Host defense mechanism and modulation of lipid metabolism by pulmonary surfactant proteins

宿主防御机制及肺表面活性蛋白对脂质代谢的调节

基本信息

批准号：
09670159
负责人：
KUROKI Yoshio
金额：
$ 2.05万
依托单位：
Sapporo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

1. Pulmonary surfactant proteins A and D (SP-A and SP-D) and mannose-binding protein A(MBP-A) are collectins in the C-type lectin superfamily. These collectins exhibit unique lipid binding properties. The structure-function relationship was investigated by using SP-ASP-D chimeras, It was found that the SP-A region of Glu^<195>- Phe^<228> is required for lipid and type II cell interactions and that the SP-D region of Cys^<261>-Phe^<355> is required for optimal lipid interactions.Monoclonal antibodies (mAbs PElO and PC6) that recognize human SP-A inhibit the interactions of SP-A with lipids and alveolar type II cells. We mapped the epitopes for antihuman SP-A mAbs by a phage display peptide library. Phage selected by mAbs displayed the consensus peptide sequences that are nearly identical to ^<184>TPVNYTNWYRG^<194> of human SP-A.Chimeric proteins were generated in which the rat SP-A region Thr^<174>-Gly^<194> was replaced with the MBP-A region Thr^<164>-Asp^<184> (rat ama4, respectively) … More . Rat ama4 bound to an affinity matrix on mannose-sepharose but lost all of the SP-A functions except carbohydrate binding and Ca^<2+> independent GalCer binding. Strikingly, the rat ama4 chimera acquired the PI binding property that MBP-A exhibits. This study demonstrates that the amino acid residues 174-194 of SP-A and the corresponding region of MBP-A are critical for SP-A-type II cell interaction and Ca^<2+>-dependent lipid binding of collectins.2. Pulmonary surfactant protein A (SP-A) plays an important part in antibody independent host defense mechanisms of the lung. SP-A bound to rough forms but not to smooth forms of LPS.When U937 cells and rat alveolar macrophages were preincubated with SP-A ; smooth LPS failed to induce TNFalpha secretion while rough LPS-induced TNFalpha secretion was modestly increased. Western blot analysis revealed that CD14 was one of the SP-A-binding proteins isolated from solubilized U937 cells. In addition, SP-A directly bound to recombinant soluble CD14 (rsCDl4). When rsCDl4 was preincubated with SP-A, the binding of rsCDl4 to smooth LPS was significantly reduced but the association of rsCDl4 with rough LPS was augmented. These results demonstrate the different actions of SP-A upon distinct serotypes of LPS, and indicate that the direct interaction of SP-A with CD14 constitutes a likely mechanism by which SP-A modulates LPS-elicited cellular responses. Less

1.肺表面活性蛋白A和D（SP-A和SP-D）和甘露糖结合蛋白A（MBP-A）是C型凝集素超家族中的凝集素，这些凝集素表现出独特的脂质结合特性。通过使用 SP-ASP-D 嵌合体研究了这种关系，发现 Glu^<195>-Phe^<228> 的 SP-A 区域是脂质和 II 型细胞相互作用所必需的，并且Cys^<261>-Phe^<355>的SP-D区域是最佳脂质相互作用所必需的。识别人SP-A的单克隆抗体(mAb PE10和PC6)抑制SP-A与脂质和II型肺泡的相互作用我们通过噬菌体展示肽库绘制了抗人 SP-A mAb 的表位图谱，该噬菌体显示了与 mAb 几乎相同的共有肽序列。人SP-A的^<184>TPVNYTNWYRG^<194>。产生嵌合蛋白，其中大鼠SP-A区域Thr^<174>-Gly^<194>被MBP-A区域Thr^<164取代>-Asp^<184>（分别为大鼠 ama4）…更多大鼠 ama4 与甘露糖琼脂糖上的亲和基质结合，但失去了所有 SP-A 功能。除了碳水化合物结合和不依赖Ca 2+ 的GalCer结合之外，引人注目的是，大鼠ama4嵌合体获得了MBP-A所表现出的PI结合特性。该研究表明SP-A的氨基酸残基174-194和相应区域。 MBP-A的表达对于SP-A-II型细胞相互作用和Ca^2+依赖性的集合素脂质结合至关重要。2．在肺的抗体独立宿主防御机制中，SP-A 与粗糙形式的 LPS 结合，但不与光滑形式的 LPS 结合。诱导的 TNFα 分泌适度增加，Western blot 分析显示 CD14 是从溶解的 U937 细胞中分离出的 SP-A 结合蛋白之一。此外，SP-A 直接结合。当rsCD14与SP-A预孵育时，rsCD14与光滑LPS的结合显着减少，但rsCD14与粗糙LPS的结合增强。这些结果证明了SP-A对不同的作用。 LPS 的血清型，并表明 SP-A 与 CD14 的直接相互作用构成了 SP-A 调节的可能机制LPS 引起的细胞反应较少。