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 (halflives Յ 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.

RH5–Basigin interaction plays a major role in the host tropism of Plasmodium falciparum

“…1A). To determine whether the EBA175 orthologs bound native GYPA directly, we screened them against GYPA extracted from human erythrocytes using a modified version of the avidity-based extracellular interaction screening (AVEXIS) assay, an approach designed to detect transient extracellular protein interactions (28,30,31). Again, both P. billcollinsi and P. reichenowi EBA175 RII orthologs bound native human GYPA in a sialic acid-dependent manner (Fig.…”

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

“…The assay is not generally suitable for multipass membrane proteins such as ion transporters or pumps since they are unlikely to be correctly folded outside the context of a plasma membrane. We have applied this to a variety of different systems and have successfully expressed extracellular proteins from zebrafish 3,16,18 , human, mouse and P. falciparum for interaction screens.…”

“…The AVEXIS method described here provides a sensitive method for detecting transient extracellular protein:protein interactions that can be implemented in a scalable manner with a low false positive rate. While other scalable methods have been developed to detect extracellular interactions [11][12][13][14][15] , one advantage of AVEXIS is that the experimental parameters such as the bait and prey activities have been quantified to detect even the weakest of interactions (t 1/2 ≤ 0.1 s) while retaining a low false positive rate 3 . In addition, the streamlined and robust sample preparation procedures have enabled this assay to be implemented on a much larger scale than other assays and we have recently described a systematic interaction screen totalling over ~16,500 potential interactions 16 .…”

“…Amplify the ectodomain fragments from all genes using these primers and clone them into the bait or prey vector. We sometimes find it helpful to first clone them into shuttle vectors before subcloning into an appropriate mammalian expression vector, we use a derivative of the pTT3 vector 3,4 . Both the bait and prey vectors contain C-terminal protein tags as follows (Fig.…”

“…The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates 3 .…”