironment. However, their application is limited by adaptive or compensatory resistance. Chondroitin sulfate (CS) regulates numerous proteins including pro-angiogenic growth factors, for whom binding affinity depends on sulfation of CS. In this study, we aimed to determine how sulfation of natural tetrasaccharides and hexasaccharides of CS affected binding to the vascular endothelial growth factor (VEGF-A) and fibroblast growth factor 2 (FGF-2). Twenty-eight CS oligosaccharide isomers were obtained by preparative HPLC, tagged with the AEAB fluorescent linker, and identified using an improved chemical derivatization strategy combined with tandem mass spectrometry. CS oligosaccharide microarrays revealed that VEGF-A and FGF-2 bound preferentially to highly sulfated CS, and the GalNAc(4 S )GlcA(2S)GalNAc(6S) sequence was found to be indispensable for binding to these proteins. By integrating glycan microarrays with computational modeling, this study revealed the relationship between the structure of CS and its interactions with pro-angiogenic factors. The degree and the specific sulfation patterns on CS should be taken into account when designing anti-angiogenic drugs.