An essential role for albumin in the interaction of endotoxin with lipopolysaccharide-binding protein and sCD14 and resultant cell activation
An essential role for albumin in the interaction of endotoxin with lipopolysaccharide-binding protein and sCD14 and resultant cell activation
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DOI：
10.1074/jbc.m206404200
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发表时间：
2002-12-06
影响因子：
4.8
通讯作者：
Weiss, JP
中科院分区：
文献类型：
--
作者：
Gioannini, TL;Zhang, DS;Weiss, JP
利用内毒素聚集体，即从代谢标记的脑膜炎奈瑟氏菌血清型B中分离的脂寡糖（LOS）的实验证明，白蛋白是脂多糖结合蛋白（LBP）和sCD 14依赖性1）LOS解聚和2）LOS激活人脐静脉内皮细胞（HUVEC）的重要组分。在Sephacryl HR S500缓冲平衡盐溶液加白蛋白中，通过凝胶筛分分离表观M（r）大于或等于2x 10（7）的LOS聚集体（LOSagg）。将LOSagg与LBP和sCD 14一起孵育，以白蛋白依赖性方式促进LOSagg解聚成含有LOS和sCD 14但不含LBP的复合物，通过Sephacryl S200色谱法测定，表观M（r）接近60，000（LOS：sCD 14）。通过凝胶过滤分离LOSagg：由LOSagg与LBP或sCD 14单独相互作用形成的蛋白质聚集体，揭示了LOS-蛋白质相互作用的顺序以及白蛋白对于生物活性LOS：sCD 14的产生所必需的步骤是特异性的。LOS：sCD 14的有效产生需要1）在与CD 14相互作用之前LOSagg与LBP相互作用，以及2）在LBP与LOSagg相互作用期间存在白蛋白。如通过IL-8产生所测量的，LOSagg对HUVEC的激活需要LBP和sCD 14两者，并且在白蛋白存在的情况下是30倍更有效。相比之下，LOS：sCD 14不需要额外的LBP、sCD 14或白蛋白来激活HLTVEC，而是依赖于白蛋白的存在，以便在形成后获得最佳溶解度/稳定性。白蛋白的作用显然是特异性的，因为卵清蛋白和明胶都不能代替白蛋白促进LBP：sCD 14依赖性的LOSagg解聚或内皮细胞活化。这些结果表明，白蛋白是LBP/sCD 14诱导的LOS解聚的重要促进剂，这是通过LOSagg激活内皮细胞所必需的。
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 (LOSagg) with an apparent M(r)greater than or equal to2x10(7) were isolated by gel sieving on Sephacryl HR S500 in buffered balanced salts solution plus albumin. Incubation of LOSagg with LBP and sCD14 promoted LOSagg disaggregation in an albumin-dependent fashion to complexes that contain LOS and sCD14, but no LBP, with an apparent M(r)similar to60,000 (LOS:sCD14) as determined by Sephacryl S200 chromatography. Isolation by gel filtration of LOSagg:protein aggregates formed by the interaction of LOSagg 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 LOSagg with LBP before interaction with CD14 and 2) the presence of albumin during the interaction of LBP with LOSagg. Activation of HUVEC by LOSagg, 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 HLTVEC 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 LOSagg 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 LOSagg.