Stelter F, Bernheiden M, Menzel R, Witt S, Jack R S, Grunwald U, Fan X, Schütt C
Institute of Immunology and Transfusion Medicine, Ernst-Moritz-Arndt-University, Greifswald, Germany.
Prog Clin Biol Res. 1998;397:301-13.
The CD14 molecule is a key receptor on myeloid lineage cells involved in the recognition of lipopolysaccharide (LPS) and Gram-negative bacteria. The application of its soluble form, sCD14, has been shown to protect mice from lethality in LPS-induced shock. Therefore the protein or its derivatives may be considered as a possible therapeutic alternative for the treatment of patients suffering from Gram-negative septic shock. In this study we performed an alanine scan of amino acids 1 to 152 of human CD14. Twenty-three substitution mutants were generated and stably transfected into CHO-cells. In each mutant five amino acids were substituted by alanine. We analyzed (a) whether mutant proteins expressed on the surface of transfectants were recognized by a panel of anti-CD14 monoclonal antibodies (mAb's), (b) the ability of mCD14-mutants to bind LPS and E. coli in a serum- or LBP-dependent manner, and (c) the capacity of soluble mutants to mediate the LPS-induced IL 6 release of U 373 astrocytoma cells. Twenty-one CD14-mutants were expressed on the surface of transfectants and 18 were present as soluble forms in the culture supernatants. We demonstrated that only CD14(39-41,43-44)A completely lacked the ability to bind LPS and E. coli. In addition, a combined mutant CD14(9-13/57,59,61-63)A had very limited capacity to interact with LPS indicating that the LPS-binding site of human CD14 is a conformational epitope. Analysis of LPS-induced activation of CD14-negative U 373 cells revealed that the regions 9-13 and 91-101 are most important for sCD14-mediated signalling.
CD14分子是髓系细胞上的一种关键受体，参与脂多糖（LPS）和革兰氏阴性菌的识别。其可溶性形式sCD14的应用已被证明可保护小鼠免受LPS诱导的休克致死。因此，该蛋白或其衍生物可被视为治疗革兰氏阴性败血症休克患者的一种可能的治疗选择。在本研究中，我们对人CD14的1至152位氨基酸进行了丙氨酸扫描。产生了23个替代突变体，并将其稳定转染到CHO细胞中。在每个突变体中，五个氨基酸被丙氨酸替代。我们分析了（a）转染细胞表面表达的突变蛋白是否能被一组抗CD14单克隆抗体（mAb）识别，（b）mCD14突变体以血清或LBP依赖方式结合LPS和大肠杆菌的能力，以及（c）可溶性突变体介导LPS诱导的U 373星形细胞瘤细胞释放IL-6的能力。21个CD14突变体在转染细胞表面表达，18个以可溶性形式存在于培养上清液中。我们证明只有CD14(39 - 41,43 - 44)A完全缺乏结合LPS和大肠杆菌的能力。此外，组合突变体CD14(9 - 13/57,59,61 - 63)A与LPS相互作用的能力非常有限，表明人CD14的LPS结合位点是一个构象表位。对LPS诱导的CD14阴性U 373细胞激活的分析表明，9 - 13和91 - 101区域对sCD14介导的信号传导最为重要。
