Multivalent recombinant proteins for probing functions of leucocyte surface proteins such as the CD200 receptor

Abstract

Summary CD200 (OX2) is a membrane glycoprotein that interacts with a structurally related receptor (CD200R) involved in the regulation of macrophage function. The interaction is of low affinity (K D $ 1 lm) but can be detected using CD200 displayed in a multivalent form on beads or with dimeric fusion proteins consisting of the extracellular region of CD200 and immunoglobulin Fc regions. We prepared putative pentamers and trimers of mouse CD200 with sequences from cartilage oligomeric matrix protein (COMP) and surfactant protein D (SP-D), respectively. The COMP protein gave high-avidity binding and was a valuable tool for showing the interaction whilst the SP-D protein gave weak binding. In vivo experiments showed that an agonistic CD200R monoclonal antibody caused some amelioration in a model of experimental autoimmune encephalomyelitis but the COMP protein was cleared rapidly and had minimal effect. Pentameric constructs also allowed detection of the rat CD48/CD2 interaction, which is of much lower affinity (K D $ 70 lm). These reagents may have an advantage over Fc-bearing hybrid molecules for probing cell surface proteins without side-effects due to the Fc regions. The CD200-COMP gave strong signals in protein microarrays, suggesting that such reagents may be valuable in high throughput detection of weak interactions.

New assay to detect low‐affinity interactions and characterization of leukocyte receptors for collagen including leukocyte‐associated Ig‐like receptor‐1 (LAIR‐1)

“…Recombinant monomeric human and mouse LAIR-1 proteins were produced as monomeric protein with domain 3 and 4 of rat CD4 as a tag as used in other studies [7]. The affinity of LAIR-1 to collagen I was measured by SPR and in addition a synthetic collagen repeat previously shown to bind LAIR-1 was tested together with control nonbinding peptide [22].…”

“…In order to increase the sensitivity of detection of weak interactions such as those between leukocyte membrane proteins, the previously used bead assay was modified [7]. Recombinant proteins corresponding to the extracellular regions of the leukocyte membrane proteins were transiently expressed and immobilized via a tag [19] that can be biotinylated, to beads coated with avidin to provide a highly avid binding reagent that enabled the recognition of new interactions by binding to cells [9,20].…”

“…For instance, typical affinities for such interactions are around 2 mM (CTLA4/CD80 K d 5 2 mM, SIRPa/CD47 K d 5 2 mM, CD2/CD58 K d 5 10 mM, CD200/CD200R K d 5 2 mM) [1][2][3][4][5][6] with fast off rates usually giving half-lives of the order of 1 s. Some highly functionally significant interactions have even lower affinities such as for CD8aa to MHC class I molecule HLA-A2 (K d $200 mm) [3]. Thus in order to detect binding to cells, recombinant proteins corresponding to the extracellular regions are often made multivalent, for example, by fusing to the Fc regions of IgG giving divalent protein, the Fc from IgM giving decavalent protein or by attaching the protein onto beads making a very avid binding reagent (discussed in [7]). These proteins, of various valencies, could be adapted and applied to screen for cells expressing receptors by flow cytometry and protein microarray [5,8,9].…”

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

“…These methods include oriented display around microbeads (Wright et al 2000;Letarte et al 2005) or tags producing dimers (such as Fcfusion 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).…”

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

“…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 .…”