Site-Specific Labeling of Neurotrophins and Their Receptors via Short and Versatile Peptide Tags

<div><p>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.</p></div

Schematic overview of the insertional mutagenesis method.

<p><b>A</b>) Cartoon depicting NGF, TrkA, and P75NTR constructs prepared for this study. The site of tag insertion is indicated in each case by a red arrow. On top of the arrow the complete amino acidic tag sequence is reported. The promoters used for expression of the receptors are depicted upstream each receptor construct. On the extracellular domain of the receptors, sites of interaction with NGF are highlighted; on the intracellular domain of the receptors, sites of receptor activity are highlighted. Abbreviations: CMV = Cytomegalovirus promoter; TRE = Tet-Responsive-Element promoter; SP = signal peptide; EC = extracellular domain; IC = intracellular domain; D5 = proximal immunoglobulin-like domain; I = NGF-interaction site 1; II = NGF-interaction site 2; DD = death domain. <b>B</b>) Crystal structure of NGF (left; PBD. n.1BET), NGF-TrkA (EC) (middle; PBD. n.2IFG), NGF-P75NTR(EC) (right; PBD. n.1SG1). The color code is the same as in panel A. Red spots highlight the sites of tag insertion in each protein. <b>C</b>) Scheme of the insertional mutagenesis procedure used in this study. Details are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113708#pone.0113708.s006" target="_blank">Text S1</a>.</p

Site-specific biotinylation of TrkA and P75NTR receptors.

<p>Western blot for the analysis of the biotinylation reaction in living cells of A1/S6-TrkA-EGFP (<b>A</b>) and A1/S6-P75NTR-EGFP constructs (<b>B</b>) using CoA-biotin substrate and AcpS or SfpS PPTases. The same biotinylation reaction is performed in parallel using untagged TrkA-EGFP (<b>A</b>) and P75NTR-EGFP (<b>B</b>) as negative controls, and ACP-TrkA (<b>A</b>) as positive control. Streptavidin-HRP is used for detection of biotin. Anti-TrkA (<b>A</b>) and anti-P75NTR (<b>B</b>) blots are loading controls together with anti-GFP (both panels). The anti-TrkA blot contains an unspecific band running over TrkA, as already shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113708#pone.0113708-Callegari1" target="_blank">[4]</a>; the actual TrkA band in each lane is highlighted by a star. At the bottom of each panel the densitometric analysis of the blot bands is reported. The biotin signal was normalized to the content of GFP (for TrkA-EGFP, A1-TrkA-EGFP, S6-TrkA-EGFP lanes), TrkA (for ACP-TrkA lanes), and P75NTR (for P75NTR-EGFP, A1-P75NTR-EGFP, S6-P75NTR-EGFP lanes), with the higher value normalized to 1. Results reported are mean±sem from 3 (panel A) and 2 (panel B) independent blots.</p

TIRFM detection of PPTase-specific A1-P75NTR and S6-TrkA fluorolabeling.

<p><b>A</b>) Scheme of the dual-color labeling protocol used in the experiment. Details are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113708#s2" target="_blank">Materials and Methods</a>. <b>B</b>) TIRF microimages analyzing PPTase-specific A1-P75NTR and S6-TrkA fluorolabeling at the SH-SY5Y plasma membrane. A1 labeling by AcpS is detected by S-Qdot525; S6 labeling by SfpS is detected by S-Qdot655. Borders of the cells basal membrane, determined through the DIC image, are highlighted in the corresponding TIRF images by a white line, while areas outside cells are grayed to simplify image interpretation. Scale bars: 10 µm. <b>C–E</b>) Quantification of the % of green and red over total particles at the basal membrane of each analyzed cell expressing A1-P75NTR (<b>C</b>), S6-TrkA (<b>D</b>) or both constructs (<b>E</b>).</p

Site-specific biotinylation of proNGF and NGF.

<p><b>A–B</b>) Western blot for the analysis of the <i>in vitro</i> biotinylation reaction of purified NGF-A4 (<b>A</b>) and proNGF-A4 (<b>B</b>) using CoA-biotin substrate and AcpS or SfpS PPTases. The same biotinylation reaction is performed in parallel using untagged wt NGF and wt proNGF as negative controls. Streptavidin-HRP is used for detection of biotin. The anti-NGF blot is the loading control. <b>C</b>) Typical DIC images obtained when performing the differentiation assay in PC12 cells using ∼50 ng/ml of wt NGF, NGF-A4 and biotinylated NGF-A4 (NGF-A4b). Untreated cells are the control. Scale bar: 20 µm.</p