title: Site-specific labeling of neurotrophins and their receptors via short and versatile peptide tags.
abstract: 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.
authors.affiliation: NEST, Scuola Normale Superiore and Istituto Nanoscienze - CNR, Pisa, Italy; BioSNS Laboratory, Scuola Normale Superiore and Istituto di Neuroscienze - CNR, Pisa, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Marchetti
authors.affiliation: BioSNS Laboratory, Scuola Normale Superiore and Istituto di Neuroscienze - CNR, Pisa, Italy.
authors.forename: Teresa
authors.initials: T
authors.lastname: De Nadai
authors.affiliation: NEST, Scuola Normale Superiore and Istituto Nanoscienze - CNR, Pisa, Italy.
authors.forename: Fulvio
authors.initials: F
authors.lastname: Bonsignore
authors.affiliation: BioSNS Laboratory, Scuola Normale Superiore and Istituto di Neuroscienze - CNR, Pisa, Italy.
authors.forename: Mariantonietta
authors.initials: M
authors.lastname: Calvello
authors.affiliation: IIT@NEST, Center for Nanotechnology Innovation, Pisa, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Signore
authors.affiliation: BioSNS Laboratory, Scuola Normale Superiore and Istituto di Neuroscienze - CNR, Pisa, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Viegi
authors.affiliation: NEST, Scuola Normale Superiore and Istituto Nanoscienze - CNR, Pisa, Italy; IIT@NEST, Center for Nanotechnology Innovation, Pisa, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Beltram
authors.affiliation: NEST, Scuola Normale Superiore and Istituto Nanoscienze - CNR, Pisa, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Luin
authors.affiliation: BioSNS Laboratory, Scuola Normale Superiore and Istituto di Neuroscienze - CNR, Pisa, Italy.
authors.forename: Antonino
authors.initials: A
authors.lastname: Cattaneo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019921:Biotinylation; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009414:Nerve Growth Factors / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D061848:Optical Imaging; D016716:PC12 Cells; D051381:Rats; D020917:Receptor, trkA / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D017475:Receptors, Nerve Growth Factor / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism
chemical_list: D009414:Nerve Growth Factors; D009842:Oligopeptides; D017475:Receptors, Nerve Growth Factor; D011994:Recombinant Proteins; D020917:Receptor, trkA