Site-specific labeling of neurotrophins and their receptors via short and versatile peptide tags

• Published in: PloS one Vol. 9; no. 11; p. e113708
• Main Authors: Marchetti, Laura, De Nadai, Teresa, Bonsignore, Fulvio, Calvello, Mariantonietta, Signore, Giovanni, Viegi, Alessandro, Beltram, Fabio, Luin, Stefano, Cattaneo, Antonino
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 26.11.2014; Public Library of Science (PLoS)
• Subjects: Acids; Amino Acid Sequence; Amino acids; Animals; Biology and Life Sciences; Biotin; Biotinylation; Cell Line; Cell surface; Cells (biology); Cloning; Cloning, Molecular; Coenzyme A; Conjugation; Gene Expression; Growth factors; Humans; Insertion; Insertional mutagenesis; Kinases; Labeling; Laboratories; Ligands; Models, Molecular; Molecular interactions; Molecular Probe Techniques; Molecular Sequence Data; Mutagenesis; Mutagenesis, Insertional; Nanotechnology; Nerve growth factor; Nerve Growth Factors - analysis; Nerve Growth Factors - genetics; Nerve Growth Factors - metabolism; Neurotrophic factors; Neurotrophins; Oligopeptides - analysis; Oligopeptides - genetics; Oligopeptides - metabolism; Optical Imaging; PC12 Cells; Peptides; Pheochromocytoma cells; Proteins; Quantum dots; Rats; Receptor, trkA - analysis; Receptor, trkA - genetics; Receptor, trkA - metabolism; Receptors; Receptors, Nerve Growth Factor - analysis; Receptors, Nerve Growth Factor - genetics; Receptors, Nerve Growth Factor - metabolism; Recombinant Proteins - analysis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Signal transduction; Stoichiometry; Substrates; Tags; TrkA protein; TrkA receptors; Italy
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0113708

We present a toolbox for the study of molecular interactions occurring between NGF and its receptors. By means of a suitable insertional mutagenesis method we show the insertion of an 8 amino acid tag (A4) into the sequence of NGF and of 12 amino acid tags (A1 and S6) into the sequence of TrkA and P75NTR NGF-receptors. These tags are shortened versions of the acyl and peptidyl carrier proteins; they are here covalently conjugated to the biotin-substituted arm of a coenzyme A (coA) substrate by phosphopantetheinyl transferase enzymes (PPTases). We demonstrate site-specific biotinylation of the purified recombinant tagged neurotrophin, in both the immature proNGF and mature NGF forms. The resulting tagged NGF is fully functional: it can signal and promote PC12 cells differentiation similarly to recombinant wild-type NGF. Furthermore, we show that the insertion of A1 and S6 tags into human TrkA and P75NTR sequences leads to the site-specific biotinylation of these receptors at the cell surface of living cells. Crucially, the two tags are labeled selectively by two different PPTases: this is exploited to reach orthogonal fluorolabeling of the two receptors co-expressed at low density in living cells. We describe the protocols to obtain the enzymatic, site-specific biotinylation of neurotrophins and their receptors as an alternative to their chemical, nonspecific biotinylation. The present strategy has three main advantages: i) it yields precise control of stoichiometry and site of biotin conjugation; ii) the tags used can be functionalized with virtually any small probe that can be carried by coA substrates, besides (and in addition to) biotin; iii) above all it makes possible to image and track interacting molecules at the single-molecule level in living systems.