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Structural Studies of Cell Surface Molecules Involved in Recognition by the Immune System
Summary: Pamela Björkman is interested in the structure and function of molecules mediating cell surface recognition.
My laboratory is interested in protein-protein interactions, particularly those mediating immune recognition. We use x-ray crystallography and biochemistry to study purified proteins, and electron and confocal microscopy to examine protein complexes in cells. We focus on three interrelated areas: (1) homologs and mimics of class I major histocompatibility complex (MHC) proteins; (2) antibody receptors, for example, the MHC-related neonatal Fc receptor (FcRn); and (3) designing antibodies with increased efficacy against pathogens.
Classical class I MHC proteins present peptide antigens derived from self- and nonself proteins to T cells during immune surveillance. The MHC structure seems ideally suited for its antigen presentation function, in that it includes a groove that is perfectly shaped to accommodate short peptide antigens. MHC homologs share similar three-dimensional structures with classical MHC molecules but have different functions, including immune functions (antibody transport by FcRn; evasion of the immune response by viral MHC mimics) and nonimmune functions (regulation of iron or lipid metabolism by HFE and ZAG; chaperoning pheromone receptors to the cell surface in the case of M10 proteins).
Our crystal structures revealed that FcRn, HFE, ZAG, and M10 do not present peptides and therefore play no role in conventional adaptive immune responses. Each of these MHC homologs uses a different structural mechanism to prevent peptide binding and accomplish its distinct function. The FcRn and HFE grooves are collapsed, and cocrystal structures with their respective protein ligands show that each uses a protein-protein interaction mode different from MHC interactions with peptides or T cell and other receptors. By contrast, the grooves of ZAG, M10, and the human cytomegalovirus MHC mimic UL18 are open and theoretically capable of antigen binding, but only UL18 associates with peptides. These results raise questions about the functions of these latter MHC homologs and about the primordial function of the MHC fold: Did it originally arise for peptide presentation/T cell interactions as part of the adaptive immune response (a relatively recent acquisition of the vertebrate immune system), or did it arise for the seemingly more ancient functions of protein transport or metabolite regulation? Perhaps surprisingly, our results (including characterization of a nonmammalian Fc receptor related to FcRn) suggest the former. Our structural, biochemical, and biophysical studies of MHC homologs provide striking examples that structure does not always dictate function: similar structures can adopt different functions, and conversely, similar functions can be accomplished by very different structures.
We have extended our characterizations of FcRn, an MHC-related receptor for IgG (immunoglobulin G) antibodies, to include cell biological studies of intracellular trafficking. FcRn is the receptor that transfers maternal IgG to the bloodstream of fetal and newborn mammals, thereby passively immunizing the neonate against pathogens likely to be encountered prior to development of its own fully functional immune system. Transfer of IgG involves trafficking of FcRn-IgG complexes in acidic intracellular vesicles across an epithelial cell barrier in the placenta (for prenatal transfer) or the intestine (for postnatal transfer). A general question exemplified by FcRn trafficking is how cargo-containing intracellular vesicles are transported to their correct ultimate locations—for example, how does the cell know that FcRn-IgG complexes should be transported across the cell for eventual release of IgG into the blood, whereas other receptor-ligand pairs should be transferred to degradative compartments?
To study the process by which FcRn-IgG complexes are correctly trafficked across cells, we are using electron tomography, a form of electron microscopy, to derive three-dimensional maps of transport vesicles in neonatal rat intestinal epithelial cells at resolutions of 4–6 nm. To facilitate these studies, we developed gold-labeling and enhancement methods to locate individual IgG fragments bound to FcRn inside intracellular vesicles. Our three-dimensional images of IgG transport reveal tangled webs of interlocking IgG-containing transport vesicles, some of which are associated with microtubule tracks to allow movement via motor proteins. Other IgG-containing vesicles include multivesicular bodies, normally associated with degradative functions but apparently functioning in IgG transport in the specialized proximal small intestinal cells of a neonate.
To complement these high-resolution, but static, studies, we are doing fluorescence imaging in live cells, which allows tracking in real time of labeled vesicles and quantification of the velocities and directions of FcRn-positive vesicles. We plan a similar combined electron/fluorescence microscopy study to characterize the intracellular trafficking pathways of two other Fc receptors: the polymeric immunoglobulin receptor (pIgR), which transports polymeric antibodies into secretions, and gE-gI, a viral Fc receptor for IgG. We discovered that gE-gI exhibits a pH-dependent affinity transition for binding IgG that is opposite that of FcRn: FcRn binds tightly to IgG at acidic, but not basic, pH, so as to bind IgG inside acidic vesicles during transport and to release IgG upon encountering the slightly basic pH of blood; by contrast, gE-gI binds IgG at the pH of blood but not at the pH of intracellular vesicles. We are testing the hypothesis that circulating IgG taken up by gE-gI by receptor-mediated endocytosis is destined for degradation after dissociating from gE-gI in acidic intracellular vesicles, which could form part of a viral mechanism to escape from antibody-mediated host immune responses. (The studies of Fc receptors are funded by the National Institutes of Health.)
In addition to studying antibody receptors, we have begun a new project to improve upon the binding and neutralization properties of antibodies themselves. This work is part of a collaboration with David Baltimore's laboratory (California Institute of Technology) to "Engineer Immunity" against HIV. The idea is to direct life-long production of specified antibodies or antibody-like proteins with desired properties; for example, neutralizing antibodies or designed antibodies engineered to bind more tightly to a pathogen or to recruit immune effector cells. The antibodies would be produced in vivo by infecting autologous hematopoietic stem cells with lentiviral vectors bearing specific antibody genes, thus allowing life-long production of anti-HIV proteins.
Our portion of the project involves designing, producing, and testing novel anti-HIV protein reagents in an effort to find proteins with increased efficacy in HIV neutralization. Although HIV has evolved to evade most or all antibodies (hence the difficulty of finding an immunogen capable of eliciting a strong neutralizing antibody response in vaccine development efforts), an attractive feature of the Engineering Immunity approach is that we are not limited to the traditional architecture of an antibody. Hence we can produce and express antibody-like proteins of different sizes (to facilitate access to hidden epitopes) and valencies (i.e., with different numbers of combining sites) and/or link antibodies to HIV-binding proteins such as the host receptor CD4.
In our initial efforts, we have developed CD4-antibody fusion proteins that cross-react to neutralize a broad range of HIV strains, and characterized a dimeric form of an anticarbohydrate antibody, 2G12, that displays a 50- to 80-fold increased potency in the neutralization of clade B HIV strains. Using a structural model for the 2G12 dimer, we designed a series of 2G12 mutants predicted to increase the dimer/monomer ratio. After expressing and testing the mutants, we found one mutation that results in an increased dimer ratio and is a candidate for a more potent reagent to be used in gene therapy or passive immunization. (Studies of anti-HIV antibodies are funded by the Bill and Melinda Gates Foundation.)
Last updated: November 4, 2008
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