title: 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.
abstract: 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.
authors.affiliation: Shanghai University of Traditional Chinese Medicine, Shanghai 201-203, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Liu
authors.forename: Shuji
authors.initials: S
authors.lastname: Mori
authors.forename: Hidenori
authors.initials: H
authors.lastname: Wake
authors.forename: Jiyong
authors.initials: J
authors.lastname: Zhang
authors.forename: Keyue
authors.initials: K
authors.lastname: Liu
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Izushi
authors.forename: Hideo K
authors.initials: HK
authors.lastname: Takahashi
authors.forename: Bo
authors.initials: B
authors.lastname: Peng
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nishibori
mesh_terms: D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002846:Chromatography, Affinity; D024243:HMGB1 Protein / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology*; D006801:Humans; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000378:metabolism*; D009418:S100 Proteins / Q000378:metabolism*; D000067616:S100A12 Protein; D015032:Zinc / Q000494:pharmacology
chemical_list: D000893:Anti-Inflammatory Agents; D024243:HMGB1 Protein; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D009418:S100 Proteins; D000067616:S100A12 Protein; C090441:S100A12 protein, human; D006493:Heparin; D015032:Zinc; D002118:Calcium