title: An essential role for albumin in the interaction of endotoxin with lipopolysaccharide-binding protein and sCD14 and resultant cell activation.
abstract: Experiments utilizing endotoxin aggregates, lipooligosaccharides (LOS) isolated from metabolically labeled Neisseria meningitidis serotype group B, demonstrate that albumin is an essential component of lipopolysaccharide binding protein- (LBP) and sCD14-dependent 1) disaggregation of LOS and 2) LOS activation of human umbilical vein endothelial cells (HUVEC). Aggregates of LOS (LOS(agg)) with an apparent M(r) >or= 2 x 10(7) were isolated by gel sieving on Sephacryl HR S500 in buffered balanced salts solution plus albumin. Incubation of LOS(agg) with LBP and sCD14 promoted LOS(agg) disaggregation in an albumin-dependent fashion to complexes that contain LOS and sCD14, but no LBP, with an apparent M(r) approximately 60,000 (LOS:sCD14) as determined by Sephacryl S200 chromatography. Isolation by gel filtration of LOS(agg):protein aggregates formed by the interaction of LOS(agg) with either LBP or sCD14 alone revealed that the sequence of LOS-protein interactions as well as the step(s) at which albumin is necessary for the production of bioactive LOS:sCD14 were specific. Efficient generation of LOS:sCD14 required 1) interaction of LOS(agg) with LBP before interaction with CD14 and 2) the presence of albumin during the interaction of LBP with LOS(agg). Activation of HUVEC by LOS(agg), as measured by IL-8 production, required both LBP and sCD14 and was thirty times more potent in the presence of albumin. In contrast, LOS:sCD14 did not require additional LBP, sCD14, or albumin to activate HUVEC but depended on the presence of albumin for optimal solubility/stability once formed. The albumin effect is apparently specific, because neither ovalbumin nor gelatin substituted for albumin in facilitating LBP:sCD14-dependent disaggregation of LOS(agg) or activation of endothelial cells. These results indicate that albumin is an essential facilitator of LBP/sCD14-induced LOS disaggregation that is required for activation of endothelial cells by LOS(agg).
authors.affiliation: Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, and Veterans Affairs Medical Center, Iowa City, Iowa 52242, USA. theresa-gioannini@uiowa.edu
authors.forename: Theresa L
authors.initials: TL
authors.lastname: Gioannini
authors.forename: DeSheng
authors.initials: D
authors.lastname: Zhang
authors.forename: Athmane
authors.initials: A
authors.lastname: Teghanemt
authors.forename: Jerrold P
authors.initials: JP
authors.lastname: Weiss
mesh_terms: D000209:Acute-Phase Proteins; D000418:Albumins / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D002845:Chromatography; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology*; D004731:Endotoxins / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D016209:Interleukin-8 / Q000378:metabolism; D050356:Lipid Metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008954:Models, Biological; D009345:Neisseria meningitidis / Q000378:metabolism; D011485:Protein Binding; D014471:Umbilical Veins / Q000166:cytology; D000099231:Lipopolysaccharide-Binding Protein
chemical_list: D000209:Acute-Phase Proteins; D000418:Albumins; D002352:Carrier Proteins; D004731:Endotoxins; D016209:Interleukin-8; D018950:Lipopolysaccharide Receptors; D008562:Membrane Glycoproteins; D000099231:Lipopolysaccharide-Binding Protein