Site-specific labeling of neurotrophins and their receptors via short and versatile peptide tags.
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 P...
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2014-01-01
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| Series: | PLoS ONE |
| Online Access: | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0113708&type=printable |
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| author | Laura Marchetti Teresa De Nadai Fulvio Bonsignore Mariantonietta Calvello Giovanni Signore Alessandro Viegi Fabio Beltram Stefano Luin Antonino Cattaneo |
| author_facet | Laura Marchetti Teresa De Nadai Fulvio Bonsignore Mariantonietta Calvello Giovanni Signore Alessandro Viegi Fabio Beltram Stefano Luin Antonino Cattaneo |
| author_sort | Laura Marchetti |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-dc685a850a494bf1ac6bc75cccddf4e7 |
| institution | OA Journals |
| issn | 1932-6203 |
| language | English |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-dc685a850a494bf1ac6bc75cccddf4e72025-08-20T02:14:50ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-01911e11370810.1371/journal.pone.0113708Site-specific labeling of neurotrophins and their receptors via short and versatile peptide tags.Laura MarchettiTeresa De NadaiFulvio BonsignoreMariantonietta CalvelloGiovanni SignoreAlessandro ViegiFabio BeltramStefano LuinAntonino CattaneoWe 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.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0113708&type=printable |
| spellingShingle | Laura Marchetti Teresa De Nadai Fulvio Bonsignore Mariantonietta Calvello Giovanni Signore Alessandro Viegi Fabio Beltram Stefano Luin Antonino Cattaneo Site-specific labeling of neurotrophins and their receptors via short and versatile peptide tags. PLoS ONE |
| title | Site-specific labeling of neurotrophins and their receptors via short and versatile peptide tags. |
| title_full | Site-specific labeling of neurotrophins and their receptors via short and versatile peptide tags. |
| title_fullStr | Site-specific labeling of neurotrophins and their receptors via short and versatile peptide tags. |
| title_full_unstemmed | Site-specific labeling of neurotrophins and their receptors via short and versatile peptide tags. |
| title_short | Site-specific labeling of neurotrophins and their receptors via short and versatile peptide tags. |
| title_sort | site specific labeling of neurotrophins and their receptors via short and versatile peptide tags |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0113708&type=printable |
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