Rapid Covalent Labeling of Membrane Proteins on Living Cells Using a Nanobody–Epitope Tag Pair

Abstract

Synthetic molecules that form a covalent bond upon binding to a targeted biomolecule (proximity-induced reactivity) are the subject of intense biomedical interest for the unique pharmacological properties imparted by irreversible binding. However, off-target covalent labeling and the lack of molecules with sufficient specificity limit more widespread applications. We describe the first example of a cross-linking platform that uses a synthetic peptide epitope and a single domain antibody (or nanobody) pair to form a covalent linkage rapidly and specifically. The rate of the cross-linking reaction between peptide and nanobody is faster than most other biocompatible cross-linking reactions, and it can be used to label live cells expressing receptor–nanobody fusions. The rapid kinetics of this system allowed us to probe the consequences on signaling for ligand cross-linking to the A2A-adenosine receptor. Our method may be generally useful to site-specifically link synthetic molecules to receptors on mammalian cell surfaces.

Highly biased agonism for GPCR ligands via nanobody tethering

“…Peptide and conjugate identity were confirmed by mass spectrometry (Supporting Tables 1 and 2). Previously described variants of PTHR1 (Figure 1C) engineered to contain a high affinity binding site for an epitope tag-binding Nb (Nb 6E binds PTHR1-6E) or an epitope peptide (6E peptide binds PTHR1-Nb 6E ) were also deployed 27,29 . Either epitope tag (6E) or Nb (Nb 6E ) were engrafted into a flexible and unstructured region of the receptor encoded by exon 2, which is known to be dispensable for high affinity ligand binding 30 .…”

“…A Human Embryonic Kidney 293 cell line (HEK 293; ATCC CRL-1573) stably transfected with luciferase-based pGlosensor-22F cAMP reporter plasmid (Glosensor, Promega Corp) has been described previously [GS22 57 ]. GS22 cells were used to generate cell line stably expressing either native human PTHR1, PTHR1-6E, or PTHR1-Nb 6E as previously described 27,29 . Plasmids encoding receptors under study have been previously defined 27 .…”

Semi‐synthetic nanobody‐ligand conjugates exhibit tunable signaling properties and enhanced transcriptional outputs at neurokinin receptor‐1

“…Supporting this hypothesis, the addition of the NK1R antagonist, spantide I (1 μM) (Folkers et al, 1984), did not abolish the sustained response (Figure S12). Prolonged signaling responses have also been observed upon covalent tethering of a GPCR ligand to its receptor (Cabalteja et al, 2022b). Compounds with F I G U R E 4 Evaluation of peptide and conjugate performance on transcription.…”

“…Supporting this hypothesis, the addition of the NK1R antagonist, spantide I (1 μM) (Folkers et al, 1984), did not abolish the sustained response (Figure S12). Prolonged signaling responses have also been observed upon covalent tethering of a GPCR ligand to its receptor (Cabalteja et al, 2022b). Compounds with a long duration of action are useful in a variety of contexts (Hothersall et al, 2016), such as long‐acting PTHR1 agonists which are used to treat hypoparathyroidism (Hothersall et al, 2016; Noda et al, 2020).…”

Semi-synthetic nanobody-ligand conjugates exhibit tunable signaling properties and enhanced transcriptional outputs at neurokinin receptor-1

“…Prolonged signaling responses have also been observed upon covalent tethering of a GPCR ligand to its receptor. 28 Compounds with a long duration of action are useful in a variety of contexts 29 , such as long-acting PTHR1 agonists which are used to treat hypoparathyroidism. 29,30 The findings here encourage exploration of Nb conjugation to facilitate the rational design of long-lasting ligands.…”