title: Analysis of leukocyte membrane protein interactions using protein microarrays.
abstract: BACKGROUND
  Protein microarrays represent an emerging class of proteomic tools to investigate multiple protein-protein interactions in parallel. A sufficient proportion of immobilized proteins must maintain an active conformation and an orientation that allows for the sensitive and specific detection of antibody and ligand binding. In order to establish protein array technology for the characterization of the weak interactions between leukocyte membrane proteins, we selected the human leukocyte membrane protein CD200 (OX2) and its cell surface receptor (hCD200R) as a model system. As antibody-antigen reactions are generally of higher affinity than receptor-ligand binding, we first analyzed the reactivity of monoclonal antibodies (mAb) to normal and mutant forms of immobilized CD200R.
  
  
  RESULTS
  Fluorescently labelled mAb DX147, DX136 and OX108 were specifically reactive with immobilized recombinant hCD200R extracellular region, over a range of 0.1-40 microg ml(-1) corresponding to a limit of sensitivity of 0.01-0.05 femtomol per spot. Orientating hCD200R using capture antibodies, showed that DX147 reacts with an epitope spatially distinct from the more closely related DX136 and OX108 epitopes. A panel of soluble recombinant proteins with mutations in hCD200R domain 1 produced by transiently transfected cells, was arrayed directly without purification and screened for binding to the three mAb. Several showed decreased binding to the blocking mAb DX136 and OX108, suggesting close proximity of these epitopes to the CD200 binding site. Binding of hCD200 to directly immobilized rat, mouse, and hCD200R was achieved with multimeric ligands, in the form of biotinylated-hCD200 coupled to FITC-labelled avidin coated beads.
  
  
  CONCLUSIONS
  We have achieved sensitive, specific and reproducible detection of immobilized CD200R with different antibodies and mapped antigenic epitopes for two mAb in the vicinity of the ligand binding site using protein microarrays. We also detected CD200 binding to its receptor, a low affinity interaction, using beads presenting multivalent ligands. Our results demonstrate the quantitative aspects of protein arrays and their potential use in detecting simultaneously multiple protein-protein interactions and in particular the weak interactions found between leukocyte membrane proteins.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK. mablab@sickkids.ca <mablab@sickkids.ca>
authors.forename: Michelle
authors.initials: M
authors.lastname: Letarte
authors.forename: Despina
authors.initials: D
authors.lastname: Voulgaraki
authors.forename: Deborah
authors.initials: D
authors.lastname: Hatherley
authors.forename: Mildred
authors.initials: M
authors.lastname: Foster-Cuevas
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Saunders
authors.forename: A Neil
authors.initials: AN
authors.lastname: Barclay
mesh_terms: D000818:Animals; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding / Q000502:physiology; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary / Q000502:physiology; D051381:Rats
chemical_list: D008565:Membrane Proteins
references.citation: Nature. 1999 Dec 16;402(6763):715-20
references.pmid: 10617183
references.citation: Nat Genet. 1999 Jan;21(1 Suppl):48-50
references.pmid: 9915501
references.citation: Immunity. 2000 Aug;13(2):233-42
references.pmid: 10981966
references.citation: Science. 2000 Dec 1;290(5497):1768-71
references.pmid: 11099416
references.citation: Genome Biol. 2001;2(2):RESEARCH0004
references.pmid: 11182887
references.citation: Immunology. 2001 Feb;102(2):173-9
references.pmid: 11260322
references.citation: Clin Chem Lab Med. 2001 Mar;39(3):209-14
references.pmid: 11350016
references.citation: Biotechniques. 2001 Jul;31(1):186-90, 192-4
references.pmid: 11464511
references.citation: Cancer Res. 2001 Oct 15;61(20):7585-93
references.pmid: 11606398
references.citation: Clin Immunol. 2001 Dec;101(3):328-34
references.pmid: 11726225
references.citation: Trends Biotechnol. 2002 Apr;20(4):160-6
references.pmid: 11906748
references.citation: Trends Immunol. 2002 Jun;23(6):285-90
references.pmid: 12072366
references.citation: Clin Immunol. 2002 Sep;104(3):256-64
references.pmid: 12217336
references.citation: Anal Biochem. 2003 Jan 15;312(2):113-24
references.pmid: 12531195
references.citation: Curr Opin Chem Biol. 2003 Feb;7(1):55-63
references.pmid: 12547427
references.citation: Proc Natl Acad Sci U S A. 2003 Apr 29;100(9):5366-71
references.pmid: 12697893
references.citation: Cancer Cell. 2003 Apr;3(4):317-25
references.pmid: 12726858
references.citation: Immunity. 2003 Aug;19(2):213-23
references.pmid: 12932355
references.citation: J Immunol. 2003 Sep 15;171(6):3034-46
references.pmid: 12960329
references.citation: Eur J Immunol. 2004 Jun;34(6):1688-94
references.pmid: 15162439
references.citation: J Virol. 2004 Jul;78(14):7667-76
references.pmid: 15220441
references.citation: J Biol Chem. 1990 Jun 25;265(18):10410-8
references.pmid: 2113054
references.citation: Immunity. 1999 Feb;10(2):219-25
references.pmid: 10072074
references.citation: Biochim Biophys Acta. 1999 Mar 25;1423(2):M17-28
references.pmid: 10214349
references.citation: Cell. 1999 Jun 11;97(6):791-803
references.pmid: 10380930
references.citation: J Pharm Biomed Anal. 1989;7(2):155-68
references.pmid: 2488616
references.citation: Ann Biol Clin (Paris). 1990;48(9):655-66
references.pmid: 2099119
references.citation: Nature. 1992 Nov 19;360(6401):232-9
references.pmid: 1279440
references.citation: Protein Eng. 1994 Apr;7(4):515-21
references.pmid: 7518084
references.citation: Trends Biochem Sci. 1994 Sep;19(9):354-8
references.pmid: 7985226
references.citation: J Biol Chem. 1995 Jan 6;270(1):369-75
references.pmid: 7529232
references.citation: Curr Opin Immunol. 1996 Apr;8(2):257-61
references.pmid: 8725949
references.citation: Nat Genet. 1996 Dec;14(4):457-60
references.pmid: 8944026
references.citation: J Protein Chem. 1997 Jul;16(5):537-44
references.pmid: 9246641
references.citation: Eur J Immunol. 1997 Aug;27(8):1911-8
references.pmid: 9295026
references.citation: J Exp Med. 1998 Dec 7;188(11):2083-90
references.pmid: 9841922
references.citation: Science. 2000 Sep 8;289(5485):1760-3
references.pmid: 10976071