Large-scale screening for novel low-affinity extracellular protein interactions

Abstract

Extracellular protein-protein interactions are essential for both intercellular communication and cohesion within multicellular organisms. Approximately a fifth of human genes encode membrane-tethered or secreted proteins, but they are largely absent from recent large-scale protein interaction datasets, making current interaction networks biased and incomplete. This discrepancy is due to the unsuitability of popular high-throughput methods to detect extracellular interactions because of the biochemical intractability of membrane proteins and their interactions. For example, cell surface proteins contain insoluble hydrophobic transmembrane regions, and their extracellular interactions are often highly transient, having half-lives of less than a second. To detect transient extracellular interactions on a large scale, we developed AVEXIS (avidity-based extracellular interaction screen), a high-throughput assay that overcomes these technical issues and can detect very transient interactions (half-lives <or= 0.1 sec) with a low false-positive rate. We used it to systematically screen for receptor-ligand pairs within the zebrafish immunoglobulin superfamily and identified novel ligands for both well-known and orphan receptors. Genes encoding receptor-ligand pairs were often clustered phylogenetically and expressed in the same or adjacent tissues, immediately implying their involvement in similar biological processes. Using AVEXIS, we have determined the first systematic low-affinity extracellular protein interaction network, supported by independent biological data. This technique will now allow large-scale extracellular protein interaction mapping in a broad range of experimental contexts.

Figure 1.

AVEXIS specifically detects low-affinity extracellular interactions with a low false-positive rate. (A) Schematic representation of AVEXIS. Biotinylated bait ectodomains (blue) are captured in an orientated manner on a streptavidin-coated microtiter plate and probed with soluble pentameric β-lactamase-tagged prey ectodomains (pink). Positive binding is detected by colorimetric enzymatic turnover of nitrocefin (yellow to red). (B) Pentamerization increases the avidity of prey ectodomains. Pentameric (squares) and monomeric (triangles) Cd200 (blue) and Cd200r (red) prey constructs were normalized for β-lactamase activity, serially diluted, and tested against the appropriate bait construct. The prey activity threshold used for AVEXIS is shown, corresponding to 50 units/mL. (C) AVEXIS interaction specificity. (Top panel) Low-affinity Cd200 (bait)–Cd200r (prey) interaction can be detected in the presence of a non-binding control antibody but is specifically blocked with an anti-Cd200 blocking monoclonal antibody; the blocking effect can be titrated by decreasing antibody concentration. The interaction specificity is independent of bait–prey orientation and can also be blocked with an anti-Cd200r antibody, bottom panel. (D) AVEXIS false-positive and -negative rate evaluation. An AVEXIS screening plate with bait proteins arrayed in rows and the prey proteins normalized to threshold levels in columns; positive binding results can be seen as red wells. Expected positive and negative wells are circled in red and black, respectively.

Figure 2.

Interaction network of zebrafish IgSF proteins as determined using AVEXIS. Proteins are identified by their current official nomenclature, with blue and red nodes representing cell surface and secreted proteins, respectively. Interactions are numbered for reference and circled if quantified by SPR. Entirely novel interactions are represented by black arrows and those previously detected using an orthologous mammalian protein with red arrows.

Figure 3.

Validation and quantification of interactions by surface plasmon resonance. (A) Reference-subtracted surface plasmon resonance traces of soluble Mpzl2-Cd4-6H protein interacting with surface-immobilized Mpzl3-Cd4-bio. The binding curves of six twofold serial dilutions (as shown) of the purified protein were injected over 1016RU of immobilized Mpzl3 as indicated by the bar. The wash-out phase of the binding was fitted to a 1:1 dissociation model, and the off-rate constant (k_off) was determined and a half-life calculated (see Methods). (B) Relative monomeric half-lives of interactions quantified by SPR as measured at 28°C. Interactions are numbered as in Fig. 2. Heterophilic interactions are placed above the scale, and homophilic interactions below. The red box represents the limits of SPR sensitivity, suggesting that several interactions have half-lives ≤ 0.1 sec.

Figure 4.

Interactions between paralogous proteins are enriched within the extracellular interaction network. A phylogenetic tree showing the relationships of the non-redundant protein ectodomains used within the screen is shown. Names and branches representing the interacting proteins from the network are highlighted in green. The line widths in the interaction network reflect the sequence identities between interacting proteins as described in the key. Homophilic interactions are shown for completeness.

Figure 5.

Expression patterns of genes encoding interacting proteins during early vertebrate development. Schematic diagrams representing the domain architecture and location of potential N-linked glycosylation sites are shown above the corresponding expression patterns. Whole-mount in situ expression patterns of zebrafish jam2 and jam3b at 18 somite stage (A), fgfrl1a, fgfrl1b, and fgfr4 at 24 hpf (B), and zgc:136455, negr1, and zgc:110372 at 48 hpf (C) showing complementary expression patterns. (D) (Upper panels) Lateral views of whole-mount embryos with anterior to the left showing Mpzl2, Mpzl3, and Cssl:d179 protein expression as detected by immunohistochemistry in the pronephric ducts at 24 hpf. (Lower panels) Immunohistochemistry of transverse 53-hpf zebrafish sections counterstained for nuclei with DAPI (blue) showing co-expression in the paired pronephric ducts (arrows) and epidermis (red). Scale bars = 50 μm.