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

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670159/
• 关键词: pulmonary surfactant; SP-A; SP-D; mannose-binding protein; collectin; lipopolysaccharides; CD14; TNFalpha; サーファクタント蛋白質A; ホスファチジルコリン; ホスファチジルイノシトール; サーファクタント蛋白質; C型レクチン; 肺胞II型細胞; 糖鎖認識領域

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）以及甘露糖结合蛋白A（MBP-A）是C型凝集素超家族中的凝集素。这些聚集素表现出独特的脂质结合特性。发现Glu^<195>- Phe^的<228>SP-A区是脂质和肺泡II型细胞相互作用所必需的，Cys^ -Phe ^的SP-D区是<261><355>最佳脂质相互作用所必需的，识别人SP-A的单克隆抗体PE 10和PC 6抑制SP-A与脂质和肺泡II型细胞的相互作用。利用噬菌体肽库技术对抗人SP-A单克隆抗体的表位进行了定位。通过单克隆抗体选择的噬菌体展示了与人SP-A的TPVNYTNWYRG^几乎相同的共有肽序列<184><194>。产生了嵌合蛋白，其中大鼠SP-A区域Thr^<174>-Gly^<194>被MBP-A区域Thr^<164>-Asp^<184>（分别为大鼠ama 4）取代。 ...更多信息 .大鼠ama 4与甘露糖-琼脂糖凝胶上的亲和基质结合，但失去了除了碳水化合物结合和Ca^2+非依赖性GalCer结合之外的所有SP-A功能。引人注目的是，大鼠ama 4嵌合体获得了MBP-A表现出的PI结合特性。本研究表明SP-A的174-194位氨基酸残基和MBP-A的相应区域对于SP-A-II型细胞相互作用和胶原凝集素的Ca^&lt;2+&gt;依赖性脂质结合是至关重要的.肺表面活性物质蛋白A（SP-A）在非抗体依赖性宿主肺防御机制中起重要作用。当U937细胞和大鼠肺泡巨噬细胞与SP-A预孵育时，光滑LPS不能诱导TNF α分泌，而粗糙LPS诱导的TNF α分泌略有增加。Western blot分析表明，CD 14是从溶解的U937细胞中分离的SP-A结合蛋白之一。此外，SP-A直接结合重组可溶性CD 14（rsCD 14）。当rsCD 14与SP-A预孵育时，rsCD 14与光滑LPS的结合显著减少，但rsCD 14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CD 14的直接相互作用构成了SP-A调节LPS引起的细胞应答的可能机制。少

项目成果

Honma T: "The mannose-binding protein A region of Glu185-Ala221 can functionally replace the surfactant protein A region of Glu195-Phe228 without loss of interaction with lipidsand alveolar type II cells." Biochemistry. 36. 7176-7184 (1997)

Honma T：“Glu185-Ala221 的甘露糖结合蛋白 A 区域可以在功能上取代 Glu195-Phe228 的表面活性蛋白 A 区域，而不会失去与脂质和 II 型肺泡细胞的相互作用。”

Shijubo N: "BAL surfactant protein A and Clara cells 10 kDa protein levels in healthy subjects." Lung. 176. 257-265 (1998)

Shijubo N：“健康受试者中 BAL 表面活性蛋白 A 和 Clara 细胞的蛋白水平为 10 kDa。”

Kuroki Y: "Surfactant proteins A and D : disease markers." Biochem.Biophys.Acta. 1408. 334-345 (1998)

Kuroki Y：“表面活性蛋白 A 和 D：疾病标志物。”

Sano H,Kuroki Y,et al.: "Analysis of chimeric proteins identifies the regions in the carbohydrate recognition domains of rat lung collectins that are essential for interactions with phospholipids,glycolipids,and alveolar typeII cells." J.Biol.Chem.273・8.

Sano H、Kuroki Y 等人：“嵌合蛋白的分析确定了大鼠肺集合素碳水化合物识别域中对于与磷脂、糖脂和肺泡 II 型细胞相互作用至关重要的区域。”・8.

Pattanajitvilai S: "Mutational analysis of Arg197 of rat surfactant protein A.His197 creates specific lipid uptake defects." J Biol Chem. 273. 5702-5707 (1998)

Pattanajitvilai S：“对大鼠表面活性蛋白 A.His197 的 Arg197 进行突变分析会产生特定的脂质摄取缺陷。”