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Study on the function of laminin by producing transgenic mice and fruit flies

转基因小鼠和果蝇层粘连蛋白功能研究

基本信息

批准号：
09460048
负责人：
KITAGAWA Yasuo
金额：
$ 2.24万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
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-09460048/
关键词：
organogenesis basement membrane Drosophila mouse laminin ショウジョウバエ

项目摘要

Laminins are heterotrimers with alpha, beta, and gamma chains held together by a triple-stranded alpha-helical coiled-coil structure. This structure is highly conserved among laminins from mammalians and invertebrates. Various isoforms of laminin chains have been found and this diversity raised the question how laminin chains are selected for the heterotrimer assembly. We here studied in vivo process of the heterotrimer assembly using four experimental systems. First, we expressed mouse laminin alpha1, beta1 or gamma1 sequence covering various region of the long arm in monkey COS1 cells and found that a common mechanism is shared by laminin chains of different animal origins. We also found that the sequences in E8 domain at C-terminal end of the long arm are crucial for the assembly. Second, we expressed mouse laminin beta1 covering either C-terminal end or whole of the long arm in mouse embryonal carcinoma F9 cells. Both fragments were disulfide-bonded to endogenous gamma1 and formed trimers with alpha1, which was actively secreted into the medium. However, beta1 sequence covering C-terminal end could not support the disulfide-bonding of alpha1 at N-terminus of the long arm. Third, we developed chain specific antibodies directed against Drosophila laminin alpha, beta and gamma and found that the disulfide-bonding between Drosophila beta and gamma is essential for alpha to from alphabetagamma heterotrimer. Fourth, we found that laminin-8 with chain composition of alpha4beta1gamma1 is the only isoform expressed in 3T3-L1 adipocytes. Northern blot showed the levels of alpha4, beta1 and gamma1 mRNAs increase 2.5-fold during the adipose conversion of the cells.

层粘连蛋白是具有 α、β 和 γ 链的异三聚体，通过三链 α 螺旋卷曲螺旋结构连接在一起。这种结构在哺乳动物和无脊椎动物的层粘连蛋白中高度保守。已经发现了层粘连蛋白链的各种亚型，这种多样性提出了如何选择层粘连蛋白链用于异源三聚体组装的问题。我们在这里使用四个实验系统研究了异源三聚体组装的体内过程。首先，我们在猴COS1细胞中表达了覆盖长臂各个区域的小鼠层粘连蛋白α1、β1或γ1序列，发现不同动物来源的层粘连蛋白链具有共同的机制。我们还发现长臂C端E8结构域的序列对于组装至关重要。其次，我们在小鼠胚胎癌 F9 细胞中表达了覆盖 C 末端或整个长臂的小鼠层粘连蛋白 beta1。两个片段均通过二硫键与内源性 γ1 结合，并与 α1 形成三聚体，α1 主动分泌到培养基中。然而，覆盖C末端的β1序列不能支持长臂N末端的α1的二硫键。第三，我们开发了针对果蝇层粘连蛋白α、β和γ的链特异性抗体，并发现果蝇β和γ之间的二硫键对于α形成字母γγ异三聚体至关重要。第四，我们发现具有α4β1γ1链组成的层粘连蛋白-8是在3T3-L1脂肪细胞中表达的唯一亚型。 Northern blot显示，在细胞脂肪转化过程中，α4、β1和γ1 mRNA的水平增加了2.5倍。