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.
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.
Yamaguchi
Keiko
K
Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Motoyama, Kita-ku, Kyoto, 603-8555, Japan.
Tamaki
Hirotoshi
H
Fukui
Shigeyuki
S
D000818:Animals; D002417:Cattle; D018946:Chemokine CCL5 / Q000378:metabolism*; D002810:Chondroitinases and Chondroitin Lyases; D051523:Fibroblast Growth Factor 7 / Q000378:metabolism*; D005456:Fluorescent Dyes; D006025:Glycosaminoglycans / Q000737:chemistry / Q000378:metabolism; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D015844:Heparin Cofactor II / Q000378:metabolism*; D019763:Heparin Lyase; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D051379:Mice; D046228:Microarray Analysis / Q000379:methods*; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism
D018946:Chemokine CCL5; C496453:FGF7 protein, human; D005456:Fluorescent Dyes; D006025:Glycosaminoglycans; C066855:HGF protein, human; D008024:Ligands; D009844:Oligosaccharides; D011994:Recombinant Proteins; D051523:Fibroblast Growth Factor 7; D017228:Hepatocyte Growth Factor; D015844:Heparin Cofactor II; D006493:Heparin; D002810:Chondroitinases and Chondroitin Lyases; D019763:Heparin Lyase