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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doaj-dc685a850a494bf1ac6bc75cccddf4e72020-11-25T02:11:56ZengPublic 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.http://europepmc.org/articles/PMC4245215?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Laura Marchetti Teresa De Nadai Fulvio Bonsignore Mariantonietta Calvello Giovanni Signore Alessandro Viegi Fabio Beltram Stefano Luin Antonino Cattaneo |
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 |
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 |
title |
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_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_sort |
site-specific labeling of neurotrophins and their receptors via short and versatile peptide tags. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2014-01-01 |
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. |
url |
http://europepmc.org/articles/PMC4245215?pdf=render |
work_keys_str_mv |
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