Rapid and sensitive large-scale screening of low affinity extracellular receptor protein interactions by using reaction induced inhibition of Gaussia luciferase

Abstract

Extracellular protein interactions mediated by cell surface receptors are essential for intercellular communication in multicellular organisms. Assays to detect extracellular interactions must account for their often weak binding affinities and also the biochemical challenges in solubilising membrane-embedded receptors in an active form. Methods based on detecting direct binding of soluble recombinant receptor ectodomains have been successful, but genome-scale screening is limited by the usual requirement of producing sufficient amounts of each protein in two different forms, usually a "bait" and "prey". Here, we show that oligomeric receptor ectodomains coupled to concatenated units of the light-generating Gaussia luciferase enzyme robustly detected low affinity interactions and reduced the amount of protein required by several orders of magnitude compared to other reporter enzymes. Importantly, we discovered that this flash-type luciferase exhibited a reaction-induced inhibition that permitted the use of a single protein preparation as both bait and prey thereby halving the number of expression plasmids and recombinant proteins required for screening. This approach was tested against a benchmarked set of quantified extracellular interactions and shown to detect extremely weak interactions (K_Ds ≥ μM). This method will facilitate large-scale receptor interaction screening and contribute to the goal of mapping networks of cellular communication.

Figure 1

Design and expression of prey constructs with different reporter enzymes. (a) Schematic diagrams of the protein constructs used in this study. Prey proteins consisting of the extracellular region of a protein of interest (Gene A) are fused at their C-terminus to the proteins tags: rat Cd4(d3 + 4) and the rat cartilage oligomeric matrix protein (COMP) followed by either five repeating units of luciferase, HRP, or beta-lactamase enzymes. Baits are expressed as monomers and consist of ectodomains of a protein of interest (Gene B) fused to the same rat Cd4(d3 + 4) tag but additionally containing a peptide sequence that is recognised by the enzyme BirA for the covalent addition of a single biotin molecule. Both baits and preys contain a terminal 6-his tag for purification. (b) Schematic representation of the AVEXIS assay involving a soluble highly avid pentameric enzyme-tagged prey protein and biotinylated bait protein immobilised on a streptavidin-coated microtitre plate. (c) Pentameric preys containing the HRP enzyme were expressed at low levels. Rat Cd200 prey constructs containing the named enzyme reporters were expressed by transient transfection of HEK293 cells and the secreted protein yield quantified after nickel affinity purification. Data points are values for three independent transfections and bars represent the means. BLac = beta-lactamase prey; GLuc = Gaussia luciferase prey; NanoLuc = Nano luciferase prey.

Figure 2

Luciferase reporters provide highly sensitive detection of the Cd200-Cd200R interaction. Highly avid rat Cd200 prey proteins containing the reporter enzymes (a) beta-lactamase (BLac), (b) Gaussia luciferase (GLuc) or (c) nano-luciferase (NanoLuc) were probed for interactions with a biotinylated rat Cd200R bait protein immobilised in streptavidin-coated microtitre plates. Prey capture was quantified using absorbance at 485 nm for hydrolysis products of the beta-lactamase colourimetric substrate nitrocefin, and luminescence using the substrates coelenterazine (Gaussia luciferase) and furimazine (nano-luciferase). Cd200 was used as a negative control bait (empty circles). The normalised signal for each data point was calculated as described in the Methods such that background signal would be 0 and the maximum signal 1. The log IC50 for each interpolated curve is displayed on each graph. Representative experiments shown with n = 3 for each concentration of prey with s.e.m. for each data point and the 95% CI for the interpolated curves indicated as dotted lines.

Figure 3

Reaction-induced inactivation of Gaussia luciferase enables the use of a single construct as both a bait and a prey. (a) Pentameric prey proteins can be used as a bait for sensitive interaction detection. Biotinylated Cd200R Gaussia luciferase-tagged pentamer (filled circles) or monomers (empty circles) were serially diluted and immobilised on a streptavidin-coated microtitre plate as bait proteins and probed for interactions with a beta-lactamase-tagged Cd200 pentamer. (b) Coelenterazine was added to a dilution series of purified Cd200 Gaussia luciferase pentamer which elicited a strong luminescence signal that was gradually lost after around an hour. A second administration of substrate did not generate the same bright luminescence signal. (c) Gaussia enzymatic activity is not restored after a nickel-affinity purification of the inactivated luciferase. Reaction inhibited Gaussia luciferase was purified using Ni–NTA agarose beads with several wash steps before addition of fresh coelenterazine (100 pmol). (d) Pentameric (non-biotinylated) Cd200 Gaussia luciferase prey was probed against biotinylated and inactivated pentameric Cd200R Gaussia luciferase bait (filled circles). The Cd200 prey was also probed against a reaction-inhibited pentameric Gaussia luciferase Cd200 protein used as a non-interacting control bait (empty circles). The inflection point for each interpolated curve is displayed as the log baits (moles) value on each graph. Representative experiments shown with n = 3 for each quantity of prey with SEM for each data point and the 95% CI for the interpolated curves.

Figure 4

Benchmarking the Gaussia luciferase single protein construct system within the zebrafish Jam family protein interactions shows it can detect low affinity interactions with a low false positive rate. The bait and prey versions of the six paralogues of the zebrafish Jam family were expressed and used in different implementations of the receptor interaction assay including both monomer and pentameric Gaussia luciferase bait formats. The indicated baits were arrayed on streptavidin-coated plates and systematically probed with the preys for a total of 36 binary interaction tests in each prey-bait combination. (a) Graphical representation of expected and observed interactions of different affinities within the zebrafish Jam family. The expected interactions within the zebrafish Jam family are shaded according to their measured dissociation half lives (black = t_1/2 > 2 s; dark grey 0.4 < t_1/2 < 2 s; light grey = t_1/2 < 0.4 s); the scale in (b) to (e) represents the normalised signal output from the assay. (b) Assay read out using the original AVEXIS assay with the beta-lactamase reporter prey against monomeric baits, and (c) using the new Gaussia luciferase reporter prey against the same array of monomeric baits. In (d), the Gaussia luciferase pentameric protein is used as a bait, inactivated, and then a same protein construct expressed in a non-biotinylated form as the prey. In (e), the same biotinylated Gaussia luciferase pentameric protein preparation is used as both a bait and a prey. Representative experiments are shown with n = 3 for each prey-bait binary interaction test and summary data are shown in Figure S1.