Design of Peptide Substrate for Sensitively and Specifically Detecting Two Aβ-Degrading Enzymes: Neprilysin and Angiotensin-Converting Enzyme

• Published in: PloS one 2016-04, Vol.11 (4), p.e0153360-e0153360
• Main Authors: Chen, Po-Ting, Chen, Chao-Long, Lin, Lilian Tsai-Wei, Lo, Chun-Hsien, Hu, Chaur-Jong, Chen, Rita P-Y, Wang, Steven S-S
• Format: Article
• Language: English
• Subjects: Acetates - chemistry; Acetates - metabolism; Alzheimer Disease - enzymology; Alzheimer Disease - metabolism; Alzheimer's disease; Amino Acid Sequence; Amyloid beta-Peptides - chemistry; Amyloid beta-Peptides - metabolism; Angiotensin; Biology and Life Sciences; Brain; Brain research; Cell Line, Tumor; Chemical engineering; Chromones - chemistry; Chromones - metabolism; Conversion; Degradation; Enzymes; Fluorescence; High-throughput screening; Humans; Insulin; Insulysin; Insulysin - metabolism; Medicine and Health Sciences; N-Terminus; Neprilysin; Neprilysin - metabolism; Neuroblastoma; Neuroblastoma cells; Neurodegeneration; Neurodegenerative diseases; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Peptides; Peptidyl-dipeptidase A; Peptidyl-Dipeptidase A - metabolism; Physical Sciences; Proteins; Proteolysis; Quenching; Research and Analysis Methods; Rodents; Somatostatin; Spectrometry, Fluorescence - methods; Substrate Specificity; Substrates
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0153360

Upregulation of neprilysin (NEP) to reduce Aβ accumulation in the brain is a promising strategy for the prevention of Alzheimer's disease (AD). This report describes the design and synthesis of a quenched fluorogenic peptide substrate qf-Aβ(12-16)AAC (with the sequence VHHQKAAC), which has a fluorophore, Alexa-350, linked to the side-chain of its C-terminal cysteine and a quencher, Dabcyl, linked to its N-terminus. This peptide emitted strong fluorescence upon cleavage. Our results showed that qf-Aβ(12-16)AAC is more sensitive to NEP than the previously reported peptide substrates, so that concentrations of NEP as low as 0.03 nM could be detected at peptide concentration of 2 μM. Moreover, qf-Aβ(12-16)AAC had superior enzymatic specificity for both NEP and angiotensin-converting enzyme (ACE), but was inert with other Aβ-degrading enzymes. This peptide, used in conjunction with a previously reported peptide substrate qf-Aβ(1-7)C [which is sensitive to NEP and insulin-degrading enzyme (IDE)], could be used for high-throughput screening of compounds that only upregulate NEP. The experimental results of cell-based activity assays using both qf-Aβ(1-7)C and qf-Aβ(12-16)AAC as the substrates confirm that somatostatin treatment most likely upregulates IDE, but not NEP, in neuroblastoma cells.