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

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集合体。这些集合具有独特的脂质结合特性。利用SP-ASP-D嵌合体研究其结构-功能关系，发现Glu^<195>- Phe^<228>的SP-A区域是脂质和II型细胞相互作用的必需区域，Cys^<261>-Phe^<355>的SP-D区域是最佳脂质相互作用的必需区域。识别人SP-A的单克隆抗体（单克隆抗体PElO和PC6）抑制SP-A与脂质和肺泡II型细胞的相互作用。我们利用噬菌体展示肽库绘制了抗人SP-A单抗的抗原表位。单克隆抗体选择的噬菌体显示的一致肽序列与人SP-A的^<184>TPVNYTNWYRG^<194>几乎相同。将大鼠SP-A区Thr^<174>-Gly^<194>替换为MBP-A区Thr^<164>-Asp^<184>（分别为大鼠ama4）的嵌合蛋白。大鼠ama4与甘露糖-sepharose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Ca^<2+>独立GalCer结合。引人注目的是，大鼠ama4嵌合体获得了MBP-A所展示的PI结合特性。本研究表明SP-A的174-194氨基酸残基和MBP-A的相应区域是SP-A型II细胞相互作用和Ca^<2+>依赖性脂质结合的关键区域。肺表面活性蛋白A （SP-A）在肺的抗体非依赖性宿主防御机制中起着重要作用。SP-A与粗糙的LPS结合，但不与光滑的LPS结合。SP-A预孵育U937细胞和大鼠肺泡巨噬细胞；光滑LPS不能诱导TNFalpha分泌，而粗糙LPS诱导的TNFalpha分泌适度增加。Western blot分析显示CD14是从溶解后的U937细胞中分离到的sp - a结合蛋白之一。此外，SP-A直接结合重组可溶性CD14 （rsCDl4）。当rsCDl4与SP-A预孵育时，rsCDl4与光滑LPS的结合显著降低，但与粗糙LPS的结合增强。这些结果证明了SP-A对不同血清型LPS的不同作用，并表明SP-A与CD14的直接相互作用构成了SP-A调节LPS诱导的细胞反应的可能机制。少

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