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| Content Provider | Directory of Open Access Journals (DOAJ) |
|---|---|
| Author | Laura Marchetti Teresa De Nadai Fulvio Bonsignore Mariantonietta Calvello Giovanni Signore Alessandro Viegi Fabio Beltram Stefano Luin Antonino Cattaneo |
| 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. |
| e-ISSN | 19326203 |
| DOI | 10.1371/journal.pone.0113708 |
| Journal | PLoS ONE |
| Issue Number | 11 |
| Volume Number | 9 |
| Language | English |
| Publisher | Public Library of Science (PLoS) |
| Publisher Date | 2014-01-01 |
| Publisher Place | United States |
| Access Restriction | Open |
| Subject Keyword | Medicine Science |
| Content Type | Text |
| Resource Type | Article |
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