Nemani S K, Xiao X, Notari S, Cali I, Lundberg K, Cracco L, Appleby B S, Surewicz W K, Sim V L, Gambetti P
Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, AB, Canada.
Division of Neurology, Department of Medicine, University of Alberta, Edmonton, AB, Canada.
Acta Neuropathol Commun. 2025 Aug 12;13(1):172. doi: 10.1186/s40478-025-02079-9.
Variably protease-sensitive prionopathy (VPSPr) is a rare and complex prion disease that differs from sporadic Creutzfeldt-Jakob disease (sCJD) in its clinical and histopathological phenotypes. VPSPr also features a variety of fragments generated by the disease-causing prion protein (PrP). However, accurately determining the number and sequence of these fragments has been challenging when relying solely on epitope mapping with existing antibodies. To address these challenges, we performed mass spectrometry analyses and designed epitope mapping experiments to determine the primary structure and verify the presence or absence of the anchor in the VPSPr proteinase K-resistant and deglycosylated PrP fragments. All three N-terminus fragments, with reported molecular weights of 20, 17, and 7 kDa, likely share Ser97 as the N-terminal amino acid. The C-terminus of the internal 7 kDa fragment is ragged, extending from Phe141 to Met154, while the 20 kDa and 17 kDa fragments differ only in the absence of the anchor in the latter. The three fragments belonging to the C-terminus group have previously been reported to have electrophoretic mobilities of 18, 12/13, and 8-9 kDa. After deglycosylation, the 18 kDa fragment was not detected. The 12 kDa component of the 12/13 kDa fragment was found to have a ragged N-terminus between Tyr162 and Asp181 and the anchor, while the 8 kDa fragment represented the anchorless version of the 12 kDa fragment. Unexpectedly, a second approximately 8 kDa fragment was identified that bore the anchor but had a shorter, ragged N-terminus ranging from Gly195 to Phe198. Calculation based on sequencing data revealed that the actual molecular masses of the 20, 17, and 12 kDa fragments are 1-2 kDa lighter. Moreover, the primary structures of the 20, 17, and 12 kDa fragments match those of the 19, 17, and 12 kDa fragments associated with sCJD type 2. Our findings provide new insights into the characteristics of the deglycosylated, PK-resistant fragments in VPSPr, which will likely assist in interpreting future high-resolution studies of amyloid fibrils in this disease.
可变蛋白酶敏感性朊病毒病（VPSPr）是一种罕见且复杂的朊病毒疾病，在临床和组织病理学表型上与散发性克雅氏病（sCJD）不同。VPSPr的特征还包括由致病朊病毒蛋白（PrP）产生的多种片段。然而，仅依靠现有抗体进行表位作图来准确确定这些片段的数量和序列一直具有挑战性。为应对这些挑战，我们进行了质谱分析并设计了表位作图实验，以确定VPSPr蛋白酶K抗性和去糖基化PrP片段的一级结构并验证锚定序列的有无。所有三个N端片段，报道的分子量分别为20、17和7 kDa，可能都以Ser97作为N端氨基酸。内部7 kDa片段的C端参差不齐，从Phe141延伸至Met154，而20 kDa和17 kDa片段的区别仅在于后者没有锚定序列。先前报道属于C端组的三个片段的电泳迁移率分别为18、12/13和8 - 9 kDa。去糖基化后，未检测到18 kDa片段。12/13 kDa片段的12 kDa组分的N端在Tyr162和Asp181之间参差不齐且有锚定序列，而8 kDa片段代表12 kDa片段的无锚定版本。出乎意料的是，鉴定出了第二个约8 kDa的片段，它带有锚定序列但N端较短且参差不齐，范围从Gly195到Phe198。根据测序数据计算表明，20、17和12 kDa片段的实际分子量轻1 - 2 kDa。此外，20、17和12 kDa片段的一级结构与2型sCJD相关的19、17和12 kDa片段的结构匹配。我们的研究结果为VPSPr中去糖基化、蛋白酶K抗性片段的特征提供了新的见解，这可能有助于解释该疾病未来淀粉样纤维的高分辨率研究。
