title: Pivotal advance: analysis of proinflammatory activity of highly purified eukaryotic recombinant HMGB1 (amphoterin).
abstract: HMGB1 (amphoterin) is a 30-kDa heparin-binding protein that mediates transendothelial migration of monocytes and has proinflammatory cytokine-like activities. In this study, we have investigated proinflammatory activities of both highly purified eukaryotic HMGB1 and bacterially produced recombinant HMGB1 proteins. Mass analyses revealed that recombinant eukaryotic HMGB1 has an intrachain disulphide bond. In mass analysis of tissue-derived HMGB1, two forms were detected: the carboxyl terminal glutamic acid residue lacking form and a full-length form. Cell culture studies indicated that both eukaryotic and bacterial HMGB1 proteins induce TNF-alpha secretion and nitric oxide release from mononuclear cells. Affinity chromatography analysis revealed that HMGB1 binds tightly to proinflammatory bacterial substances. A soluble proinflammatory substance was separated from the bacterial recombinant HMGB1 by chloroform-methanol treatment. HMGB1 interacted with phosphatidylserine in both solid-phase binding and cell culture assays, suggesting that HMGB1 may regulate phosphatidylserine-dependent immune reactions. In conclusion, HMGB1 polypeptide has a weak proinflammatory activity by itself, and it binds to bacterial substances, including lipids, that may strengthen its effects.
authors.affiliation: Neuroscience Center, and Institute of Biotechnology, Viikinkaari 4, PL 56, University of Helsinki, Helsinki 00014, Finland. ari.rouhiainen@helsinki.fi
authors.forename: Ari
authors.initials: A
authors.lastname: Rouhiainen
authors.forename: Sarka
authors.initials: S
authors.lastname: Tumova
authors.forename: Leena
authors.initials: L
authors.lastname: Valmu
authors.forename: Nisse
authors.initials: N
authors.lastname: Kalkkinen
authors.forename: Heikki
authors.initials: H
authors.lastname: Rauvala
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D024243:HMGB1 Protein / Q000378:metabolism / Q000494:pharmacology*; D007249:Inflammation / Q000188:drug therapy*; D015850:Interleukin-6 / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D024243:HMGB1 Protein; D015850:Interleukin-6; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide