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

Extracellular protein interactions such as those made between secreted and membrane-tethered proteins are important for diverse cellular behaviors, such as initiating differentiation pathways, directing migration and pathfinding routes, and mediating intricate recognition processes. Approximately a fifth of human genes encode extracellular and membrane-associated proteins, but despite their importance and abundance, they are significantly underrepresented in recent large-scale protein interaction datasets (Futschik et al. 2007). This discrepancy is remarkable since their accessibility to systemically delivered drugs makes extracellular proteins excellent therapeutic targets (Clark et al. 2003). There are several reasons for this disparity. First, cell surface proteins are biochemically difficult to manipulate; for example, their hydrophobic membrane-spanning region renders them insoluble. Second, functionally important post-translational modifications such as disulfide bonds and large hydrophilic glycans are not added in commonly used expression systems such as bacteria and cell-free systems. Finally, interactions between cell surface proteins have very fast dissociation rates and are therefore often highly transient, having half-lives of fractions of a second (van der Merwe and Barclay 1994); this makes purification protocols involving wash steps impractical. The transient nature of these interactions and necessity for posttranslational modifications makes current high-throughput protein interaction assays, such as yeast-two-hybrid and biochemical purification methods, generally unsuitable to identify this important class of interactions. Existing methods to directly detect extracellular transient interactions often rely on experimentally increasing the overall avidity of the interaction by multimerizing a soluble recombinant protein, mirroring the way these interactions occur in vivo through arrayed proteins on apposing membranes. These methods include oriented display around microbeads (Wright et al. 2000; Letarte et al. 2005) or tags producing dimers (such as Fc-fusion proteins), trimers, and, often most potently, pentamers (Holler et al. 2000; Voulgaraki et al. 2005). No broad assessment for the suitability of any of these techniques to be used in systematic high-throughput screening has been made, since only individual interactions have been reported (Lin et al. 2003; Gonzalez et al. 2005). To address the need for a high-throughput technique that can detect low-affinity extracellular protein interactions, we have developed a novel assay termed AVEXIS (avidity-based extracellular interaction screen) and used it to identify novel extracellular receptor–ligand pairs within the zebrafish immunoglobulin superfamily (IgSF). Independent support for identified interactions was provided by quantifying interaction strengths, performing a phylogenetic analysis, and showing that genes encoding interacting pairs were expressed in either the same or adjacent tissues.