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

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.

Peptide-tags for site-specific protein labelling in vitro and in vivo

“…Labelling with 1-10 mM CoA-derivative for 20-40 min; reaction temperature: 37 1C 38,39 In vitro, in vivo (cell surface labelling with fixed or live cells)…”

“…38 The S6/A1 pair of tags was also used to enable labelling of the tropomyosin receptor kinase TrkA and the p75 neurotrophin co-receptor (P75NTR) in SH-SY5Y cells. 39 The A1-P75NTR construct was first labelled with biotin-CoA using AcpS and then stained with streptavidin-QDot525. Subsequently, the S6-TrkA construct was biotinylated by using Sfp followed by labelling with streptavidin-QDot655.…”

Ligand-Induced Dynamics of Neurotrophin Receptors Investigated by Single-Molecule Imaging Approaches

“…In detail, the TrkA cDNA sequence was engineered by adding the ACP (acyl carrier protein) tag sequence [ 95 ] to the extracellular domain of TrkA downstream the signal peptide ( Figure 2 ) [ 100 ]. More recently, shortened versions of the ACP tag were also inserted that are only 12 amino acids long but yield the same labeling of the full length tag [ 128 ]. Together with its cognate PCP (peptidyl carrier protein), the ACP tag is the ancestor of a family of protein and peptide chemical tags, which can be covalently conjugated to virtually any small-probe substituted phosphopantetheinyl (PP) arm of Coenzyme A (CoA) substrate by post-translational modification enzymes named PP transferases (PPTases) [ 57 , 61 , 95 ].…”

Phospholipase Cβ-TRAX Association Is Required for PC12 Cell Differentiation

“…When treated with nerve growth factor (NGF), proliferation ceases and differentiation begins as seen by the onset of neurite growth as well as accumulation of Ca 2ϩ vesicles (2). NGF binds to TrkA growth factor receptors (3,4) leading to changes in transcription of a number of pro-teins that are associated with the differentiated state (5). Differentiation of PC12 cells is generally considered complete when the length of the neurites is three-four times the length of the cell body.…”