Establishment of in vitro binding assay of high mobility group box-1 and S100A12 to receptor for advanced glycation endproducts: heparin's effect on binding.
Establishment of in vitro binding assay of high mobility group box-1 and S100A12 to receptor for advanced glycation endproducts: heparin's effect on binding.
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DOI：
10.18926/amo/31812
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发表时间：
2009-08
影响因子：
0.5
通讯作者：
Rui Liu;S. Mori;H. Wake;Jiyong Zhang;Keyue Liu;Yasuhisa Izushi;Hideo Takahashi;Bo Peng;M. Nishibori
中科院分区：
文献类型：
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作者：
Rui Liu;S. Mori;H. Wake;Jiyong Zhang;Keyue Liu;Yasuhisa Izushi;Hideo Takahashi;Bo Peng;M. Nishibori
晚期糖基化终产物受体（RAGE）与其配体之间的相互作用与各种炎症疾病的发病机制有关。本研究建立了重组人高迁移率组盒1 （rhHMGB1）或重组人S100A12 （rhS100A12）固定在微孔板上与重组可溶性RAGE （rsRAGE）结合的体外结合实验。rsRAGE与rhHMGB1和rhS100A12的结合是饱和的，并且依赖于固定化配体。rsRAGE与rhS100A12的结合依赖于Ca2+和Zn2+，而与rhHMGB1的结合不依赖于Ca2+和Zn2+。Scatchard图分析显示rsRAGE对rhHMGB1的亲和力高于rhS100A12。rsRAGE被证明可以与肝素结合，rhS100A12在Ca2+存在下也可以与肝素结合。我们研究了不同分子大小的肝素制剂——未分离天然肝素（UFH）、低分子量肝素（LMWH） 5000Da和低分子量肝素3000Da——对rsRAGE与rhHMGB1和rhS100A12结合的影响。3种制剂均能以浓度依赖性抑制rsRAGE与rhHMGB1的结合，且抑制程度大于rhS100A12。这些结果表明，肝素的抗炎作用可以部分解释为其阻断HMGB1或S100A12与RAGE之间的相互作用。另一方面，肝素可能是一种有希望的有效治疗rage相关炎症性疾病的药物。
Interaction between the receptor for advanced glycation end products (RAGE) and its ligands has been implicated in the pathogenesis of various inflammatory disorders. In this study, we establish an in vitro binding assay in which recombinant human high-mobility group box 1 (rhHMGB1) or recombinant human S100A12 (rhS100A12) immobilized on the microplate binds to recombinant soluble RAGE (rsRAGE). The rsRAGE binding to both rhHMGB1 and rhS100A12 was saturable and dependent on the immobilized ligands. The binding of rsRAGE to rhS100A12 depended on Ca2+ and Zn2+, whereas that to rhHMGB1 was not. Scatchard plot analysis showed that rsRAGE had higher affinity for rhHMGB1 than for rhS100A12. rsRAGE was demonstrated to bind to heparin, and rhS100A12, in the presence of Ca2+, was also found to bind to heparin. We examined the effects of heparin preparations with different molecular sizes - unfractionated native heparin (UFH), low molecular weight heparin (LMWH) 5000Da, and LMWH 3000Da - on the binding of rsRAGE to rhHMGB1 and rhS100A12. All 3 preparations concentration-dependently inhibited the binding of rsRAGE to rhHMGB1 to a greater extent than did rhS100A12. These results suggested that heparin's anti-inflammatory effects can be partly explained by its blocking of the interaction between HMGB1 or S100A12 and RAGE. On the other hand, heparin would be a promising effective remedy against RAGE-related inflammatory disorders.