Madampage Claudia Avis, Marciniuk Kristen, Määttänen Pekka, Cashman Neil R, Potter Andrew, Lee Jeremy S, Napper Scott
Vaccine and Infectious Disease Organization; University of Saskatchewan; Saskatoon, SK Canada; Department of Biochemistry; University of Saskatchewan; Saskatoon, SK Canada.
Vaccine and Infectious Disease Organization; University of Saskatchewan; Saskatoon, SK Canada.
Prion. 2013 Nov-Dec;7(6):511-9. doi: 10.4161/pri.27502. Epub 2014 Jan 8.
Species, as well as individuals within species, have unique susceptibilities to prion infection that are likely based on sequence differences in cellular prion protein (PrP(C)). Species barriers to transmission also reflect PrP(C) sequence differences. Defining the structure-activity relationship of PrP(C)/PrP(Sc) with respect to infectivity/susceptibility will benefit disease understanding and assessment of transmission risks. Here, nanopore analysis is employed to investigate genotypes of sheep PrP(C) corresponding to differential susceptibilities to scrapie infection. Under non-denaturing conditions scrapie resistant (ARR) and susceptible (VRQ) genotypes display similar, type I (bumping) predominant event profiles, suggesting a conserved folding pattern. Under increasingly denaturing conditions both proteins shift to type II (intercalation/translocation) events but with different sensitivities to unfolding. Specifically, when pre-incubated in 2M Gdn-HCl, the VRQ variant had more of type II events as compared with the ARR protein, suggesting a more flexible unfolding pattern. Addition of PrP(Sc)-specific polyclonal antibody (YML) to the ARR variant, pre-incubated in 2M Gdn-HCl, reduced the number of type II events with no clear intercalation/translocation peak, whereas for VRQ, type II events above blockades of 90 pA bound YML. A second PrP(Sc)-specific antibody (SN6b) to a different cryptic epitope reduced type II events for VRQ but not the ARR variant. Collectively, the event patterns associated with sequential denaturation, as well as interactions with PrP(Sc)-specific antibodies, support unique patterns and/or propensities of misfolding between the genotypes. Overall, nanopore analysis identifies intermediate conformations that occur during the unfolding pathways of ARR and VRQ genotypes and may help to understand the correlation of structural properties that induce protein misfolding.
物种以及物种内的个体对朊病毒感染具有独特的易感性，这可能基于细胞朊蛋白（PrP(C)）的序列差异。传播的物种屏障也反映了PrP(C)序列差异。确定PrP(C)/PrP(Sc)在感染性/易感性方面的结构-活性关系将有助于疾病的理解和传播风险评估。在此，采用纳米孔分析来研究与痒病感染不同易感性相对应的绵羊PrP(C)基因型。在非变性条件下，抗痒病（ARR）和易感（VRQ）基因型显示出相似的、以I型（碰撞）为主的事件图谱，表明存在保守的折叠模式。在逐渐增加的变性条件下，两种蛋白质都转变为II型（插入/易位）事件，但对解折叠的敏感性不同。具体而言，当在2M盐酸胍中预孵育时，与ARR蛋白相比，VRQ变体有更多的II型事件，表明其解折叠模式更灵活。将PrP(Sc)特异性多克隆抗体（YML）添加到在2M盐酸胍中预孵育的ARR变体中，减少了II型事件的数量，且没有明显的插入/易位峰，而对于VRQ，高于90 pA阻断的II型事件与结合的YML有关。针对不同隐蔽表位的第二种PrP(Sc)特异性抗体（SN6b）减少了VRQ的II型事件，但对ARR变体没有影响。总的来说，与顺序变性相关的事件模式以及与PrP(Sc)特异性抗体的相互作用支持了基因型之间错误折叠的独特模式和/或倾向。总体而言，纳米孔分析确定了在ARR和VRQ基因型解折叠途径中出现的中间构象，并可能有助于理解诱导蛋白质错误折叠的结构特性之间的相关性。
