期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 113, 期 45, 页码 12774-12779出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1611642113
关键词
nerve highlighting; laminin; proximity-based labeling; molecular imaging; surgery with molecular navigation
资金
- NIH/National Cancer Institute (NCI) [5R01CA158448]
- NIH/National Institute of Neurological Disorders and Stroke (NINDS) [2RO1NS027177]
- NIH/National Institute of Biomedical Imaging and Bioengineering (NIBIB) [1R01EB014929]
- Swiss National Science Foundation [31003A_160259]
- Special Opportunity Project HDL-X from the Swiss Initiative in Systems Biology SystemsX.ch
- Swiss National Science Foundation (SNF) [31003A_160259] Funding Source: Swiss National Science Foundation (SNF)
Target-blind activity-based screening ofmolecular libraries is often used to develop first-generation compounds, but subsequent target identification is rate-limiting to developing improved agents with higher specific affinity and lower off-target binding. A fluorescently labeled nerve-binding peptide, NP41, selected by phage display, highlights peripheral nerves in vivo. Nerve highlighting has the potential to improve surgical outcomes by facilitating intraoperative nerve identification, reducing accidental nerve transection, and facilitating repair of damaged nerves. To enable screening of molecular target-specific molecules for higher nerve contrast and to identify potential toxicities, NP41' s binding target was sought. Laminin-421 and -211 were identified by proximity-based labeling using singlet oxygen and by an adapted version of TRICEPS-based ligand-receptor capture to identify glycoprotein receptors via ligand cross-linking. In proximity labeling, photooxidation of a ligand-conjugated singlet oxygen generator is coupled to chemical labeling of locally oxidized residues. Photooxidation of methylene blue-NP41-bound nerves, followed by biotin hydrazide labeling and purification, resulted in light-induced enrichment of laminin subunits alpha 4 and alpha 2, nidogen 1, and decorin ( FDR-adjusted P value < 10(-7)) and minor enrichment of laminin-.1 and collagens I and VI. Glycoprotein receptor capture also identified laminin-alpha 4 and -gamma 1. Laminins colocalized with NP41 within nerve sheath, particularly perineurium, where laminin-421 is predominant. Binding assays with phage expressing NP41 confirmed binding to purified laminin-421, laminin-211, and laminin-alpha 4. Affinity for these extracellular matrix proteins explains the striking ability of NP41 to highlight degenerated nerve ghosts months posttransection that are invisible to the unaided eye but retain hollow laminin-rich tubular structures.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据