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 labeledNeisseria 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 > 2 ؋ 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 ϳ 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 LOSprotein 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 .

Molecular Basis of Reduced Potency of Underacylated Endotoxins

“…However, it has also been demonstrated that functional differences between an endotoxin species that is a potent agonist and a partially deacylated derivative that is a potent antagonist involve interactions that occur after CD14 (22,31,32). Because, at least in solution, the active form of endotoxin complexed to CD14 is a monomeric protein:endotoxin complex (7,24), these findings suggest variables that are independent of differences in the aggregate properties of the different endotoxin species. We have previously speculated that, at least for some endotoxin species, decreased acylation would decrease endotoxin agonist properties not by effects on endotoxin aggregate properties, but by altering properties of monomeric protein:endotoxin complexes that regulate delivery of endotoxin to MD-2 and/or activation of TLR4 (8,12).…”

“…2A), making it likely that some residual hexa-acylated (wt) LOS remains in LOS AOAH preparations (26). Nevertheless, LOS AOAH : MD-2 complexes have the identical fatty acid composition as LOS AOAH aggregates, also indicating that LBP, sCD14, and These transfer reactions in aqueous solution also depend on albumin (12,24). We propose that hexa-acylated, but not underacylated, endotoxins promote conformational/ configurational changes in MD-2 and/or TLR4 that lead to oligomerization of TLR4, an event necessary for cellular activation (36 -38).…”

Endotoxin-binding Proteins Modulate the Susceptibility of Bacterial Endotoxin to Deacylation by Acyloxyacyl Hydrolase

“…The susceptibility of LOS to AOAH was markedly increased by pretreatment of LOS agg with LBP ϩ sCD14 to convert LOS agg to monomeric LOS-sCD14 complex (Fig. 1A) (23). Incubation of LOS agg with LBP alone also markedly increased the susceptibility of LOS to AOAH at specific LBP:LOS ratios (Fig.…”

Biochemical and Functional Characterization of Membrane Blebs Purified from Neisseria meningitidis Serogroup B

“…7, A and B, both the rate and extent of accumulation of radiolabeled M r ϳ60,000 species was greater from pLOS agg than from membrane blebs. These products were not generated from either pLOS agg or blebs when LBP or sCD14 was added alone (data not shown (7,25). The product derived from pLOS agg is LOS⅐sCD14 (7).…”

“…Complex-Maximal potency of LBP-and sCD14-dependent cell activation of endothelial cells by endotoxin requires formation of monomeric endotoxin⅐sCD14 complex (7,9). Thus, differences in cell activation by pLOS agg and by membrane blebs could reflect differences in the efficiency of LBP/sCD14-dependent extraction and transfer of endotoxin to sCD14 from purified endotoxin aggregates versus purified membranes.…”