Kato Dai, Takahashi Yoshiaki, Iwata Haruto, Hatakawa Yusuke, Lee Seon Hwa, Oe Tomoyuki
Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aramaki-aoba, Aoba-ku, Sendai, 980-8578, Japan.
Biochem Biophys Rep. 2022 May 9;30:101268. doi: 10.1016/j.bbrep.2022.101268. eCollection 2022 Jul.
Amyloid beta (Aβ) proteins are produced from amyloid precursor protein cleaved by β- and γ-secretases, and are the main components of senile plaques pathologically found in Alzheimer's disease (AD) patient brains. Therefore, the relationship between AD and Aβs has been well studied for both therapeutic and diagnostic purposes. Several enzymes have been reported to degrade Aβs , with neprilysin (NEP) and insulysin (insulin-degrading enzyme, IDE) being the most prominent. In this article, we describe the mass spectrometric characterization of peptide fragments generated using NEP and IDE, and clarify the differences in digestion specificities between these two enzymes for non-aggregated Aβ, aggregated Aβ, and Aβ peptide fragments, including Aβ. Our results allowed identification of all the peptide fragments from non-aggregated Aβ: NEP, 23 peptide fragments consisting of 2-11 amino-acid residues, 17 cleavage sites; IDE, 23 peptide fragments consisting of 6-33 amino-acid residues, 15 cleavage sites. Also, we confirmed that IDE can digest only whole Aβ, whereas NEP can digest both Aβ and partial structures such as Aβ and peptide fragments generated by the digestion of Aβ by IDE. Furthermore, we confirmed that IDE and NEP are unable to digest aggregated Aβ.
淀粉样β蛋白（Aβ）由淀粉样前体蛋白经β-分泌酶和γ-分泌酶切割产生，是阿尔茨海默病（AD）患者大脑中病理发现的老年斑的主要成分。因此，出于治疗和诊断目的，AD与Aβ之间的关系已得到充分研究。据报道，有几种酶可降解Aβ，其中中性内肽酶（NEP）和胰岛素降解酶（IDE）最为突出。在本文中，我们描述了使用NEP和IDE产生的肽片段的质谱表征，并阐明了这两种酶对非聚集Aβ、聚集Aβ以及包括Aβ在内的Aβ肽片段的消化特异性差异。我们的结果使得能够鉴定出非聚集Aβ的所有肽片段：NEP产生23个由2至11个氨基酸残基组成的肽片段，有17个切割位点；IDE产生23个由6至33个氨基酸残基组成的肽片段，有15个切割位点。此外，我们证实IDE只能消化完整的Aβ，而NEP既能消化Aβ，也能消化部分结构，如Aβ以及由IDE消化Aβ产生的肽片段。此外，我们还证实IDE和NEP均无法消化聚集的Aβ。
