Akins D R, Purcell B K, Mitra M M, Norgard M V, Radolf J D
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
Infect Immun. 1993 Apr;61(4):1202-10. doi: 10.1128/iai.61.4.1202-1210.1993.
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 [3H]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.
一种针对梅毒螺旋体17-kDa主要膜免疫原的鼠单克隆抗体，被用于从梅毒螺旋体基因组文库中筛选表达该分子的重组大肠杆菌克隆。对该免疫原（命名为tpp17）的结构基因进行序列分析，发现一个468 bp的开放阅读框，编码一个由156个氨基酸组成的多肽，计算分子量为16,441 Da。推导的氨基酸序列包括一个假定的前导肽，其末端是用于原核脂蛋白修饰和加工的共有四肽。用[3H]棕榈酸酯放射性标记的克隆免疫原的免疫沉淀证实它是一种脂蛋白。氨基酸序列还预测，除了N端经脂质修饰的半胱氨酸外，成熟蛋白还含有四个半胱氨酸残基。通过对在有和没有2-巯基乙醇存在下溶解的梅毒螺旋体进行免疫印迹，证明了天然免疫原有二硫键连接的多聚体形式。从谷胱甘肽S-转移酶融合蛋白上切割下来的17-kDa免疫原的非脂质形式的Triton X-114相分配表明，脂质修饰是免疫原疏水特性的原因。同样的免疫原非脂质形式也被用于证明脂质修饰对于该分子刺激小鼠巨噬细胞产生肿瘤坏死因子α的能力至关重要。我们得出结论，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，还赋予这些分子激活免疫效应细胞的能力。
