Reference

Contributed by Roger Y. Tsien, August 3, 2016 (sent for review November 16, 2015; reviewed by Joshua E. Elias and Jeff W. Lichtman) Heather L. Glasgow^a,¹, Michael A. Whitney^a, Larry A. Gross^b, Beth Friedman^a, Stephen R. Adams^a, Jessica L. Crisp^b, Timon Hussain^c, Andreas P. Frei^d, Karel Novy^d, Bernd Wollscheid^d, Quyen T. Nguyen^c, and Roger Y. Tsien^a,^b,^e,²  

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-adjusted P value < 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.

Author

Heather L. Glasgow ^aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093; Michael A. Whitney ^aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093; Larry A. Gross ^bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093; Beth Friedman ^aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093; Stephen R. Adams ^aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093; Jessica L. Crisp ^bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093; Timon Hussain ^cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093; Andreas P. Frei ^dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland; Karel Novy ^dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland; Bernd Wollscheid ^dInstitute of Molecular Systems Biology at the Department of Health Sciences and Technology, CH-8093 Zurich, Switzerland; Quyen T. Nguyen ^cDivision of Otolaryngology–Head and Neck Surgery, University of California, San Diego, La Jolla, CA 92093; Roger Y. Tsien ^aDepartment of Pharmacology, University of California, San Diego, La Jolla, CA 92093; ^bHoward Hughes Medical Institute, University of California, San Diego, La Jolla, CA 92093; ^eDepartment of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093