|Budget Amount *help
¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 2004 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 2003 : ¥2,300,000 (Direct Cost : ¥2,300,000)
Heparan sulfate (HS) are known to interact with numerous proteins, such as heparin-binding growth factors (HB-GFs), extracellular matrix proteins, protease inhibitors and various pathogens. These interactions have been shown to play pivotal role in various patho-physiological phenomenons as well as in development. It is important to survey the compounds to control the function of HS.
1. Preparation of octasaccharides n by recombinant enzymes and its inhibitory activity
We first generated 2-O-sulfated oligosaccharides and 6-O-sulfated octasaccharides from completely desulfated 1V-resulfated heparin-derived octasaccharide by in vitro reaction with heparan sulfate-2-O-sulfotransfease (HS2ST) and heparansulfate-6-O-sulfotransferase (HS6ST). Both 2-O-, and 6-O-sulfated octasaccharides were prepared by limited cleavage of heparin. Using this oligosaccharide library, we investigated systematically investigated the specific binding structures for various heparin-binding growth factors (FGF-2, -4
, -7, -8, -10, and -18, HGF, BMP-, and VEGF) by affinity chromatography and surface plasmon resonance. The results indicated that the above listed HB-GFs could be classified roughly into five groups on the basis of the difference in affinity with the oligosaccharides. In particular, FGF-2 and FGF-10 bound to specific oligosaccharide, and the former bound to only 6-0-sulfated octasaccharide and the latter to 2-O-sulfated octasaccharide. Next, we examined the effects of these oligosaccharides and modified heparin on the activities of VEGF_<165>, FGF-1 and FGF-2 by phosphorylation of their receptors. The activity of VEGF_<165> was enhanced by addition of heparin in cultured endothelial cells (HUVEC), but was not affected by any oligosaccharides). In cultured fibroblasts, FGF-1 dependen phosphorylation was increased at low concentration of heparin but decreased at higher concentration of heparin. As FGF-1 bound strongly to some oligosaccharides, we are investigating the effects of oligosaccharides on the activity of FGF-1.
2. Screening of chemical inhibitor for HS2ST and HS6ST
We first surveyed the chemical inhibitors of HS2ST and HS6ST from acceptor substrate analogs. Metyl-β-GlcA(2SO_4) inhibited the activity of HS6ST in vitro, but not Metyl-α-GlcA(2SO_4). Next, to examine whether this compound inhibit the activity of HS6ST in cultured cell, we analyzed the effects of this compound on phosphorylation of FGFR-1 and VEGFR-2 induced with FGF-2 and VEGF_<165>, respectively. More than 3 mM Metyl-β-GlcA(2SO_4) showed inhibitory effect for phosphorylation of VEGFR-2. However, disaccharide compositions of HS generated in the presence of Metyl-β-GlcA(2SO_4) did not change significantly. Therefore, it remains to be clarified whether the inhibitory effect of this compound is due to the mechanism mediated by HS. Less