Expression and Circular Dichroism Studies of the Extracellular Domain of the alpha Subunit of the Nicotinic Acetylcholine Receptor

To provide material suitable for structural studies of the nicotinic acetylcholine receptor, we have expressed and purified the NH2-terminal extracellular domain of the mouse muscle alpha subunit. Several constructs were initially investigated using Xenopus oocytes as a convenient small scale expression system. A fusion protein (alpha210GPI) consisting of the 210 NH2-terminal amino acids of the alpha subunit and a glycosylphosphatidylinositol anchorage sequence conferred surface alpha-bungarotoxin binding in oocytes. Coexpression of alpha210GPI with an analogous construct made from the delta subunit showed no evidence of heterodimer formation. The alpha210GPI protein was chosen for large scale expression in transfected Chinese hamster ovary cells. The alpha210GPI protein was cleaved from these cells and purified on an immunoaffinity column. Gel and column chromatography show that the purified protein is processed as expected and exists as a monomer. The purified protein also retains the two distinct, conformation-specific binding sites expected for the correctly folded alpha subunit. Circular dichroism studies of alpha210GPI suggest that this region of the receptor includes considerable beta-sheet secondary structure, with a small proportion of alpha-helix

Model for the Interaction of T-cell Receptors with Peptide/MHC Complexes

The immune response against a viral infection is mediated by two different types of cells known as B and T lymphocytes. The receptor on the B cell is the well-characterized antibody molecule, which exists in a membrane-bound form and in a secreted form involved in the initiation of complement-mediated killing and the inactivation of viral particles by direct binding. The recognition molecule on T cells is the membrane-bound T-cell antigen receptor, which has specificity for a combination of foreign antigen with a molecule of the major histocompatibility complex (MHC), as first demonstrated by Zinkernagel and Doherty (1974). MHC proteins exist in two closely related forms called class I and class II MHC molecules, both of which are cell-surface glycoproteins that are highly polymorphic in the human population. In general, class II MHC molecules are involved in interactions with T-helper cells, which cooperate with B cells to make antibody

Toward Mapping the Detailed Density Structure of Classical Be Circumstellar Disks

We present the preliminary results of near contemporaneous optical and infrared spectroscopic observations of select classical Be stars. We find strong evidence of oppositely oriented V/R hydrogen line profiles in the optical versus infrared spectra of zeta Tau, and briefly discuss how sustained contemporaneous optical and infrared spectroscopic observations might enable us to trace the detailed density structure of classical Be circumstellar disks

Hydrophobic Ligand Binding by Zn-α_2-glycoprotein, a Soluble Fat-depleting Factor Related to Major Histocompatibility Complex Proteins

Zn-alpha2-glycoprotein (ZAG) is a member of the major histocompatibility complex (MHC) class I family of proteins and is identical in amino acid sequence to a tumor-derived lipid-mobilizing factor associated with cachexia in cancer patients. ZAG is present in plasma and other body fluids, and its natural function, like leptin's, probably lies in lipid store homeostasis. X-ray crystallography has revealed an open groove between the helices of ZAG's alpha1 and alpha2 domains, containing an unidentified small ligand in a position similar to that of peptides in MHC proteins (Sanchez, L. M., Chirino, A. J., and Bjorkman, P. J. (1999) Science 283, 1914-1919). Here we show, using serum-derived and bacterial recombinant protein, that ZAG binds the fluorophore-tagged fatty acid 11-(dansylamino)undecanoic acid (DAUDA) and, by competition, natural fatty acids such as arachidonic, linolenic, eicosapentaenoic, and docosahexaenoic acids. Other MHC class I-related proteins (FcRn, HFE, HLA-Cw*0702) showed no such evidence of binding. Fluorescence and isothermal calorimetry analysis showed that ZAG binds DAUDA with Kd in the micromolar range, and differential scanning calorimetry showed that ligand binding increases the thermal stability of the protein. Addition of fatty acids to ZAG alters its intrinsic (tryptophan) fluorescence emission spectrum, providing a strong indication that ligand binds in the expected position close to a cluster of exposed tryptophan side chains in the groove. This study therefore shows that ZAG binds small hydrophobic ligands, that the natural ligand may be a polyunsaturated fatty acid, and provides a fluorescence-based method for investigating ZAG-ligand interactions

2-D and 3-D Radiation Transfer Models of High-Mass Star Formation

2-D and 3-D radiation transfer models of forming stars generally produce bluer 1-10 micron colors than 1-D models of the same evolutionary state and envelope mass. Therefore, 1-D models of the shortwave radiation will generally estimate a lower envelope mass and later evolutionary state than multidimensional models. 1-D models are probably reasonable for very young sources, or longwave analysis (wavelengths > 100 microns). In our 3-D models of high-mass stars in clumpy molecular clouds, we find no correlation between the depth of the 10 micron silicate feature and the longwave (> 100 micron) SED (which sets the envelope mass), even when the average optical extinction of the envelope is >100 magnitudes. This is in agreement with the observations of Faison et al. (1998) of several UltraCompact HII (UCHII) regions, suggesting that many of these sources are more evolved than embedded protostars. We have calculated a large grid of 2-D models and find substantial overlap between different evolutionary states in the mid-IR color-color diagrams. We have developed a model fitter to work in conjunction with the grid to analyze large datasets. This grid and fitter will be expanded and tested in 2005 and released to the public in 2006.Comment: 10 pages, 8 figures, to appear in the proceedings of IAU Symp 227, Massive Star Birth: A Crossroads of Astrophysics, (Cesaroni R., Churchwell E., Felli M., Walmsley C. editors

2-D Radiative Transfer in Protostellar Envelopes: I. Effects of Geometry on Class I Sources

We present 2-D radiation transfer models of Class I Protostars and show the effect of including more realistic geometries on the resulting spectral energy distributions and images. We begin with a rotationally flattened infalling envelope as our comparison model, and add a flared disk and bipolar cavity. The disk affects the spectral energy distribution most strongly at edge-on inclinations, causing a broad dip at about 10 um (independent of the silicate feature) due to high extinction and low scattering albedo in this wavelength region. The bipolar cavities allow more direct stellar+disk radiation to emerge into polar directions, and more scattering radiation to emerge into all directions. The wavelength-integrated flux, often interpreted as luminosity, varies with viewing angle, with pole-on viewing angles seeing 2-4 times as much flux as edge-on, depending on geometry. Thus, observational estimates of luminosity should take into account the inclination of a source. The envelopes with cavities are significantly bluer in near-IR and mid-IR color-color plots than those without cavities. Using 1-D models to interpret Class I sources with bipolar cavities would lead to an underestimate of envelope mass and an overestimate of the implied evolutionary state. We compute images at near-, mid-, and far-IR wavelengths. We find that the mid-IR colors and images are sensitive to scattering albedo, and that the flared disk shadows the midplane on large size scales at all wavelengths plotted. Finally, our models produce polarization spectra which can be used to diagnose dust properties, such as albedo variations due to grain growth. Our results of polarization across the 3.1 um ice feature agree well with observations for ice mantles covering 5% of the radius of the grains.Comment: Accepted for publication in ApJ, 37 pages, 13 figures (several figures reduced in quality; find original version at http://gemelli.colorado.edu/~bwhitney/preprints.html

Interaction of hemojuvelin with neogenin results in iron accumulation in human embryonic kidney 293 cells

Type 2 hereditary hemochromatosis (HH) or juvenile hemochromatosis is an early onset, genetically heterogeneous, autosomal recessive disorder of iron overload. Type 2A HH is caused by mutations in the recently cloned hemojuvelin gene (HJV; also called HFE2) (Papanikolaou, G., Samuels, M. E., Ludwig, E. H., MacDonald, M. L., Franchini, P. L., Dube, M. P., Andres, L., MacFarlane, J., Sakellaropoulos, N., Politou, M., Nemeth, E., Thompson, J., Risler, J. K., Zaborowska, C., Babakaiff, R., Radomski, C. C., Pape, T. D., Davidas, O., Christakis, J., Brissot, P., Lockitch, G., Ganz, T., Hayden, M. R., and Goldberg, Y. P. (2004) Nat. Genet. 36, 77–82), whereas Type 2B HH is caused by mutations in hepcidin. HJV is highly expressed in both skeletal muscle and liver. Mutations in HJV are implicated in the majority of diagnosed juvenile hemochromatosis patients. In this study, we stably transfected HJV cDNA into human embryonic kidney 293 cells and characterized the processing of HJV and its effect on iron homeostasis. Our results indicate that HJV is a glycosylphosphatidylinositol-linked protein and undergoes a partial autocatalytic cleavage during its intracellular processing. HJV co-immunoprecipitated with neogenin, a receptor involved in a variety of cellular signaling processes. It did not interact with the closely related receptor DCC (deleted in Colon Cancer). In addition, the HJV G320V mutant implicated in Type 2A HH did not co-immunoprecipitate with neogenin. Immunoblot analysis of ferritin levels and transferrin-55Fe accumulation studies indicated that the HJV-induced increase in intracellular iron levels in human embryonic kidney 293 cells is dependent on the presence of neogenin in the cells, thus linking these two proteins to intracellular iron homeostasis

Distinct Intracellular Trafficking Patterns of Host IgG by Herpes Virus Fc-Receptors

Members of both alpha and beta herpes viruses affects 50–98% of people around the world. They cause severe symptoms in congenitally infected newborns, a lifelong latent infection that is lethal in immunocompromised individuals, and are associated with several types of cancer. Human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) viruses express proteins (HCMV gp68 and gp34; HSV-1 gE-gI) that function as Fc receptors (FcRs) by binding to the Fc regions of human IgG. In addition to binding free IgG, these viral FcRs can bind to IgG complexed with an antigen to form an antibody bipolar bridged (ABB) complex. Although HCMV gp68 and HSV-1 gE-gI have an overlapping binding site on Fc, the finding that the gp68/Fc interaction is stable at pH values between 5.6 and 8.1 but that gE-gI binds only at neutral or basic pH suggests distinct pH-based downstream events after IgG is internalized via receptor-mediated endocytosis into intracellular compartments. Here we developed a cell-based in vitro model system to define the fates of ABB complexes formed by the two types of viral FcRs. We found that alpha (HSV-1) and beta (HCMV) herpes virus FcRs displayed distinct intracellular trafficking patterns to target internalized ligands: HSV-1 gE-gI dissociates from its IgG-antigen ligand in acidic endosomal compartments and recycles back to the cell surface, whereas HCMV FcRs (gp68) are transported together with IgG-antigen complexes to lysosomes for degradation. In both cases, anti-viral IgGs and their viral targets are selectively degraded, a potential immune evasion strategy allowing herpes viruses to escape from IgG-mediated immune responses

Comparison of the Interactions of Transferrin Receptor and Transferrin Receptor 2 with Transferrin and the Hereditary Hemochromatosis Protein HFE

The transferrin receptor (TfR) interacts with two proteins important for iron metabolism, transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. A second receptor for Tf, TfR2, was recently identified and found to be functional for iron uptake in transfected cells (Kawabata, H., Germain, R. S., Vuong, P. T., Nakamaki, T., Said, J. W., and Koeffler, H. P. (2000) J. Biol. Chem. 275, 16618-16625). TfR2 has a pattern of expression and regulation that is distinct from TfR, and mutations in TfR2 have been recognized as the cause of a non-HFE linked form of hemochromatosis (Camaschella, C., Roetto, A., Cali, A., De Gobbi, M., Garozzo, G., Carella, M., Majorano, N., Totaro, A., and Gasparini, P. (2000) Nat. Genet. 25, 14-15). To investigate the relationship between TfR, TfR2, Tf, and HFE, we performed a series of binding experiments using soluble forms of these proteins. We find no detectable binding between TfR2 and HFE by co-immunoprecipitation or using a surface plasmon resonance-based assay. The affinity of TfR2 for iron-loaded Tf was determined to be 27 nM, 25-fold lower than the affinity of TfR for Tf. These results imply that HFE regulates Tf-mediated iron uptake only from the classical TfR and that TfR2 does not compete for HFE binding in cells expressing both forms of TfR