title: Multivalent recombinant proteins for probing functions of leucocyte surface proteins such as the CD200 receptor.
abstract: 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) approximately 1 microm) 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) approximately 70 microm). 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.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
authors.forename: Despina
authors.initials: D
authors.lastname: Voulgaraki
authors.forename: Rita
authors.initials: R
authors.lastname: Mitnacht-Kraus
authors.forename: Michelle
authors.initials: M
authors.lastname: Letarte
authors.forename: Mildred
authors.initials: M
authors.lastname: Foster-Cuevas
authors.forename: Marion H
authors.initials: MH
authors.lastname: Brown
authors.forename: A Neil
authors.initials: AN
authors.lastname: Barclay
mesh_terms: D000818:Animals; D000915:Antibody Affinity / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology; D000954:Antigens, Surface / Q000276:immunology*; D018801:CD2 Antigens / Q000276:immunology; D000071178:CD48 Antigen; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000276:immunology; D016326:Extracellular Matrix Proteins / Q000276:immunology*; D006023:Glycoproteins / Q000276:immunology*; D007141:Immunoglobulin Fc Fragments / Q000276:immunology; D007962:Leukocytes / Q000276:immunology*; D064235:Matrilin Proteins; D008562:Membrane Glycoproteins / Q000276:immunology; D051379:Mice; D040081:Protein Array Analysis / Q000379:methods; D037663:Pulmonary Surfactant-Associated Protein D / Q000276:immunology*; D051381:Rats; D011971:Receptors, Immunologic / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology
chemical_list: D015703:Antigens, CD; D000954:Antigens, Surface; D018801:CD2 Antigens; D000071178:CD48 Antigen; C495490:Cd200 protein, rat; C000604066:Cd48 protein, mouse; C000604067:Cd48 protein, rat; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D007141:Immunoglobulin Fc Fragments; C577540:Matn1 protein, mouse; D064235:Matrilin Proteins; D008562:Membrane Glycoproteins; D037663:Pulmonary Surfactant-Associated Protein D; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; C042739:antigens, CD200