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

层粘连蛋白是具有通过三链α-螺旋卷曲螺旋结构保持在一起的α、β和γ链的异源三聚体。这种结构在来自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同种型，并且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。我们在这里研究了在体内过程中的异源三聚体组装使用四个实验系统。首先，我们在猴COS 1细胞中表达了覆盖长臂不同区域的小鼠层粘连蛋白α 1、β 1或γ 1序列，并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-末端的E8结构域中的序列对组装至关重要。第二，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β 1。这两个片段都与内源性γ 1二硫键结合，并与α 1形成三聚体，α 1被主动分泌到培养基中。第三，我们研制了针对果蝇层粘连蛋白α、β和γ的链特异性抗体，发现果蝇层粘连蛋白β和γ之间的二硫键是α形成α-γ异源三聚体所必需的。第四，我们发现，层粘连蛋白-8与α 4 β 1 γ 1的链组成是唯一的异构体表达在3 T3-L1脂肪细胞。北方印迹显示，在细胞的脂肪转化过程中，α 4、β 1和γ 1 mRNA的水平增加2.5倍。

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.