Structure of an HIV-1-neutralizing antibody target, the lipid-bound gp41 envelope membrane proximal region trimer.

The membrane proximal external region (MPER) of HIV-1 glycoprotein (gp) 41 is involved in viral-host cell membrane fusion. It contains short amino acid sequences that are binding sites for the HIV-1 broadly neutralizing antibodies 2F5, 4E10, and 10E8, making these binding sites important targets for HIV-1 vaccine development. We report a high-resolution structure of a designed MPER trimer assembled on a detergent micelle. The NMR solution structure of this trimeric domain, designated gp41-M-MAT, shows that the three MPER peptides each adopt symmetric α-helical conformations exposing the amino acid side chains of the antibody binding sites. The helices are closely associated at their N termini, bend between the 2F5 and 4E10 epitopes, and gradually separate toward the C termini, where they associate with the membrane. The mAbs 2F5 and 4E10 bind gp41-M-MAT with nanomolar affinities, consistent with the substantial exposure of their respective epitopes in the trimer structure. The traditional structure determination of gp41-M-MAT using the Xplor-NIH protocol was validated by independently determining the structure using the DISCO sparse-data protocol, which exploits geometric arrangement algorithms that guarantee to compute all structures and assignments that satisfy the data.

In planta production of ELPylated spidroin-based proteins results in non-cytotoxic biopolymers.

BACKGROUND: Spider silk is a tear-resistant and elastic biopolymer that has outstanding mechanical properties. Additionally, exiguous immunogenicity is anticipated for spider silks. Therefore, spider silk represents a potential ideal biomaterial for medical applications. All known spider silk proteins, so-called spidroins, reveal a composite nature of silk-specific units, allowing the recombinant production of individual and combined segments. RESULTS: In this report, a miniaturized spidroin gene, named VSO1 that contains repetitive motifs of MaSp1 has been synthesized and combined to form multimers of distinct lengths, which were heterologously expressed as elastin-like peptide (ELP) fusion proteins in tobacco. The elastic penetration moduli of layered proteins were analyzed for different spidroin-based biopolymers. Moreover, we present the first immunological analysis of synthetic spidroin-based biopolymers. Characterization of the binding behavior of the sera after immunization by competitive ELISA suggested that the humoral immune response is mainly directed against the fusion partner ELP. In addition, cytocompatibility studies with murine embryonic fibroblasts indicated that recombinant spidroin-based biopolymers, in solution or as coated proteins, are well tolerated. CONCLUSION: The results show that spidroin-based biopolymers can induce humoral immune responses that are dependent on the fusion partner and the overall protein structure. Furthermore, cytocompatibility assays gave no indication of spidroin-derived cytotoxicity, suggesting that recombinant produced biopolymers composed of spider silk-like repetitive elements are suitable for biomedical applications.

Differential accessibility of a rotavirus VP6 epitope in trimers comprising type I, II, or III channels as revealed by binding of a human rotavirus VP6-specific antibody.

Previous human antibody studies have shown that the human VH1-46 antibody variable gene segment encodes much of the naturally occurring human B cell response to rotavirus and is directed to virus protein 6 (VP6). It is currently unknown why some of the VH1-46-encoded human VP6 monoclonal antibodies inhibit viral transcription while others do not. In part, there are affinity differences between antibodies that likely affect inhibitory activity, but we also hypothesize that there are differing modes of binding to VP6 that affect the ability to block the transcriptional pore on double-layered particles. Here, we used a hybrid method approach for antibody epitope mapping, including single-particle cryo-electron microscopy (cryo-EM) and enhanced amide hydrogen-deuterium exchange mass spectrometry (DXMS) to determine the location and mode of binding of a VH1-46-encoded antibody, RV6-25. The structure of the RV6-25 antibody-double-layered particle (DLP) complex indicated a very complex binding pattern that revealed subtle differences in accessibility of the VP6 epitope depending on its position in the type I, II, or III channels. These subtle variations in the presentation or accessibility of the RV VP6 capsid layer led to position-specific differences in occupancy for binding of the RV6-25 antibody. The studies also showed that the location of binding of the noninhibitory antibody RV6-25 on the apical surface of RV VP6 head domain does not obstruct the transcription pore upon antibody binding, in contrast to binding of an inhibitory antibody, RV6-26, deeper in the transcriptional pore.

[Glycopolymers of microorganisms: achievements and future research (review)].

This review presents the current literature data on the structure of peptidoglycans, lipopolysaccharides, teichoic acids, the mechanism of biological action of lipopolysaccharides, and the possibility of uising oligosaccharides for creation of glycoconjugate vaccines, as well as promising areas for further research of glycopolymers of microorganisms.

Gamma-delta T-cell responses during subcutaneous Mycobacterium avium subspecies paratuberculosis challenge in sensitized or naive calves using matrix biopolymers.

We have developed a model to explore the early immune response against Mycobacterium avium subspecies paratuberculosis (Map) infection in the bovine calf using subcutaneously placed liquid gel matrix biopolymer (matrigel) containing live Map. Matrigel rapidly polymerizes in vivo, retains recruited cellular infiltrates and soluble immune mediators, and can be rapidly removed 48 hours later and depolymerized for analysis. In this study, we examined early host immune events at matrigel/Map sites; recruited cells were evaluated by histopathology and flow cytometry, and cytokines were measured by flow cytometry, enzyme-linked immunosorbent assay, and Luminex bead immunoassay. Our results demonstrate earlier recruitment of gamma-delta (γδ) T cells to matrigel/Map challenge sites compared to CD4+ T cells. We also show that significantly more γδ T cells were recruited to matrigel/Map sites postinfection day 7 compared to postinfection day 30 and that these cells produced significant amounts of the cytokine interferon gamma. We also provide evidence that peripheral blood-derived γδ T-cell subsets in cattle differentially generate interferon gamma, suggesting distinct roles for these cells. These data provide unique insight into initial antimycobacterial host cellular immune responses following Map infection in calves.

Co-expression of two distinct polysialic acids, α2,8- and α2,9-linked polymers of N-acetylneuraminic acid, in distinct glycoproteins and glycolipids in sea urchin sperm.

Naturally occurring polysialic acid (polySia) structures have a large diversity, primarily arising from the diversity in the sialic acid components as well as in the intersialyl linkages. In 2004, we demonstrated the presence of a new type of polySia, 8-O-sulfated N-acetylneuraminic acid (Neu5Ac) capped α2,9-linked polyNeu5Ac, on the O-glycans of a major 40-80 kDa sialoglycoprotein, flagellasialin, in sea urchin sperm. In this study, we demonstrated that another type of polySia, the α2,8-linked polyNeu5Ac, exclusively occurs on O-glycans of a 190 kDa glycoprotein (190 kDa-gp), whereas the α2,9-linked polyNeu5Ac is exclusively present on flagellasialin. The 190 kDa-gp is localized in both flagellum and head of sperm. We also demonstrated that polysialogangliosides containing the α2,8-linked polyNeu5Ac are present in sperm head. Thus, this study shows two novel features of the occurrence of polySia in nature, the co-localization of polySia with different intersialyl linkages, the α2,8- and α2,9-linkages, in a single cell and the occurrence of α2,8-linked polyNeu5Ac in glycolipids. Anti-α2,8-linked polyNeu5Ac antibody had no effect on fertilization, which contrasted with the previous results that anti-α2,9-linked polyNeu5Ac antibody inhibited sperm motility and fertilization. Based on these properties, distinct functions of α2,8- and α2,9-polySia structures are implicated in fertilization.

Immunostructural evidence for the template mechanism of microtubule nucleation.

Two opposing models have been proposed to explain how the gamma-tubulin ring complex (gammaTuRC) induces microtubule nucleation. In the 'protofilament' model, the gammaTuRC induces nucleation as a partially or completely straightened protofilament that is incorporated longitudinally into the wall of the nascent microtubule, whereas the 'template' model proposes that the gammaTuRC acts as a helical template that constitutes the base of the newly-formed polymer. Here we appraise these two models, using high-resolution structural and immunolocalization methods. We show that components of the gammaTuRC localize to a narrow zone at the extreme minus end of the microtubule and that these ends terminate in a pointed cap. Together, these results strongly favour the template model of microtubule nucleation.

Structure of the gamma-tubulin ring complex: a template for microtubule nucleation.

The gamma-tubulin ring complex (gammaTuRC) is a protein complex of relative molecular mass approximately 2.2 x 10(6) that nucleates microtubules at the centrosome. Here we use electron-microscopic tomography and metal shadowing to examine the structure of isolated Drosophila gammaTuRCs and the ends of microtubules nucleated by gammaTuRCs and by centrosomes. We show that the gammaTuRC is a lockwasher-like structure made up of repeating subunits, topped asymmetrically with a cap. A similar capped ring is also visible at one end of microtubules grown from isolated gammaTuRCs and from centrosomes. Antibodies against gamma-tubulin label microtubule ends, but not walls, in centrosomes. These data are consistent with a template-mediated mechanism for microtubule nucleation by the gammaTuRC.

Immune safety evaluation of polymerized porcine hemoglobin (pPolyHb): a potential red blood cell substitute.

Polymerized Porcine Hemoglobin (pPolyHb), a hemoglobin-based oxygen carrier (HBOC), was developed as a potential red blood substitute for clinical applications. Assessment of its effects on the immune system is an important component of the overall safety evaluation of HBOC. For this purpose, we assessed three inflammation indicators, including complement C3a, IL-6, and TNF-? in cultured cells and in a rat model when pPolyHb was incubated or administrated with the cells/animals. Our results suggested that the levels of these three indicators were not statistically changed upon pPolyHb stimulation, indicating that pPolyHb is not immunotoxic to cells and animals in this aspect.

Analysis of the expression of peptide-major histocompatibility complexes using high affinity soluble divalent T cell receptors.

Understanding the regulation of cell surface expression of specific peptide-major histocompatibility complex (MHC) complexes is hindered by the lack of direct quantitative analyses of specific peptide-MHC complexes. We have developed a direct quantitative biochemical approach by engineering soluble divalent T cell receptor analogues (TCR-Ig) that have high affinity for their cognate peptide-MHC ligands. The generality of this approach was demonstrated by specific staining of peptide-pulsed cells with two different TCR-Ig complexes: one specific for the murine alloantigen 2C, and one specific for a viral peptide from human T lymphocyte virus-1 presented by human histocompatibility leukocyte antigens-A2. Further, using 2C TCR- Ig, a more detailed analysis of the interaction with cognate peptide-MHC complexes revealed several interesting findings. Soluble divalent 2C TCR-Ig detected significant changes in the level of specific antigenic-peptide MHC cell surface expression in cells treated with gamma-interferon (gamma-IFN). Interestingly, the effects of gamma-IFN on expression of specific peptide-MHC complexes recognized by 2C TCR-Ig were distinct from its effects on total H-2 Ld expression; thus, lower doses of gamma-IFN were required to increase expression of cell surface class I MHC complexes than were required for upregulation of expression of specific peptide-MHC complexes. Analysis of the binding of 2C TCR-Ig for specific peptide-MHC ligands unexpectedly revealed that the affinity of the 2C TCR-Ig for the naturally occurring alloreactive, putatively, negatively selecting, complex, dEV-8-H-2 Kbm3, is very low, weaker than 71 microM. The affinity of the 2C TCR for the other naturally occurring, negatively selecting, alloreactive complex, p2Ca-H-2 Ld, is approximately 1000-fold higher. Thus, negatively selecting peptide-MHC complexes do not necessarily have intrinsically high affinity for cognate TCR. These results, uniquely revealed by this analysis, indicate the importance of using high affinity biologically relevant cognates, such as soluble divalent TCR, in furthering our understanding of immune responses.

Immunomodulatory effects of Aureobasidium pullulans SM-2001 exopolymers on the cyclophosphamide-treated mice.

The immunomodulatory effects of exopolymers of Aureobasidium pullulans SM-2001 containing beta-1,3/1,6-glucan were evaluated on the cyclophosphamide (CPA)-treated mice. To induce immunosuppress, 150 and 110 mg/kg of CPA were intraperitoneally injected at 1 and 3 days before start of test material administrations, respectively. Exopolymers were subcutaneously or orally administered in a volume of 10 ml/kg, 4 times; 12-hr intervals from 24 hrs after second treatment of CPA. After treatment of exopolymers, the changes of thymus and spleen weights, splenic amounts of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-10, thymic and splenic CD3+, CD4+, CD8+ and TNF-alpha+ cells were monitored in CPA-treated mice. As results of CPA treatment, dramatical decreases of the CD3+, CD4+, CD8+ and TNF-alpha+ cells were detected in thymus and spleen with decreases of thymus and spleen weights. In addition, decreases of splenic TNF-alpha, IL-1beta and IL-10 contents were also detected at flow cytometrical observations. However, oral and subcutaneous treatment of exopolymers effectively reduced the immunosuppressive changes induced by CPA. Therefore, it is concluded that exopolymers of A. pullulans can be effectively prevent the immunosuppress mediated, at least partially, recruitment of T cells and TNF-alpha+ cells or enhancement of their activity, and can provide effective prevention or treat regimes for the immunosuppress and related diseases such as cancer, sepsis and high-dose chemotherapy or radiotherapy.

The supramolecular organization of fibrillin-rich microfibrils.

We propose a new model for the alignment of fibrillin molecules within fibrillin microfibrils. Automated electron tomography was used to generate three-dimensional microfibril reconstructions to 18.6-A resolution, which revealed many new organizational details of untensioned microfibrils, including heart-shaped beads from which two arms emerge, and interbead diameter variation. Antibody epitope mapping of untensioned microfibrils revealed the juxtaposition of epitopes at the COOH terminus and near the proline-rich region, and of two internal epitopes that would be 42-nm apart in unfolded molecules, which infers intramolecular folding. Colloidal gold binds microfibrils in the absence of antibody. Comparison of colloidal gold and antibody binding sites in untensioned microfibrils and those extended in vitro, and immunofluorescence studies of fibrillin deposition in cell layers, indicate conformation changes and intramolecular folding. Mass mapping shows that, in solution, microfibrils with periodicities of <70 and >140 nm are stable, but periodicities of approximately 100 nm are rare. Microfibrils comprise two in-register filaments with a longitudinal symmetry axis, with eight fibrillin molecules in cross section. We present a model of fibrillin alignment that fits all the data and indicates that microfibril extensibility follows conformation-dependent maturation from an initial head-to-tail alignment to a stable approximately one-third staggered arrangement.

Probing the Origin of the Toxicity of Oligomeric Aggregates of α-Synuclein with Antibodies.

The aggregation of α-synuclein, a protein involved in neurotransmitter release at presynaptic terminals, is associated with a range of highly debilitating neurodegenerative conditions, most notably Parkinson's disease. Intraneuronal inclusion bodies, primarily composed of α-synuclein fibrils, are the major histopathological hallmarks of these disorders, although small oligomeric assemblies are believed to play a crucial role in neuronal impairment. We have probed the mechanism of neurotoxicity of α-synuclein oligomers isolated in vitro using antibodies targeting the N-terminal region of the protein and found that the presence of the antibody resulted in a substantial reduction of the damage induced by the aggregates when incubated with primary cortical neurons and neuroblastoma cells. We observed a similar behavior in vivo using a strain of C. elegans overexpressing α-synuclein, where the aggregation process itself is also partially inhibited as a result of incubation with the antibodies. The similar effects of the antibodies in reducing the toxicity of the aggregated species formed in vitro and in vivo provide evidence for a common origin of cellular impairment induced by α-synuclein aggregates.

Anti-oligomeric single chain variable domain antibody differentially affects huntingtin and alpha-synuclein aggregates.

Huntington's and Parkinson's diseases are both neurodegenerative disorders caused at least in part by misfolding and aggregation of huntingtin (htt) and alpha-synuclein, respectively. Here we use a single chain antibody fragment (scFv) isolated against oligomeric alpha-synuclein to probe similarities and differences between the aggregation and toxic mechanisms of htt and alpha-synuclein. When incubated with htt, the scFv both blocks formation of and promotes dissociation of fibrillar aggregates, but stabilizes formation of cytotoxic oligomeric aggregates. Previous studies with monomeric alpha-synuclein showed the scFv prevented fibrillar aggregation, but blocked toxicity of oligomeric aggregates. These divergent effects suggest the toxic mechanisms of oligomeric aggregates differ among amyloidogenic protein species.

An antigenic epitope of influenza virus nucleoprotein (NP) associated with polymeric forms of NP.

Intracellular influenza virus nucleoprotein (NP) is characterized by a high efficiency of homo-polymers formation, however their antigenic structure is still incompletely known. Herein, we report that RNase-resistant intracellular NP homo-polymers have a highly ordered conformational antigenic epitope, which depends on inter-subunit interactions of monomeric NPs. Our studies have shown that in radioimmunoprecipitation (RIPA) intracellular NP polymers bind mAb N5D3 and RNase does not prevent their mAb binding. In contrast to NP polymers, NP monomeric subunits, obtained by thermo-dissociation of NP polymers, fail to bind the mAb N5D3 in RIPA. At the same time, the in vitro concentration of thermo-denatured monomeric NPs in both soluble and immobilized forms results in NP-NP association, accompanied by renaturation of the N5D3 epitope. The same results were detected by Western blotting, where the pre-denatured NP monomers were concentrated on nitrocellulose into a single 56 kDa band, which then caused NP-NP self-association as well as N5D3 epitope renaturation. Thus, the in vitro renaturation of N5D3 epitope is markedly dependent on NP monomers concentration. The results obtained suggest that in vivo formation and in vitro renaturation of the N5D3 epitope depend on inter-subunit interactions of monomeric NPs and NP-NP interactions influence the antigenic structure of the influenza virus NP polymers.

Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography.

Nucleophosmin (NPM1) is a multifunctional phosphoprotein which plays important roles in diverse biological processes. NPM1 can form homo- or hetero-oligomers through its N-terminal region, and bind DNA and RNA through its C-terminal region. However, the monomer-oligomer distribution of NPM1, and the extent of NPM1 binding and unbinding to RNA in living cells, are not fully understood. In this work, we analysed molecular complexes of NPM1 using size exclusion chromatography. We found that a substantial fraction of NPM1 behaves as an oligomer in HeLa cells. Furthermore, we identified three distinct oligomeric states of NPM1 using molecular characterization techniques such as subcellular localization and RNA binding. Finally, we found that heterozygous expression of a leukemia-associated NPM1 mutant significantly decreases the RNA binding level. Our data demonstrate that size exclusion chromatography provides a powerful tool for analysing NPM1 oligomers.

Linear α-(1 → 6)-d-glucan from Bifidobacterium bifidum BIM В-733D is low molecular mass biopolymer with unique immunochemical properties.

Role of microorganisms in induction of/protection from autoimmune diseases is proven though molecular mechanisms and bacterial/viral/yeast biopolymers responsible for these effects are in the research stage. Autoantobodies (AAbs) to thyroid peroxidase (anti-TPO) and thyroglobulin (anti-Tg) as well as AAbs to transglutaminase 2 (anti-TG2) and antibodies to gliadins (anti-gliadins) are serological markers of autoimmune thyroid disease and celiac disease, respectively, and players in pathogenesis of these autoimmune diseases. In current study, biopolymer of Bifidobacterium bifidum BIM В-733D that interacts selectively with anti-gliadins (Bb-Ganti-gliadins) was isolated by affinity chromatography with anti-gliadins, purified by size exclusion chromatography on TSK 40 gel and identified by NMR as linear α-(1 → 6)-d-glucan with molecular mass about 5000 Da. It was proven that compounds Bb-Ganti-gliadins and Bb-Ganti-TPO/Bb-Ganti-Tg isolated early from the same strain [Kiseleva, E. P. et al., Benef Microbes.2013, 4, 375 -391] are the same substance designated GBb. Its unique immunochemical property is the ability to interact selectively with anti-TPO, anti-Tg, anti-TG2 and anti-gliadins in presence of no less than 10-fold excess of total immunoglobulins of class G (tIgG), as it was proven by ELISA. Synthesis of GBb-bovine serum albumin (GBb-BSA) conjugate is an example of increasing the reliability and reproducibility of ELISA results by mediated immobilization of a polysaccharide covalently attached to a well-adsorbed protein. Taking into account that there are population of bispecific anti-gliadins (anti-gliadins and anti-TG2 simultaneously) we regard our data as first argument in favor of hypothesis that GBb differentiates between human AAbs per se and other human Ig (e.g. antibodies to antigens of infectious agents) due to its binding with a yet unidentified site which is present in the molecules of all AAbs (independently on their specificity) and absent in other human Igs.

Chemical characteristics and immuno-modulating activities of exo-biopolymers produced by Grifola frondosa during submerged fermentation process.

The immuno-modulating activities and chemical characteristics of exo-biopolymer (EX-GF) produced by a submerged mycelial culture of Grifola frondosa were studied. The EX-GF was fractionated into EX-GF-Fr.I, II, and III by Sephadex G-100 gel chromatography. Anti-complementary activity of EX-GF-Fr.III was highest (71.1%) among them, and its activation system occurred through both classical and alternative pathways, where the classical pathway found to be major one. Lysosomal enzyme activity and nitric oxide production ability of macrophage were also found to be mediated by EX-GF-Fr.III. The molecular weight of the EX-GF-Fr.I, II, and III was estimated to be about 163, 40, and 2.8 kDa, respectively. Total sugar and protein contents of the three fractions were 80.3, 61.9 and 89.3%, and 17.3, 35.2, and 10.7%, respectively. The sugar and amino acid compositions of the EX-GF-Fr.I, II, and III were also analyzed in detail.

[Immunogenicity of surface and capsular antigens of Burkholderia].

Study showed that five (C3, C6, C9, C10, C11) out of ten chromatographic fractions of surface and capsular antigens of B. mallei significantly stimulated cell-mediated immunity that manifested in activation of delayed hypersensivity reactions (DHS) and phagocyteability of noncapsulated avirulent strain of B. mallei with added surface and capsular antigenic complexes. Other fractions did not stimulate cell-mediated immunity, furthermore, fraction C8, which contained capsular biopolymer with mass of 200 kD (Ar8), was characterized by immunosuppressive effect on DHS and phagocytosis. Observed stimulation of cell-mediated immunity by fractions referred above has been confirmed by assessment of their protective effects on the model of experimental melioidosis in white rats. Relationship between markers of humoral and cell-mediated immunity, including markers of specific response, was not observed.

Chemical characteristics and immuno-stimulating properties of biopolymers extracted from Acanthopanax sessiliflorus.

During our search for macrophage stimulating compounds from medicinal plants, we isolated biopolymers from Acanthopanax sessiliflorus. Isolated fraction AS-5 showed maximum potential, and stimulated lysosonal enzymatic activity by 230% at 300 microg/ml. The nitric oxide (NO) producing ability of AS-5 100 microg/ml was 58 microM when treated with interferon-gamma and lipopolysaccharide 20 micro/ml. The lymphocyte proliferating effects of isolated biopolymer fractions were also investigated. Highest lymphoproliferative activity (a 2.8-fold enhancement compared to salines treated group was exhibited by AS-3 at 200 micro/ml followed by AS-5 and AS-6. The AS-3 fraction stimulated only T-lymphocytes and had little or no effect on B-lymphocyte proliferation. Partially methylated alditol acetates were prepared to elucidate the glycosyl linkage-compositions of the AS-3 and AS-5 biopolymers, and were analyzed by GC-MS. The AS-3 and AS-5 biopolymer fractions were found to contain 2,3,4-tri-O-methyl-D-glucitol, 2,3,4-tri-O-methyl-D-galacitol 3,4,6-tri-O-methyl-galacitol, 2-O-methyl-arabinitol and 2,4,6-tri-O-methyl-D-glucitol, 2,3,6-tri-O-methyl-D-galacitol linkages, respectively.