Abstract：
OBJECTIVE: The aggregation and deposition of amyloid-β (Aβ) peptides in the brain is thought to be the initial driver in the pathogenesis of Alzheimer\'s disease (AD). Aside from full-length Aβ peptides starting with an aspartate residue in position 1, both N-terminally truncated and elongated Aβ peptides are produced by various proteases from the amyloid precursor protein (APP) and have been detected in brain tissues and body fluids. Recently, we demonstrated that the particularly abundant N-terminally truncated Aβ4-x peptides are generated by ADAMTS4, a secreted metalloprotease that is exclusively expressed in the oligodendrocyte cell population. In this study, we investigated whether ADAMTS4 might also be involved in the generation of N-terminally elongated Aβ peptides.
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.
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.
摘要：
目的：淀粉样β（Aβ）肽在大脑中的聚集和沉积被认为是阿尔茨海默病（AD）发病的最初驱动因素。除了从位置1的天冬氨酸残基开始的全长Aβ肽之外,N-末端截短和延长的Aβ肽都由来自淀粉样前体蛋白(APP)的各种蛋白酶产生,并且已经在脑组织和体液中检测到。最近,我们证明了特别丰富的N末端截短的Aβ4-x肽是由ADAMTS4产生的，ADAMTS4是一种分泌的金属蛋白酶，仅在少突胶质细胞群中表达。在这项研究中,我们研究了ADAMTS4是否也可能参与N末端延长的Aβ肽的产生。
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。
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。
