Kalluri R, Sun M J, Hudson B G, Neilson E G
Penn Center for Molecular Studies of Kidney Diseases, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 1996 Apr 12;271(15):9062-8. doi: 10.1074/jbc.271.15.9062.
The family of type IV collagen comprises six chains numbered alpha1 through alpha6. The alpha3(IV) NC1 domain is the primary target antigen for autoantibodies from patients with anti-basement membrane disease and Goodpasture syndrome. Earlier peptide studies suggested that the last 36 amino acids of the alpha3 NC1 domain probably contains one recognition site for Goodpasture autoantibodies, and an algorithm analysis of secondary structure from a later study predicted a second possible upstream epitope near the triple helix junction. We have used several analytic approaches to evaluate the likelihood of two immunologic epitopes for the Goodpasture antigen. In our first set of studies, peptide antibodies directed against these two putative regions co-inhibited Goodpasture autoantibodies binding to denatured human alpha3(IV) NC1 monomer by nearly 80%, with the helix-junction region of the alpha3 NC1 domain contributing 26% of the binding sites and the C-terminal region contributing the remaining 50%. Second, both of these candidate regions are normally sequestered within the associated alpha3(IV) NC1 hexamer but become more visible for binding by anti-peptide antibodies upon their dissociation, a property that is shared by the Goodpasture autoantibodies. Third, segment deletions of recombinant alpha3 NC1 domain further confirmed the presence of two serologic binding sites. Finally, we looked more closely at the C-terminal binding region of the alpha3(IV) NC1 domain. Since the lysines in that region have been previously advanced as possible contact sites, we created several substitutions within the C-terminal epitope of the alpha3 NC1 domain. Substitution of lysines to alanines revealed lysines 219 and 229 as essential for antibody binding to this distal site; no lysines were present in the NC1 part of the helix-NC1 junction region. Substitutions involving arginine and cysteines to alanines in the same C-terminal region did not produce significant reductions in antibody binding. In summary, our findings characterize two Goodpasture epitopes confined to each end of the alpha3 NC1 domain; one is lysine-dependent, and the other is not. We propose, as a hypothetical model, that these two immunologically privileged regions fold to form an optimal pathogenic structure within the NC1 domain of the alpha3 chain. These sites are subsequently concealed by NC1 hexamer assembly of type IV collagen.
IV型胶原家族由编号为α1至α6的六条链组成。α3（IV）NC1结构域是抗基底膜病和Goodpasture综合征患者自身抗体的主要靶抗原。早期的肽研究表明，α3 NC1结构域的最后36个氨基酸可能包含一个Goodpasture自身抗体的识别位点，后来一项研究的二级结构算法分析预测在三螺旋交界处附近有第二个可能的上游表位。我们使用了几种分析方法来评估Goodpasture抗原的两个免疫表位的可能性。在我们的第一组研究中，针对这两个假定区域的肽抗体共同抑制Goodpasture自身抗体与变性的人α3（IV）NC1单体的结合，抑制率近80%，其中α3 NC1结构域的螺旋交界处区域贡献了26%的结合位点，C末端区域贡献了其余的50%。其次，这两个候选区域通常被隔离在相关的α3（IV）NC1六聚体内，但在解离后对肽抗体的结合变得更易见，这是Goodpasture自身抗体共有的特性。第三，重组α3 NC1结构域的片段缺失进一步证实了两个血清学结合位点的存在。最后，我们更仔细地研究了α3（IV）NC1结构域的C末端结合区域。由于该区域的赖氨酸先前被认为是可能的接触位点，我们在α3 NC1结构域的C末端表位内进行了几处替换。将赖氨酸替换为丙氨酸显示，赖氨酸219和229对于抗体结合该远端位点至关重要；螺旋-NC1交界处区域的NC1部分不存在赖氨酸。在同一C末端区域将精氨酸和半胱氨酸替换为丙氨酸不会显著降低抗体结合。总之，我们的研究结果确定了α3 NC1结构域两端的两个Goodpasture表位；一个依赖赖氨酸，另一个不依赖。作为一个假设模型，我们提出这两个免疫特权区域折叠形成α3链NC1结构域内的最佳致病结构。这些位点随后被IV型胶原的NC1六聚体组装所掩盖。
