METHODS: We used cell-free and cell-based assays in combination with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) and electrochemiluminescence sandwich immunoassays to identify and quantify N-terminally elongated Aβ peptide variants. Antibodies against these Aβ variants were characterised by peptide microarrays and employed for the immunohistochemical analyses of human brain samples.
RESULTS: In this study, we discovered additional ADAMTS4 cleavage sites in APP. These were located N-terminal to Asp-(1) in the Aβ peptide sequence between residues Glu-(-7) and Ile-(-6) as well as Glu-(-4) and Val-(-3), resulting in the release of N-terminally elongated Aβ-6-x and Aβ-3-x peptides, of which the latter serve as a component in a promising Aβ-based plasma biomarker. Aβ-6/-3-40 peptides were detected in supernatants of various cell lines and in the cerebrospinal fluid (CSF), and ADAMTS4 enzyme activity promoted the release of Aβ-6/-3-x peptides. Furthermore, by immunohistochemistry, a subset of AD cases displayed evidence of extracellular and vascular localization of N-terminally elongated Aβ-6/-3-x peptides.
CONCLUSIONS: The current findings implicate ADAMTS4 in both the pathological process of Aβ peptide aggregation and in the early detection of amyloid pathology in AD.
方法:我们使用无细胞和基于细胞的测定与基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF)和电化学发光夹心免疫测定相结合,以鉴定和定量N-末端延长的Aβ肽变体。针对这些Aβ变体的抗体通过肽微阵列进行表征,并用于人脑样品的免疫组织化学分析。
结果:在这项研究中,我们在APP中发现了额外的ADAMTS4切割位点。它们位于Aβ肽序列中Asp-(1)的N端,位于Glu-(-7)和Ile-(-6)以及Glu-(-4)和Val-(-3)之间,导致N末端延长的Aβ-6-x和Aβ-3-x肽的释放,其中后者作为有前途的基于Aβ的血浆生物标志物的组成部分。在各种细胞系的上清液和脑脊液(CSF)中检测到Aβ-6/-3-40肽,和ADAMTS4酶活性促进Aβ-6/-3-x肽的释放。此外,通过免疫组织化学,一组AD病例显示了N末端延长的Aβ-6/-3-x肽的细胞外和血管定位的证据。
结论:目前的发现表明ADAMTS4参与了AD中Aβ肽聚集的病理过程和淀粉样蛋白病理的早期检测。