Interaction of peptide-bound beads with lipopolysaccharide and lipoproteins
Interaction of peptide-bound beads with lipopolysaccharide and lipoproteins
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DOI：
10.1016/j.mimet.2014.02.018
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发表时间：
2014-05-01
影响因子：
2.2
通讯作者：
Takagi, Takashi
中科院分区：
文献类型：
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作者：
Suzuki, Masatsugu M.;Matsumoto, Megumi;Takagi, Takashi
我们先前报道了通过噬菌体展示和化学修饰产生脂多糖（LPS）结合肽。其中，命名为Li 5 -025的十二肽（K ′ YSSSSIRAC ′; K ′和C ′分别表示D-赖氨酸和D-半胱氨酸）显示出对LPS的高结合亲和力，并且对蛋白酶消化具有抗性（Suzuki等人，2010年）。在本研究中，产生了Li 5 -025结合的二氧化硅珠粒（下文称为P-珠粒），并且发现其缺乏LPS中和活性。因此，LPS结合的Lathe P-珠可直接用于鲎变形细胞裂解物（LAL）测定。定量分析溶解在乙醇、pH 4、pH 10和0.5M NaCl溶液中的P-珠结合的LPS和结合至P-珠的LPS。观察到该测定的灵敏度约为0.1 pg/mL LPS。P-珠结合溶于抗凝血酶III（AT III）溶液中的LPS，该溶液是LAL试验中活化因子C和B以及凝血酶的强抑制剂;用25%乙腈洗涤P-珠后，AT III的抑制作用完全逆转。这被用作使用LAL测定法检测血浆中游离LPS的第一步。在0 ℃下将LPS加入人血浆中，然后施加到P-珠上，随后用25%乙腈洗涤，导致低LPS活性，如通过LAL测定所检测的。然而，用25%乙腈中的0.1%Triton X100进一步洗涤P-珠导致高LPS活性。这是使用LAL测定法定量检测血浆中游离LPS的第一个实例，并且观察到该方法的灵敏度为1 pg/mL LPS。使用十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析在0.1%Triton X-100洗涤液中洗脱的蛋白质。主要观察到28 kDa和18 kDa的两条蛋白带。质谱分析显示，28 kDa和18 kDa的条带分别对应于载脂蛋白A-1（apoA-I）和载脂蛋白A-II（apoA-II）。ApoA-I和ApoA-II是高密度脂蛋白（HDL）的组分。因此，很可能P-珠结合的LPS被HDL螯合，导致其毒性被中和。该研究表明，通过使用P-珠，血浆中的游离LPS可以通过LAL测定在1 pg/ml的浓度下定量测量。(c)© 2014 Elsevier B. V.保留所有权利。
We previously reported the generation of lipopolysaccharide (LPS)-binding peptides by phage display and chemical modification. Among them, a dodecapeptide designated Li5-025 (K'YSSSISSIRAC'; K' and C' denote D-lysine and D-cysteine, respectively) showed a high binding affinity for LPS and was resistant to protease digestion (Suzuki et al., 2010). In the current study, Li5-025-bound silica beads, hereafter referred to as P-beads, were generated and found to be devoid of LPS-neutralizing activity. Thus, LPS bound Lathe P-beads could be directly used in the Limulus amebocyte lysate (LAL) assay. P-beads bound LPS dissolved in solutions of ethanol, pH 4, pH 10, and 0.5 M NaCl and LPS bound to the P-beads was quantitatively assayed. The sensitivity of this assay was observed to be approximately 0.1 pg/mL LPS. P-beads bound LPS dissolved in antithrombin III (AT III) solution which is a strong inhibitor of activated factors C and B as well as the clotting enzyme in the LAL assay; the inhibitory effect of AT III was completely reversed upon washing the P-beads with 25% acetonitrile. This was employed as the first step for the detection of free LPS in plasma using the LAL assay. LPS added to human plasma at 0 C followed by application to the P-beads and subsequent washing with 25% acetonitrile resulted in low LPS activity as detected by the LAL assay. However, further washing of the P-beads with 0.1% Triton X100 in 25% acetonitrile resulted in high LPS activity. This is the first instance of quantitative detection of free LPS in plasma using the LAL assay, and the sensitivity of this method was observed to be 1 pg/mL of LPS. The proteins eluted in the 0.1% Triton X-100 wash were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis. Two protein bands of 28 kDa and 18 kDa were predominantly observed. Mass spectrometry analysis revealed that the 28 kDa and 18 kDa bands corresponded to apolipoprotein A-1 (apoA-I) and apolipoprotein A-II (apoA-II), respectively. ApoA-I and apoA-II are components of high density lipoprotein (HDL). Thus, it is likely that the P-beads-bound LPS was sequestered by HDL, resulting in neutralization of its toxicity. This study showed that by using P-beads, free LPS in plasma can be quantitatively measured by the LAL assay at a concentration of 1 pg/ml. (c) 2014 Elsevier B.V. All rights reserved.