Analysis of leukocyte membrane protein interactions using protein microarrays

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 μg 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. CONCLUSION: 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

Crystal structure of signal regulatory protein gamma (SIRPγ) in complex with an antibody Fab fragment

BACKGROUND Signal Regulatory Protein γ (SIRPγ) is a member of a closely related family of three cell surface receptors implicated in modulating immune/inflammatory responses. SIRPγ is expressed on T lymphocytes where it appears to be involved in the integrin-independent adhesion of lymphocytes to antigen-presenting cells. Here we describe the first full length structure of the extracellular region of human SIRPγ. RESULTS We obtained crystals of SIRPγ by making a complex of the protein with the Fab fragment of the anti-SIRP antibody, OX117, which also binds to SIRPα and SIRPβ. We show that the epitope for FabOX117 is formed at the interface of the first and second domains of SIRPγ and comprises residues which are conserved between all three SIRPs. The FabOX117 binding site is distinct from the region in domain 1 which interacts with CD47, the physiological ligand for both SIRPγ and SIRPα but not SIRPβ. Comparison of the three domain structures of SIRPγ and SIRPα showed that these receptors can adopt different overall conformations due to the flexibility of the linker between the first two domains. SIRPγ in complex with FabOX117 forms a dimer in the crystal. Binding to the Fab fixes the position of domain 1 relative to domains 2/3 exposing a surface which favours formation of a homotypic dimer. However, the interaction appears to be relatively weak since only monomers of SIRPγ were observed in sedimentation velocity analytical ultracentrifugation of the protein alone. Studies of complex formation by equilibrium ultracentrifugation showed that only a 1:1 complex of SIRPγ: FabOX117 was formed with a dissociation constant in the low micromolar range (Kd = 1.2 +/- 0.3 μM). CONCLUSION The three-domain extracellular regions of SIRPs are structurally conserved but show conformational flexibility in the disposition of the amino terminal ligand-binding Ig domain relative to the two membrane proximal Ig domains. Binding of a cross-reactive anti-SIRP Fab fragment to SIRPγ stabilises a conformation that favours SIRP dimer formation in the crystal structure, though this interaction does not appear sufficiently stable to be observed in solution

Structures of CD6 and Its Ligand CD166 Give Insight into Their Interaction

SummaryCD6 is a transmembrane protein with an extracellular region containing three scavenger receptor cysteine rich (SRCR) domains. The membrane proximal domain of CD6 binds the N-terminal immunoglobulin superfamily (IgSF) domain of another cell surface receptor, CD166, which also engages in homophilic interactions. CD6 expression is mainly restricted to T cells, and the interaction between CD6 and CD166 regulates T-cell activation. We have solved the X-ray crystal structures of the three SRCR domains of CD6 and two N-terminal domains of CD166. This first structure of consecutive SRCR domains reveals a nonlinear organization. We characterized the binding sites on CD6 and CD166 and showed that a SNP in CD6 causes glycosylation that hinders the CD6/CD166 interaction. Native mass spectrometry analysis showed that there is competition between the heterophilic and homophilic interactions. These data give insight into how interactions of consecutive SRCR domains are perturbed by SNPs and potential therapeutic reagents

Structural basis of ligand recognition by Myeloid Paired Receptors

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Structure of Signal-regulatory Protein α: A LINK TO ANTIGEN RECEPTOR EVOLUTION*

Signal-regulatory protein α (SIRPα) is a myeloid membrane receptor that interacts with the membrane protein CD47, a marker of self. We have solved the structure of the complete extracellular portion of SIRPα, comprising three immunoglobulin superfamily domains, by x-ray crystallography to 2.5 Å resolution. These data, together with previous data on the N-terminal domain and its ligand CD47 (possessing a single immunoglobulin superfamily domain), show that the CD47-SIRPα interaction will span a distance of around 14 nm between interacting cells, comparable with that of an immunological synapse. The N-terminal (V-set) domain mediates binding to CD47, and the two others are found to be constant (C1-set) domains. C1-set domains are restricted to proteins involved in vertebrate antigen recognition: T cell antigen receptors, immunoglobulins, major histocompatibility complex antigens, tapasin, and β2-microglobulin. The domains of SIRPα (domains 2 and 3) are structurally more similar to C1-set domains than any cell surface protein not involved in antigen recognition. This strengthens the suggestion from sequence analysis that SIRP is evolutionarily closely related to antigen recognition proteins

Reconstitution of immune cell interactions in free-standing membranes

The spatiotemporal regulation of signalling proteins at the contacts formed between immune cells and their targets determines how and when immune responses begin and end. Therapeutic control of immune responses therefore relies on thorough elucidation of the molecular processes occurring at these interfaces. However, the detailed investigation of each component's contribution to the formation and regulation of the contact is hampered by the complexities of cell composition and architecture. Moreover, the transient nature of these interactions creates additional challenges, especially in the use of advanced imaging technology. One approach that circumvents these problems is to establish in vitro systems that faithfully mimic immune cell interactions, but allow complexity to be ‘dialled-in’ as needed. Here, we present an in vitro system that makes use of synthetic vesicles that mimic important aspects of immune cell surfaces. Using this system, we began to explore the spatial distribution of signalling molecules (receptors, kinases and phosphatases) and how this changes during the initiation of signalling. The GUV/cell system presented here is expected to be widely applicable.The spatiotemporal regulation of signalling proteins at the contacts formed between immune cells and their targets determines how and when immune responses begin and end. Therapeutic control of immune responses therefore relies on thorough elucidation of the molecular processes occurring at these interfaces. However, the detailed investigation of each component's contribution to the formation and regulation of the contact is hampered by the complexities of cell composition and architecture. Moreover, the transient nature of these interactions creates additional challenges, especially in the use of advanced imaging technology. One approach that circumvents these problems is to establish in vitro systems that faithfully mimic immune cell interactions, but allow complexity to be ‘dialled-in’ as needed. Here, we present an in vitro system that makes use of synthetic vesicles that mimic important aspects of immune cell surfaces. Using this system, we began to explore the spatial distribution of signalling molecules (receptors, kinases and phosphatases) and how this changes during the initiation of signalling. The GUV/cell system presented here is expected to be widely applicable

Effects of a local auxiliary protein on the two-dimensional affinity of a TCR-peptide MHC interaction

The affinity of T-cell receptors (TCRs) for major histocompatibility complex molecules (MHCs) presenting cognate antigens likely determines whether T cells initiate immune responses, or not. There exist few measurements of two-dimensional (2D) TCR-MHC interactions, and the effect of auxiliary proteins on binding is unexplored. Here, Jurkat T-cells expressing the MHC molecule HLA-DQ8-glia-α1 and the ligand of an adhesion protein (rat CD2) were allowed to bind supported lipid bilayers (SLBs) presenting fluorescently labelled L3-12 TCR and rat CD2, allowing measurements of binding unconfounded by cell signaling effects or co-receptor binding. The 2D Kd for L3-12 TCR binding to HLA-DQ8-glia-α1, of 14±5 molecules/μm2 (mean±s.d.), was only marginally influenced by including CD2 up to ∼200 bound molecules/μm2 but higher CD2 densities reduced the affinity up to 1.9-fold. Cell-SLB contact size increased steadily with ligand density without affecting binding for contacts at up to ∼20% of total cell area, but beyond this lamellipodia appeared, giving an apparent increase in bound receptors of up to 50%. Our findings show how parameters other than the specific protein-protein interaction can influence binding behavior at cell-cell contacts