Figure 2

Overview of binding assay to analyze CNTN–amyloid interactions.A, domain organization of CNTNs and amyloid proteins. CNTN1–6 include six immunoglobulin (Ig) domains, four FN domains, and are tethered to the cell membrane by a GPI anchor. APP and its homologs, APLP1 and APLP2, include two globular domains in their extracellular region designed E1 and E2. The E1 domain itself includes two subdomains, a growth factor–like domain followed by a copper-binding domain. The ectodomains of amyloid proteins are followed by single transmembrane helix (TM) followed by an intracellular domain (ICD). B, design of constructs used in the binding assay. The ectodomains of CNTNs and amyloids are fused to the Fc domain of human IgG₁ or a protein designated FcYTS that includes (i) the Cν3 and Cν4 domains of chicken IgY and (ii) a Twin-Strep (TS) tag for detection with streptactin. C, in AlphaScreen assays, excitation of donor beads at 680 nm triggers the release of a singlet oxygen. An acceptor bead hit by this highly reactive molecule emits a signal between 520 and 620 nm. Because the half-life of the singlet oxygen is limited, a luminescent signal is only obtained when the donor beads and acceptor beads are within 200 nm. Here, the binding of the FcYTS fusion protein immobilized on donor beads to the Fc fusion protein bound to acceptor beads brings the two beads in proximity. D, results of the binding assay shown in a heat map representation. Raw signals are provided in Fig. S1. The scale indicates the value of the signal for the protein pair protein 1-FcYTS/protein 2-Fc divided by the signal measured for the protein 1-FcYTS/Fc only pair. E, summary of the interactions identified in the extracellular interaction screen. Black and red lines denote known and novel interactions, respectively. The positive controls, GABBRI and PTPRG, have been omitted for clarity. APLP1, amyloid beta precursor like protein 1; APLP2, amyloid beta precursor like protein 2; APP, amyloid precursor protein; CNTN, contactin; FN, fibronectin type III repeat; GABBRI, gamma-aminobutyric acid type B receptor subunit 1; GPI, glycosylphosphatidylinositol; PTPRG, protein tyrosine phosphatase receptor type G.