Improvement of carbohydrate microarray and the search for biological active ligands by using oligosaccharides library.

这种新型的糖脂技术非常适合用于高通量检测和碳水化合物-蛋白质相互作用特异性分配的微阵列设计。皮肤硫酸酯（DS）由于能够结合生长因子和趋化因子，并在炎症和损伤反应过程中调节其生物活性而发挥重要作用。我们利用软骨素酶ACI完全消化DS制备了多种富含伊糖醛酸的片段，并研究了DS结合蛋白如肝细胞生长因子/散射因子（HGF/SF）、RANTES、角化细胞生长因子或成纤维细胞生长因子-7 （KGF/FGF-7）和肝素辅助因子- ii （HCII）能否在新糖脂芯片上检测到它们的低聚糖配体。首先，将软骨素寡糖与肝素寡糖的结合信号强度进行比较，表明我们的微阵列系统不仅可以分离出寡糖配体，而且可以检测出与静电相互作用无关的内在相互作用与非特异性静电相互作用之间的差异。其次，HGF/SF、KGF/FGF-7和HCII优先结合长度大于8米的富含伊杜醛酸的DS寡糖片段。相反，RANTES结合似乎只依赖于负电荷；它们对DS寡糖的结合强度比HGF/SF、KGF/FGF-7和HCII的结合强度稍强。第三，在这些依赖糖胺聚糖的反应中，使用聚乙烯吡咯烷酮（PVP）、卵清蛋白（OV）和Tween 20代替牛血清白蛋白（BSA）作为印迹剂是有用的，可以最大限度地减少因非特异性相互作用而产生的背景。

The 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 the 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 Hepatocyte growth factor/scatter factor (HGF/SF), RANTES, Keratinocyte growth factor or fibroblast growth factor-7 (KGF/FGF-7) and Heparin cofactor-II (HCII), can detect their oligosaccharide ligands in 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 preferentially binding to iduronic acid-rich fragments of DS oligosaccharides that are greater than 8-mers in length. 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 (PVP), ovalbumin (OV) and Tween 20 in place of BSA as a blotting agent was useful in these glycosaminoglycan dependent reactions for minimizing background due to non-specific interactions.