Prion Research Center, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado 80523, USA.
J Biol Chem. 2012 Oct 26;287(44):37219-32. doi: 10.1074/jbc.M112.395921. Epub 2012 Sep 4.
Whereas prion replication involves structural rearrangement of cellular prion protein (PrP(C)), the existence of conformational epitopes remains speculative and controversial, and PrP transformation is monitored by immunoblot detection of PrP(27-30), a protease-resistant counterpart of the pathogenic scrapie form (PrP(Sc)) of PrP. We now describe the involvement of specific amino acids in conformational determinants of novel monoclonal antibodies (mAbs) raised against randomly chimeric PrP. Epitope recognition of two mAbs depended on polymorphisms controlling disease susceptibility. Detection by one, referred to as PRC5, required alanine and asparagine at discontinuous mouse PrP residues 132 and 158, which acquire proximity when residues 126-218 form a structured globular domain. The discontinuous epitope of glycosylation-dependent mAb PRC7 also mapped within this domain at residues 154 and 185. In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized mAbs whose epitopes also reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments. Our studies also address the paradox of how conformational epitopes remain functional following denaturing treatments and indicate that cellular PrP and PrP(27-30) both renature to a common structure that reconstitutes the globular domain.
虽然朊病毒的复制涉及细胞朊蛋白（PrP(C)）的结构重排，但构象表位的存在仍然是推测性的和有争议的，并且 PrP 的转化是通过免疫印迹检测 PrP(27-30)来监测的，PrP(27-30)是致病瘙痒形式（PrP(Sc)）的蛋白酶抗性对应物。我们现在描述了针对随机嵌合 PrP 产生的新型单克隆抗体（mAb）的特定氨基酸在构象决定因素中的参与。两种 mAb 的表位识别依赖于控制疾病易感性的多态性。一种被称为 PRC5 的 mAb 的检测需要在不连续的小鼠 PrP 残基 132 和 158 处具有丙氨酸和天冬酰胺，当残基 126-218 形成结构球状结构域时，这些残基就会接近。依赖于糖基化的 mAb PRC7 的不连续表位也映射在该结构域内，位于残基 154 和 185 处。与它们的构象依赖性一致，三级结构的干扰会影响 PRC5、PRC7 以及先前表征的 mAb 的识别，这些 mAb 的表位也位于球状结构域内，而该区域附近或远处的构象非依赖性表位则对这种不稳定处理有抗性。我们的研究还解决了构象表位在变性处理后如何保持功能的悖论，并表明细胞 PrP 和 PrP(27-30)都能重新形成一种共同的结构，从而重新形成球状结构域。
