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

Laura Marchetti,Fabio Beltram,Fulvio Bonsignore,Mariantonietta Calvello,Stefano Luin,Antonino Cattaneo,Teresa De Nadai,Giovanni Signore,Alessandro Viegi,Lucio Annunziato

doi:10.1371/journal.pone.0113708

Laura Marchetti, Fabio Beltram + Show 8 more

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https://doi.org/10.1371/journal.pone.0113708

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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.

Highlights

Neurotrophic factors, whose prototype member is nerve growth factor (NGF) [1], are a family of secreted proteins that crucially regulate neuronal development, survival and plasticity both in the central and in the peripheral nervous system
We previously demonstrated that the insertion of the acyl carrier protein (ACP) tag [3] at the extracellular domain of TrkA makes it possible to label the receptor at the cell surface when the construct is transfected in living cells [4,5]
ACP and peptidyl carrier protein (PCP) tags belong to a family of protein and peptide tags that can be covalently conjugated to virtually any small-probe-substituted PP arm of a Coenzyme A (CoA) substrate by post-translational modification enzymes named PP transferases (PPTases) [6]

Summary

Introduction

Neurotrophic factors, whose prototype member is nerve growth factor (NGF) [1], are a family of secreted proteins that crucially regulate neuronal development, survival and plasticity both in the central and in the peripheral nervous system. While most of the signaling cascades activated by NGF binding to TrkA and P75NTR receptors were identified, the impact on receptor dynamics caused by TrkA and P75NTR engagement by NGF and the regulation of their cellular traffic are far from being understood In this context, techniques that make it possible to investigate the TrkA-NGF-P75NTR dynamic interplay in a physiological context (e.g. the intact plasma membrane and endosomes in living neuronal cells) can be decisive to unveil the molecular mechanisms governing their functional interactions. Techniques that make it possible to investigate the TrkA-NGF-P75NTR dynamic interplay in a physiological context (e.g. the intact plasma membrane and endosomes in living neuronal cells) can be decisive to unveil the molecular mechanisms governing their functional interactions To date these issues were poorly explored largely owing to the lack of suitable experimental tools. Labeling strategies should have the following properties: i) if labeling relies on the use of tagged constructs of the proteins of interest, tags should be as small as possible, in order to minimally interfere with protein functionality and with the formation of molecular complexes; ii) a 1:1 stoichiometry between the labeled protein and the probe should be obtained; iii) they should be versatile, in order to yield molecular species that can be derivatized with different probes, depending on the experimental needs (e.g. biotin, fluorophores, gold or magnetic nanoparticles); iv) they should allow the simultaneous differential labeling of at least two molecules that are supposed to form a complex (i.e. neurotrophin and one of its receptors, or two neurotrophin receptors)

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