Hori H, Kanamori T, Mizuta T, Yamaguchi N, Liu Y, Nagai Y
Department of Tissue Physiology, Tokyo Medical and Dental University.
J Biochem. 1994 Dec;116(6):1212-9. doi: 10.1093/oxfordjournals.jbchem.a124666.
We have produced four monoclonal antibodies (mAbs) against human placenta laminin purified by immunoaffinity chromatography. Three clones, 2D9, 3DM, and 4F1, recognized 320 kDa (M) chain of the laminin and the other one, 3DB, recognized B2 chain. One cDNA clone (HLM-1, 3.5 kb) was immunoscreened from human placenta cDNA library using 2D9, and three further overlapping cDNA clones (HLM-2, HLM-3, and HLM-4) covering 2.0 kb were isolated. Nucleotide sequencing and fusion protein analysis demonstrated that the amino acid sequence deduced from HLM-1 coincided with that of the G-domain of human merosin chain, and HLM-2, HLM-3, and HLM-4 encoded the long-arm and EGF-like domain of M chain. The binding regions of 2D9 and 3DM to M chain were defined as homologous repeating sequences of G2-G3 region of G-domain and the carboxy-terminal region of the long-arm, respectively. The extents of identity of amino acid sequences of the long-arm and EGF-like domains between human M chain and A chain were about 37% and 52%, respectively, which were lower than between mouse and human A chains. Northern blot analysis revealed that M chain mRNA, 8.6 kb, was highly expressed in heart and placenta, but less highly expressed in skeletal muscle, brain, and lung. Immunostaining showed selective distributions of M chain in nerve fibers in the dermis and mesangial matrix of the kidney, and B2 chain in subepidermal and kidney glomerular basement membranes.
我们通过免疫亲和层析法纯化了人胎盘层粘连蛋白，并制备了四种抗人胎盘层粘连蛋白的单克隆抗体（mAb）。三个克隆，2D9、3DM和4F1，识别层粘连蛋白的320 kDa（M）链，另一个克隆3DB识别B2链。使用2D9从人胎盘cDNA文库中免疫筛选出一个cDNA克隆（HLM - 1，3.5 kb），并分离出另外三个覆盖2.0 kb的重叠cDNA克隆（HLM - 2、HLM - 3和HLM - 4）。核苷酸测序和融合蛋白分析表明，从HLM - 1推导的氨基酸序列与人肌纤连蛋白链G结构域的序列一致，HLM - 2、HLM - 3和HLM - 4编码M链的长臂和EGF样结构域。2D9和3DM与M链的结合区域分别定义为G结构域G2 - G3区域的同源重复序列和长臂的羧基末端区域。人M链和A链的长臂和EGF样结构域的氨基酸序列同一性程度分别约为37%和52%，低于小鼠和人A链之间的同一性程度。Northern印迹分析显示，8.6 kb的M链mRNA在心脏和胎盘中高表达，但在骨骼肌、脑和肺中表达较低。免疫染色显示M链在真皮神经纤维和肾系膜基质中选择性分布，B2链在表皮下和肾小球基底膜中分布。
