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。肺表面活性剂蛋白A和D（SP-A和SP-D）和甘露糖结合蛋白A（MBP-A）是C-Type超级家族中的collectins。这些集合蛋白具有独特的脂质结合特性。 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抑制SP-A与脂质和牙槽II型细胞的相互作用。我们通过噬菌体展示肽库映射了抗人SP-A mAb的表位。通过mAb选择的噬菌体显示了与^<184> tpvnytnwyrg^<194>几乎相同的人类sp-a.Chimeric蛋白的共识序列，其中将大鼠SP-A区域Thr^<174> -glly^<194>与Mbp-a^<194>更换了大鼠SP-A区域Thr^<174> -GASP-a^<194> <164> - <184> <184大鼠AMA4与甘露糖 - 塞术上的亲和力基质结合，但丢失了除碳水化合物结合和Ca^<2+>独立的galcer结合以外的所有SP-A功能。令人惊讶的是，大鼠AMA4嵌合体获得了MBP-A所表现出的PI结合特性。这项研究表明，氨基酸保留了SP-A的174-194，MBP-A的相应区域对于SP-A型II细胞相互作用和CA^<2+> - 依赖性脂质结合至关重要。2。肺表面活性剂蛋白A（SP-A）在肺的抗体独立宿主防御机制中起重要作用。 SP-A与粗糙形式结合，但不能平滑LP的形式。当U937细胞和大鼠肺泡巨噬细胞被SP-A预孵育时。光滑的LP无法诱导TNFalpha分泌，而粗糙的LPS诱导的TNFALPHA分泌则适度增加。 Western印迹分析表明，CD14是从可溶性U937细胞中分离出的SP-A结合蛋白之一。另外，SP-A直接与重组固体CD14（RSCDL4）结合。当RSCDL4与SP-A预孵育时，RSCDL4与光滑LPS的结合显着降低，但RSCDL4与粗糙LPS的关联增加了。这些结果证明了SP-A对LPS不同血清型的不同作用，并表明SP-A与CD14的直接相互作用构成了SP-A调节LPS渗透的细胞反应的可能机制。较少的

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.

Sano H: "Pulmonary surfactant protein A modulates the cellular response to smooth and rough lipopolysaccharides by intertaction with CD14" J Immunol. (in press). (1999)

Sano H：“肺表面活性蛋白 A 通过与 CD14 相互作用来调节细胞对光滑和粗糙脂多糖的反应”J 免疫学杂志。