Site-specific labeling of neurotrophins and their receptors via short and versatile peptide tags.

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  • Additional Information
    • Source:
      Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: eCollection Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE
    • Publication Information:
      Original Publication: San Francisco, CA : Public Library of Science
    • Subject Terms:
      Nerve Growth Factors/*analysis ; Oligopeptides/*analysis ; Receptors, Nerve Growth Factor/*analysis; Amino Acid Sequence ; Animals ; Biotinylation ; Cell Line ; Cloning, Molecular ; Gene Expression ; Humans ; Models, Molecular ; Molecular Probe Techniques ; Molecular Sequence Data ; Mutagenesis, Insertional ; Nerve Growth Factors/genetics ; Nerve Growth Factors/metabolism ; Oligopeptides/genetics ; Oligopeptides/metabolism ; Optical Imaging ; PC12 Cells ; Rats ; Receptor, trkA/analysis ; Receptor, trkA/genetics ; Receptor, trkA/metabolism ; Receptors, Nerve Growth Factor/genetics ; Receptors, Nerve Growth Factor/metabolism ; Recombinant Proteins/analysis ; Recombinant Proteins/genetics ; Recombinant Proteins/metabolism
    • 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.
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    • Accession Number:
      0 (Nerve Growth Factors)
      0 (Oligopeptides)
      0 (Receptors, Nerve Growth Factor)
      0 (Recombinant Proteins)
      EC 2.7.10.1 (Receptor, trkA)
    • Publication Date:
      Date Created: 20141127 Date Completed: 20151221 Latest Revision: 20181113
    • Publication Date:
      20221213
    • Accession Number:
      PMC4245215
    • Accession Number:
      10.1371/journal.pone.0113708
    • Accession Number:
      25426999