Turner N, Forstová J, Rees A, Pusey C D, Mason P J
Department of Medicine, Royal Postgraduate Medical School, London, United Kingdom.
J Biol Chem. 1994 Jun 24;269(25):17141-5.
The Goodpasture antigen is the target of anti-basement membrane autoantibodies in Goodpasture's disease, a severe human autoimmune disease characterized by glomerulonephritis and lung hemorrhage. It has been identified as the NC1 domain of the alpha 3 chain of type IV collagen (alpha 3(IV)NC1), a minority component of glomerular basement membrane (GBM). Protocols for obtaining pure human antigen are laborious and low yielding and require cadaver kidneys. Recombinant alpha 3(IV)NC1 produced in Escherichia coli has been insoluble and poorly recognized by patients' autoantibodies. We have used the baculovirus expression system to produce the antigen as a soluble product in Sf9 cells. A transfer vector was constructed from cDNAs encoding the leader peptide, NH2 terminus, and 7 S domain of the human alpha 1 chain of type IV collagen and was joined inframe to the NC1 domain and COOH terminus of the human alpha 3 chain under the control of the polyhedrin promoter. It therefore encodes a hybrid "mini"-collagen chain from which the majority of the central triple helical region has been deleted. The recombinant antigen was seen on SDS-polyacrylamide gel electrophoresis and Western blots of supernatants at its predicted molecular size of 41 kDa and as dimers of 82 kDa. It reacted strongly with human autoantibodies by Western blotting and enzyme-linked immunosorbent assay, inhibited binding of autoantibodies to human GBM, and bound two monoclonal antibodies known to recognize human alpha 3(IV)NC1. A common alternatively spliced variant alpha 3(IV)NC1 mRNA, leading to a truncated NC1 domain of 60 amino acids, was expressed as a fusion protein with the same alpha 1 NH2-terminal sequence. It failed to be exported from the cell and was not recognized by autoantibodies. Other NC1 domains could be expressed in the same way. These recombinant molecules should prove invaluable for the in vitro study of the immunopathogenesis of Goodpasture's disease, and the approach provides a means by which interactions between the different type IV collagen chains found in GBM could be studied in vitro.
Goodpasture抗原是Goodpasture病中抗基底膜自身抗体的靶抗原，Goodpasture病是一种严重的人类自身免疫性疾病，其特征为肾小球肾炎和肺出血。它已被鉴定为IV型胶原α3链的NC1结构域（α3(IV)NC1），是肾小球基底膜（GBM）的一种次要成分。获取纯人类抗原的方案既费力又产量低，且需要尸体肾脏。在大肠杆菌中产生的重组α3(IV)NC1不溶，且患者自身抗体对其识别不佳。我们利用杆状病毒表达系统在Sf9细胞中产生可溶性抗原产物。构建了一种转移载体，其由编码IV型胶原α1链的前导肽、NH2末端和7S结构域的cDNA组成，并在多角体蛋白启动子的控制下与人类α3链的NC1结构域和COOH末端读框内连接。因此，它编码一种杂交“微型”胶原链，其中大部分中央三螺旋区域已被删除。重组抗原在SDS-聚丙烯酰胺凝胶电泳和上清液的Western印迹中以其预测的41 kDa分子大小出现，并以82 kDa的二聚体形式出现。通过Western印迹和酶联免疫吸附测定，它与人自身抗体强烈反应，抑制自身抗体与人GBM的结合，并结合已知识别人类α3(IV)NC1的两种单克隆抗体。一种常见的可变剪接变体α3(IV)NC1 mRNA，导致产生一个60个氨基酸的截短NC1结构域，被表达为具有相同α1 NH2末端序列的融合蛋白。它未能从细胞中分泌出来，也未被自身抗体识别。其他NC1结构域也可以以同样的方式表达。这些重组分子对于Goodpasture病免疫发病机制的体外研究应具有极高价值，并且该方法提供了一种在体外研究GBM中发现的不同IV型胶原链之间相互作用的手段。
