Demonstration of the hepatocyte growth factor signaling pathway in the in vitro neuritogenic activity of chondroitin sulfate from ray fish cartilage
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概要
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Chondroitin sulfate (CS) was isolated from ray fish cartilage, an industrial waste, after protease digestion, and its structure and neurite outgrowth-promoting (NOP) activity were analyzed to investigate a potential application to nerve regeneration. A disaccharide analysis using chondroitinase ABC revealed that the major unit in the CS preparation was GlcUA-GalNAc(6-O-sulfate) (63%), where GlcUA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively. Small proportions of other disaccharide units, GlcUA-GalNAc(4-O-sulfate) (25%), GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate) (7%), and GlcUA-GalNAc (5%), were also detected. The average molecular mass of CS was estimated to be 142 kDa by gel-filtration chromatography. The prepration showed NOP activity in vitro, which was eliminated by digestion with chondroitinase ABC, suggesting that a polymeric structure is required for the activity. Antibodies against hepatocyte growth factor (HGF) and its receptor c-Met suppressed the NOP activity, suggesting the involvement of the HGF signaling pathway in the in vitro NOP activity of the CS preparation. Since the specific binding of HGF to the CS preparation was also demonstrated by surface plasmon resonance spectroscopy, the CS chains were fractionated using an HGF-immobilized column into unbound and bound fractions accounting for 44 and 56% of the total yield, respectively. The latter contained a higher proportion of the GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate) unit, and showed greater NOP activity than the former, indicating that the HGF-binding domain contains GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate) and is involved in the NOP activity. CS from ray cartilage may have potential pharmaceutical applications.
著者
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Sugahara Kazuyuki
神戸薬科大学 生化
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Sugahara Kazuyuki
Hokkaido Univ. Sapporo
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Yamada Shuhei
Department Of Biochemistry Kobe Pharmaceutical University
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Sugahara Kazuyuki
Dept. Of Biochemistry. Kobe Pharmaceutical University
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Yamada S
Department Of Bioscience And Biotechnology Fuculty Of Engineering Okayama University
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