Detection of oligosaccharide ligands for Hepatocyte growth factor/Scatter factor (HGF/SF), Keratinocyte growth factor (KGF/FGF-7), RANTES and Heparin cofactor II by neoglycolipid microarrays of glycosaminoglycan-derived oligosaccharide fragments |
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Authors: | Keiko Yamaguchi Hirotoshi Tamaki Shigeyuki Fukui |
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Institution: | (1) Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Motoyama, Kita-ku, Kyoto 603-8555, Japan;(2) CREST, Japan Science and Technology Agency, Japan;(3) Shionogi & Co., LTD, Hikari-machi, Higashi-ku, Hiroshima 732-0052, Japan;(4) Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Kamigamo-Motoyama, Kita-ku, Kyoto 603-8555, Japan |
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Abstract: | Neoglycolipid technology is eminently adaptable for microarray design for high-throughput detection and specificity assignments
of carbohydrate-protein interactions. Dermatan sulfate (DS) is known to play an important role because of its ability to bind
growth factors as well as chemokines and to modulate their biological activities during inflammation and response to injury.
We prepared various iduronic acid-rich fragments from DS by complete digestion with chondroitinase ACI, and investigated whether
the DS-binding proteins, such as HGF/SF, RANTES, KGF/FGF-7 and HCII, can detect their oligosaccharide ligands in a neoglycolipid
microarray. First, a comparison of the intensity of binding signals obtained from chondroitin oligosaccharides with those
of heparin oligosaccharides showed that our microarray system is feasible not only to single-out the oligosaccharide ligands,
but also to detect the difference between an intrinsic interaction unrelated only to electrostatic interaction and non-specific
electrostatic interaction. Second, HGF/SF, KGF/FGF-7 and HCII showed preferential binding to iduronic acid-rich fragments
of DS oligosaccharides that are greater than 8-mers in lengths. In contrast, RANTES binding seemed to depend only on the negative
charges; their binding intensity towards the DS oligosaccharides was somewhat stronger than the binding of HGF/SF, KGF/FGF-7
and HCII. Third, the use of polyvinylpyrrolidone-40 (PVP-40), ovalbumin (OV) and Tween 20 in place of BSA as a blotting agent
was useful in these glycosaminoglycan dependent reactions to minimize background due to non-specific interactions. |
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Keywords: | Oligosaccharide-microarray Hepatocyte growth factor/scatter factor Keratinocyte growth factor RANTES Heparin cofactor II Dermatan sulfate Neoglycolipid |
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