Biotinylation is a widely used covalent protein modification. In particular, the biotinylation of neurotrophins and their receptors is of great interest. Chemical conjugation of biotin to proteins can be readily achieved, but displays almost no control in number and type of biotinylated residues, possibly leading to impairment of biological activity. Alternatively, peptide tags, containing a residue that is a specific target of a biotinylating enzyme, can be fused in frame to the protein of interest. Despite current efforts to reduce the size of such tags, to minimally interfere with protein structure and function, their use is still limited. Here we present an insertional mutagenesis method for the insertion of a 8 aminoacids tag (A4) into the sequence of nerve growth factor (NGF) and of 12 aminoacids tags (A1 and S6) into the sequence of the two TrkA and P75NTR NGF-receptors. These tags contain a serine residue that is target of specific transfer of a pantetheinyl arm bearing one biotin moiety, from a CoA-biotin substrate, by post-translational modification enzymes called phosphopantetheinyl transferases (PPTases). We demonstrate that, upon insertion of A4 tag at the C-terminus of human NGF sequence, a site-specific biotinylation is achieved for the purified recombinant neurotrophin, both as immature proNGF and mature NGF forms. The obtained tagged NGF is able to promote PC12 cells differentiation, as well as TF-1 cells proliferation, to a similar extent to wt recombinant human NGF. Furthermore, we show that the insertion of A1 and S6 tags downstream the signal peptide at the N terminus of both human TrkA and P75NTR sequences, successfully leads to the site-specific biotinylation of these receptors at the cell surface of living cells. Notably, by using Tet-ON inducible lentiviral vectors, we achieve a tight regulation of the tagged receptors expression level in the cells, which allows retaining their full functional NGF-responsiveness and signalling activation. Overall, this work provides a first alternative to the previously used chemical biotinylation of neurotrophins and their receptors. Our strategy has the advantage of a precise control of stoichiometry and site of biotin conjugation. Moreover, given the properties of orthogonal labeling displayed by these tags, our work paves the way to the simultaneous labeling of TrkA and P75NTR receptors in the same cell by using two different PPTases and two spectrally distinct streptavidin-conjugated fluorophores. Single particle tracking studies will also be possible by using this strategy.

Site-specific biotinylation of neurotrophins and their receptors by the insertion of short versatile peptide tags

DE NADAI, TERESA;MARCHETTI, LAURA;BONSIGNORE, FULVIO;PORCIANI, DAVID;BELTRAM, Fabio;LUIN, Stefano;CATTANEO, ANTONINO
2013

Abstract

Biotinylation is a widely used covalent protein modification. In particular, the biotinylation of neurotrophins and their receptors is of great interest. Chemical conjugation of biotin to proteins can be readily achieved, but displays almost no control in number and type of biotinylated residues, possibly leading to impairment of biological activity. Alternatively, peptide tags, containing a residue that is a specific target of a biotinylating enzyme, can be fused in frame to the protein of interest. Despite current efforts to reduce the size of such tags, to minimally interfere with protein structure and function, their use is still limited. Here we present an insertional mutagenesis method for the insertion of a 8 aminoacids tag (A4) into the sequence of nerve growth factor (NGF) and of 12 aminoacids tags (A1 and S6) into the sequence of the two TrkA and P75NTR NGF-receptors. These tags contain a serine residue that is target of specific transfer of a pantetheinyl arm bearing one biotin moiety, from a CoA-biotin substrate, by post-translational modification enzymes called phosphopantetheinyl transferases (PPTases). We demonstrate that, upon insertion of A4 tag at the C-terminus of human NGF sequence, a site-specific biotinylation is achieved for the purified recombinant neurotrophin, both as immature proNGF and mature NGF forms. The obtained tagged NGF is able to promote PC12 cells differentiation, as well as TF-1 cells proliferation, to a similar extent to wt recombinant human NGF. Furthermore, we show that the insertion of A1 and S6 tags downstream the signal peptide at the N terminus of both human TrkA and P75NTR sequences, successfully leads to the site-specific biotinylation of these receptors at the cell surface of living cells. Notably, by using Tet-ON inducible lentiviral vectors, we achieve a tight regulation of the tagged receptors expression level in the cells, which allows retaining their full functional NGF-responsiveness and signalling activation. Overall, this work provides a first alternative to the previously used chemical biotinylation of neurotrophins and their receptors. Our strategy has the advantage of a precise control of stoichiometry and site of biotin conjugation. Moreover, given the properties of orthogonal labeling displayed by these tags, our work paves the way to the simultaneous labeling of TrkA and P75NTR receptors in the same cell by using two different PPTases and two spectrally distinct streptavidin-conjugated fluorophores. Single particle tracking studies will also be possible by using this strategy.
2013
Neuroscience 2013
San Diego, California, USA
November 9-13, 2013
Society for Neuroscience Abstract Viewer and Itinerary Planner
NERVE GROWTH FACTOR; NGF receptor; Biotinylation
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11384/39708
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