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Redundancy and specified function of the serine protease inhibitors, SLPI and elafin, in the airway tracts

丝氨酸蛋白酶抑制剂 SLPI 和 elafin 在气道中的冗余和特定功能

基本信息

批准号：
10470150
负责人：
NUKIWA Toshihiro
金额：
$ 8.38万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10470150/
关键词：
protease inhibitor SLPI elafin targeting airway defense system WAP motif mouse EST database multi-disulfide bridge structure 気道防御

项目摘要

While SLPI (secretory leukoprotease inhibitor) was found as a protease inhibitor (PI) produced in respiratory tracts or reproductive organs, SLPI belongs to the proteins that have characteristic WAP (whey acidic protein) motif of multi-disulfide bridge. The aim of this study is to clarify the physiologic role of both SLPI and elafin, PIs and WAP motif proteins, by molecular cloning and targeting of mouse SLPI and elafin genes. The mouse SLPI cDNA was cloned by usage of the SLPI was expressed in non-epithelial cells in the germinal center of mouse spleen as well as neutrophils and macrophages in the pneumonic alveoli. It is of interest in this context that we have also confirmed human recombinant SLPI induces multi-organotrophic factor, HGF, in human lung fibroblasts, which opens a novel fields of SLPI function. We obtained the genomic mouse SLPI gene structure and identified the location of both human SLPI gene (chromosome 20q12-13.2) and mouse SLPI gene (chromosome 2H). With the infor … More mation of mouse SLPI genomic structure, we constructed a targeting plasmid with PGKNEO and DTA for mouse SLPI gene, transfected with 129/SvJ ES cells, and obtained chimeric SLPI +/- mice and then SLPI -/- mice for the study of their characteristic phenotype. As for the mouse elafin cDNA, failure of the cloning by the same strategy using homologous base sequence among species lead us to access the mouse EST database with WAP motif and found novel ELM1 and ELM2 genes. By way of 5'RACE method we obtained full length cDNA of ELM1 and ELM2, and subsequently revealed the genomic structure of ELM1 and ELM2. Northern blot analysis revealed high expression of ELM1 in kidney, and similar induction pattern of lung ELM1 gene as that of SLP1 gene upon bacterial pneumoria. Because antimicrobial peptides including defensins are also among WAP motif proteins with growing concern for their role in the innate immunity through evolution, our study pioneered the way to understand WAP motif PIs of SLPI and elafin in the context of their physiologic functions. Less

分泌型白蛋白水解酶抑制物(SLPI)是一种产生于呼吸道或生殖器官的蛋白酶抑制物，而SLPI属于具有多二硫键WAP(乳清酸性蛋白)特征基序的蛋白质。本研究的目的是通过小鼠SLPI和elafin基因的分子克隆和靶向，阐明SLPI和elafin、PI和WAP基序蛋白的生理作用。小鼠脾生发中心的非上皮性细胞和肺泡内的中性粒细胞和巨噬细胞均有表达。在此背景下，我们还证实了重组人SLPI可在人肺成纤维细胞中诱导多器官营养因子，这开辟了SLPI功能的新领域。我们获得了小鼠SLPI基因的基因组结构，并对人SLPI基因(染色体20q12-13.2)和小鼠SLPI基因(染色体2H)进行了定位。使用InFor…为了进一步了解小鼠SLPI的基因组结构，我们构建了针对小鼠SLPI基因的靶向表达载体pGKNEO和DTA，并将其导入129/SVJ ES细胞，获得嵌合的SLPI+/-小鼠和SLPI-/-小鼠，以研究其特征表型。对于小鼠elafin基因的克隆，采用物种间同源碱基序列的方法克隆失败，导致我们访问带有WAP基序的小鼠EST数据库，发现了新的ELM1和ELM2基因。通过5‘RACE方法获得了ELM1和ELM2的全长cDNA，揭示了ELM1和ELM2的基因组结构。Northern印迹分析显示ELM1基因在肾脏中高表达，肺ELM1基因的诱导模式与SLP1基因在细菌性肺炎中的诱导模式相似。由于包括防御素在内的抗菌肽也是WAP基序蛋白之一，人们越来越关注它们通过进化在先天性免疫中的作用，我们的研究开创了从生理功能的角度理解SLPI和elafin的WAP基序PI的方法。较少