LIPID MODIFICATION OF THE 17-KILODALTON MEMBRANE IMMUNOGEN OF TREPONEMA-PALLIDUM DETERMINES MACROPHAGE ACTIVATION AS WELL AS AMPHIPHILICITY
LIPID MODIFICATION OF THE 17-KILODALTON MEMBRANE IMMUNOGEN OF TREPONEMA-PALLIDUM DETERMINES MACROPHAGE ACTIVATION AS WELL AS AMPHIPHILICITY
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DOI：
10.1128/iai.61.4.1202-1210.1993
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发表时间：
1993-04-01
影响因子：
3.1
通讯作者：
RADOLF, JD
中科院分区：
文献类型：
--
作者：
AKINS, DR;PURCELL, BK;RADOLF, JD
使用对梅毒螺旋体 17-kDa 主要膜免疫原具有特异性的鼠单克隆抗体，从梅毒螺旋体基因组文库中选择表达该分子的重组大肠杆菌克隆。对免疫原（指定为 tpp17）结构基因的序列分析揭示了一个 468 bp 的开放阅读框，编码 156 个氨基酸的多肽，计算分子量为 16,441 Da。推导的氨基酸序列包括由共有四肽终止的推定前导肽，用于原核脂蛋白的修饰和加工。用[H-3]棕榈酸酯放射性标记的克隆免疫原的免疫沉淀证实它是脂蛋白。氨基酸序列还预测成熟蛋白除了N端脂质修饰的半胱氨酸外还含有四个半胱氨酸残基。通过在存在和不存在 2-巯基乙醇的情况下溶解的梅毒螺旋体进行免疫印迹，证明了天然免疫原的二硫键多聚体形式的存在。 Triton X-114 对从谷胱甘肽 S-转移酶融合蛋白裂解的非脂质化形式的 17-kDa 免疫原进行相分配，证明脂质修饰是免疫原疏水特性的原因。相同的非脂质形式的免疫原也被用来证明脂质修饰对于该分子刺激鼠巨噬细胞产生肿瘤坏死因子α的能力至关重要。我们得出的结论是，共价连接的脂肪酸不仅将梅毒螺旋体脂蛋白锚定在螺旋体膜上，而且还赋予这些分子激活免疫效应细胞的能力。
A murine monoclonal antibody specific for a 17-kDa major membrane immunogen of Treponema pallidum was used to select recombinant Escherichia coli clones expressing the molecule from a T. pallidum genomic library. Sequence analysis of the structural gene for the immunogen (designated tpp17) revealed a 468-bp open reading frame encoding a polypeptide of 156 amino acids with a calculated molecular mass of 16,441 Da. The deduced amino acid sequence included a putative leader peptide terminated by a consensus tetrapeptide for the modification and processing of prokaryotic lipoproteins. Immunoprecipitation of the cloned immunogen radiolabeled with [H-3]palmitate confirmed that it was a lipoprotein. The amino acid sequence also predicted that the mature protein contains four cysteine residues in addition to the lipid-modified cysteine of the N terminus. The existence of disulfide-bonded multimeric forms of the native immunogen was demonstrated by immunoblotting T. pallidum solubilized in the presence and absence of 2-mercaptoethanol. Triton X-114 phase partitioning of a nonlipidated form of the 17-kDa immunogen cleaved from a glutathione S-transferase fusion protein demonstrated that lipid modification is responsible for the immunogen's hydrophobic character. The same nonlipidated form of the immunogen also was used to demonstrate that lipid modification is essential for the molecule's ability to stimulate production of tumor necrosis factor alpha by murine macrophages. We conclude that covalently attached fatty acids not only anchor T. pallidum lipoproteins to spirochetal membranes but also confer upon these molecules the ability to activate immune effector cells.