The BioID method uses a promiscuous biotin ligase to detect protein-protein associations as well as proximate proteins in living cells. Here we report improvements to the BioID method centered on ...BioID2, a substantially smaller promiscuous biotin ligase. BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, and exhibits enhanced labeling of proximate proteins. Thus BioID2 improves the efficiency of screening for protein-protein associations. We also demonstrate that the biotinylation range of BioID2 can be considerably modulated using flexible linkers, thus enabling application-specific adjustment of the biotin-labeling radius.
Envelope glycoprotein (Env) spikes on AIDS retroviruses initiate infection of host cells and are therefore targets for vaccine development. Though crystal structures for partial Env subunits are ...known, the structure and distribution of native Env spikes on virions is obscure. We applied cryoelectron microscopy tomography to define ultrastructural details of spikes. Virions of wild-type human immunodeficiency virus 1 (HIV-1) and a mutant simian immunodeficiency virus (SIV) had approximately 14 and approximately 73 spikes per particle, respectively, with some clustering of HIV-1 spikes. Three-dimensional averaging showed that the surface glycoprotein (gp120) 'head' of each subunit of the trimeric SIV spike contains a primary mass, with two secondary lobes. The transmembrane glycoprotein 'stalk' of each trimer is composed of three independent legs that project obliquely from the trimer head, tripod-like. Reconciling available atomic structures with the three-dimensional whole spike density map yields insights into the orientation of Env spike structural elements and possible structural bases of their functions.
Influence of high pressure processing (HPP) at 450 and 600MPa, 30°C for various holding times (0, 30, 60,180, 300 and 600s) on almond milk amandin was investigated. The immunoreactivity of pressure ...treated almond milk was compared with raw and thermally processed (TP) almond milk (72, 85 and 99°C for 0 to 300s) using a sandwich enzyme-linked immunosorbent assay (ELISA), Western blot and dot blot. Monoclonal antibodies (mAbs) targeting linear (4F10) and conformational (4C10) epitopes on amandin were used to assess amandin immunoreactivity. To determine the aggregation of almond proteins, almond milk protein solubility was quantified after 300s of HPP (up to 600MPa, 30°C) and TP (at 72, 85 and 99°C, 0.1MPa). After HPP (for all holding times), amandin can no longer be detected by the anti-conformational mAb in ELISA while signal generated from the anti-linear epitopes mAb was reduced by half (P<0.05). On the other hand, most TP samples did not show significant reductions in immunoreactivity (P>0.05) unless processed at 85 and 99°C for 300s. Western blot and dot blot also confirmed the loss of immunoreactivity by both antibodies for HPP almond milk. The reduced band intensity of the 61 and 63kDa polypeptides and concomitant appearance of high molecular weight polypeptides in Western blot indicated that the observed decrease in immunoreactivity was partly due to the aggregation of amandin. The tested HPP and TP treatments respectively caused a maximum of ~70% and ~75% reduction in protein solubility. The study demonstrated that the loss of protein solubility, rather than the epitope destruction, may be responsible for the observed decrease in amandin immunoreactivity.
•Pressure effect on almond milk protein solubility and immunoreactivity was studied.•Pressure and thermal treatments decreased protein solubility due to aggregation.•High pressure processing destroyed some conformational epitopes.•Pressure treated samples had lower residual immunoreactivity than thermal treatment.
The human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) envelope spike (Env) mediates viral entry into host cells. The V3 loop of the gp120 component of the Env trimer ...contributes to the coreceptor binding site and is a target for neutralizing antibodies. We used cryo-electron tomography to visualize the binding of CD4 and the V3 loop monoclonal antibody (MAb) 36D5 to gp120 of the SIV Env trimer. Our results show that 36D5 binds gp120 at the base of the V3 loop and suggest that the antibody exerts its neutralization effect by blocking the coreceptor binding site. The antibody does this without altering the dynamics of the spike motion between closed and open states when CD4 is bound. The interaction between 36D5 and SIV gp120 is similar to the interaction between some broadly neutralizing anti-V3 loop antibodies and HIV-1 gp120. Two conformations of gp120 bound with CD4 are revealed, suggesting an intrinsic dynamic nature of the liganded Env trimer. CD4 binding substantially increases the binding of 36D5 to gp120 in the intact Env trimer, consistent with CD4-induced changes in the conformation of gp120 and the antibody binding site. Binding by MAb 36D5 does not substantially alter the proportions of the two CD4-bound conformations. The position of MAb 36D5 at the V3 base changes little between conformations, indicating that the V3 base serves as a pivot point during the transition between these two states.
Glycoprotein spikes on the surfaces of SIV and HIV are the sole targets available to the immune system for antibody neutralization. Spikes evade the immune system by a combination of a thick layer of polysaccharide on the surface (the glycan shield) and movement between spike domains that masks the epitope conformation. Using SIV virions whose spikes were "decorated" with the primary cellular receptor (CD4) and an antibody (36D5) at part of the coreceptor binding site, we visualized multiple conformations trapped by the rapid freezing step, which were separated using statistical analysis. Our results show that the CD4-induced conformational dynamics of the spike enhances binding of the antibody.
Allergy to peanuts has become a common and severe problem, especially in westernized countries. In this study, we evaluated the target and epitope specificity of the capture and detection mouse ...monoclonal antibodies (mAbs) used in a commercial peanut allergen detection platform. We first identified the target of these antibodies as Ara h 3 and then used an overlapping peptide array of Ara h 3 to determine the antibody-binding epitopes. Further amino acids critical for the binding via alanine substitutions at individual amino acid residues within the epitope were mapped. Finally, inhibition ELISA and inhibition immunoblotting using a recombinant Ara h 3 protein were performed to confirm these results. Surprisingly, the capture and detection mAbs showed identical binding characteristics and were presumed to represent two isolates of the same clone, a notion supported by both isoelectric focusing electrophoresis and Liquid chromatography-mass spectrometry experiments. The simultaneous binding of a pair of identical mAbs to an individual allergen such as Ara h3 is attributed to the multivalency of the analyte and has implications for developing diagnostic assays for additional multimeric allergens.
Background The low-affinity receptor for IgE, FcεRII (CD23), contributes to allergic inflammation through allergen presentation to T cells, regulation of IgE responses, and enhancement of ...transepithelial allergen migration. Objective We sought to investigate the interaction between CD23, chimeric monoclonal human IgE, and the corresponding birch pollen allergen Bet v 1 at a molecular level. Methods We expressed 4 CD23 variants. One variant comprised the full extracellular portion of CD23, including the stalk and head domain; 1 variant was identical with the first, except for an amino acid exchange in the stalk region abolishing the N-linked glycosylation site; and 2 variants represented the head domain, 1 complete and 1 truncated. The 4 CD23 variants were purified as monomeric and structurally folded proteins, as demonstrated by gel filtration and circular dichroism. By using a human IgE mAb, the corresponding allergen Bet v 1, and a panel of antibodies specific for peptides spanning the CD23 surface, both binding and inhibition assays and negative stain electron microscopy were performed. Results A hitherto unknown IgE-binding site was mapped on the stalk region of CD23, and the non–N-glycosylated monomeric version of CD23 was superior in IgE binding compared with glycosylated CD23. Furthermore, we demonstrated that a therapeutic anti-IgE antibody, omalizumab, which inhibits IgE binding to FcεRI, also inhibited IgE binding to CD23. Conclusion Our results provide a new model for the CD23-IgE interaction. We show that the stalk region of CD23 is crucially involved in IgE binding and that the interaction can be blocked by the therapeutic anti-IgE antibody omalizumab.
New influenza A viruses with pandemic potential periodically emerge due to viral genomic reassortment. In the face of pandemic threats, production of conventional egg-based vaccines is time consuming ...and of limited capacity. We have developed in this study a novel DNA vaccine in which viral hemagglutinin (HA) is bivalently targeted to MHC class II (MHC II) molecules on APCs. Following DNA vaccination, transfected cells secreted vaccine proteins that bound MHC II on APCs and initiated adaptive immune responses. A single DNA immunization induced within 8 d protective levels of strain-specific Abs and also cross-reactive T cells. During the Mexican flu pandemic, a targeted DNA vaccine (HA from A/California/07/2009) was generated within 3 wk after the HA sequences were published online. These results suggest that MHC II-targeted DNA vaccines could play a role in situations of pandemic threats. The vaccine principle should be extendable to other infectious diseases.
Electron tomography is a technique for three-dimensional reconstruction, that is widely used for imaging macromolecules, macromolecular assemblies or whole cells. Combined with cryo-electron ...microscopy, it is capable of visualizing structural detail in a state close to in vivo conditions in the cell. In electron tomography, micrographs are taken while tilting the specimen to different angles about a fixed axis. Due to mechanical constraints, the angular tilt range is limited. As a consequence, the reconstruction of a 3D image is missing data, which for a single axis tilt series is called the “missing wedge”, a region in reciprocal space where Fourier coefficients cannot be obtained experimentally. Tomographic data is analyzed by extracting subvolumes from the raw tomograms, by alignment of the extracted subvolumes, multivariate data analysis, classification, and class-averaging, which results in an increased signal-to-noise ratio and substantial data reduction. Subvolume analysis is a valuable tool to discriminate heterogeneous populations of macromolecules, or conformations of a macromolecule or macromolecular assembly as well as to characterize interactions between macromolecules. However, this analysis is hampered by the lack of data in the original tomograms caused by the missing wedge. Here, we report enhancements of our subvolume processing protocols in which the problem of the missing data in reciprocal space is addressed by using constrained correlation and weighted averaging in reciprocal space. These procedures are applied to the analysis of myosin V and simian immunodeficiency virus (SIV) envelope spikes. We also investigate the effect of the missing wedge on image classification and establish limits of reliability by model calculations with generated phantoms.
A detailed understanding of the morphology of the HIV-1 envelope (Env) spike is key to understanding viral pathogenesis and for informed vaccine design. We have previously presented a cryoelectron ...microscopic tomogram (cryoET) of the Env spikes on SIV virions. Several structural features were noted in the gp120 head and gp41 stalk regions. Perhaps most notable was the presence of three splayed legs projecting obliquely from the base of the spike head toward the viral membrane. Subsequently, a second 3D image of SIV spikes, also obtained by cryoET, was published by another group which featured a compact vertical stalk. We now report the cryoET analysis of HIV-1 virion-associated Env spikes using enhanced analytical cryoET procedures. More than 2,000 Env spike volumes were initially selected, aligned, and sorted into structural classes using algorithms that compensate for the "missing wedge" and do not impose any symmetry. The results show varying morphologies between structural classes: some classes showed trimers in the head domains; nearly all showed two or three legs, though unambiguous three-fold symmetry was not observed either in the heads or the legs. Subsequently, clearer evidence of trimeric head domains and three splayed legs emerged when head and leg volumes were independently aligned and classified. These data show that HIV-1, like SIV, also displays the tripod-like leg configuration, and, unexpectedly, shows considerable gp41 leg flexibility/heteromorphology. The tripod-like model for gp41 is consistent with, and helps explain, many of the unique biophysical and immunological features of this region.
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