title: Laminin targeting of a peripheral nerve-highlighting peptide enables degenerated nerve visualization.
abstract: Target-blind activity-based screening of molecular 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 α4 and α2, nidogen 1, and decorin (FDR-adjustedPvalue < 10-7) and minor enrichment of laminin-γ1 and collagens I and VI. Glycoprotein receptor capture also identified laminin-α4 and -γ1. 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-α4. 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.
authors: Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093; hlglasgow@gmail.com.
authors: Heather L
authors: HL
authors: Glasgow
authors: Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093.
authors: Michael A
authors: MA
authors: Whitney
authors: Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093.
authors: Larry A
authors: LA
authors: Gross
authors: Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093.
authors: Beth
authors: B
authors: Friedman
authors: Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093.
authors: Stephen R
authors: SR
authors: Adams
authors: Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093.
authors: Jessica L
authors: JL
authors: Crisp
authors: Division of Otolaryngology-Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093.
authors: Timon
authors: T
authors: Hussain
authors: Institute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland.
authors: Andreas P
authors: AP
authors: Frei
authors: Institute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland.
authors: Karel
authors: K
authors: Novy
authors: Institute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland.
authors: Bernd
authors: B
authors: Wollscheid
authors: Division of Otolaryngology-Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093.
authors: Quyen T
authors: QT
authors: Nguyen
authors: Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093.|Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093.|Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093.
authors: Roger Y
authors: RY
authors: Tsien
keywords: laminin; molecular imaging; nerve highlighting; proximity-based labeling; surgery with molecular navigation