Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite-promoting site.
Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite-promoting site.
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DOI：
10.1083/jcb.103.6.2457
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发表时间：
1986-12
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单克隆抗体针对从胎盘分离的人层粘连蛋白的截短形式制备。分离并表征了12种抗体。所有的抗体染色胎盘基底膜和免疫沉淀层粘连蛋白培养的绒毛膜癌细胞的培养基。三种抗体，3E 5，4C 7和4 E10，部分阻断了层粘连蛋白的神经突促进活性。添加第二种抗体，山羊抗小鼠IgG，引起更完全的阻断活性。两个封闭抗体，4C 7和4 E10，与层粘连蛋白长臂末端的球状结构域内的表位反应，第三个，3E 5，在与球状结构域相邻的长臂的杆状部分的末端反应，如旋转阴影后的电子显微镜所示。在相同的测试中使用的五种非阻断抗体与该分子的其他结构域中的表位反应。阻断抗体3E 5和4 E10可用于免疫印迹，两种抗体均与人层粘连蛋白胃蛋白酶片段中的相同多肽反应，主要多肽约为400 kD。当使用人羊膜的粗提取物作为完整层粘连蛋白的来源时，4 E10抗体检测到约400 kD的单一多肽。一个非封闭性抗体，2 E8，在层粘连蛋白交叉的中心反应，主要与人层粘连蛋白片段中的200 kD多肽反应，并专门与羊膜提取物和大鼠层粘连蛋白中的200 kD多肽反应。我们对人层粘连蛋白的研究结果与埃德加，D.，R. Timpl和H. Thoenen，1984，EMBO（Eur.摩尔Biol. Organ.）J.，3：1463-1468，其中小鼠层粘连蛋白的促神经突活性位于长臂的末端，其也是肝素结合的位点。然而，由于人层粘连蛋白的活性片段不与肝素结合，因此神经突促进位点应该不同于肝素结合位点。我们的研究结果进一步表明，轴突促进网站可能包含在或接近层粘连蛋白的400 kD组分。
Monoclonal antibodies were made against a truncated form of human laminin isolated from placenta. 12 antibodies were isolated and characterized. All antibodies stained basement membranes in placenta and immunoprecipitated laminin from media of cultured choriocarcinoma cells. Three antibodies, 3E5, 4C7, and 4E10, partially blocked the neurite-promoting activity of laminin. Addition of a second antibody, goat anti-mouse IgG, caused more complete blocking of the activity. Two of the blocking antibodies, 4C7 and 4E10, reacted with epitopes within the globular domain at the end of the long arm of laminin, and the third one, 3E5, reacted at the end of the rod-like portion of the long arm adjacent to the globular domain, as shown by electron microscopy after rotary shadowing. Five nonblocking antibodies used in the same test reacted with epitopes in other domains of the molecule. Blocking antibodies 3E5 and 4E10 could be used in immunoblotting and both antibodies reacted with the same polypeptides in pepsin fragments of human laminin, the predominant polypeptides being approximately 400 kD. When a crude extract of human amnion was used as a source of intact laminin, the 4E10 antibody detected a single polypeptide of approximately 400 kD. A nonblocking antibody, 2E8, which reacted at the center of the laminin cross, reacted predominantly with a 200-kD polypeptide in human laminin fragments and exclusively with a 200-kD polypeptide in amnion extract and in rat laminin. Our results with human laminin match the results by Edgar, D., R. Timpl, and H. Thoenen, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1463-1468, in which the neurite-promoting activity of mouse laminin resides at the end of the long arm, which is also the site for heparin binding. However, since the active fragments of human laminin did not bind to heparin, the neurite-promoting site should be different from the heparin-binding site. Our results further suggest that the neurite-promoting site may be contained in or close to the 400-kD component of laminin.