Molecular Basis for the High Degree of Antigenic Cross-Reactivity between Hepatitis B Virus Capsids (HBcAg) and Dimeric Capsid-Related Protein (HBeAg): Insights into the Enigmatic Nature of the e-Antigen

The hepatitis B virus core gene codes for two closely related antigens: a 21-kDa protein that forms dimers that assemble as multimegadalton capsids, and a 17-kDa protein that also forms dimers but that do not assemble. The proteins, respectively referred to as core antigen (HBcAg) and e-antigen (HBeAg), share a sequence of 149 residues but have different amino- and carboxy-termini. Their structural and serological relationship has long been unclear. With insights gained from recent structural studies on immune complexes of the capsids, the relationship was reassessed using recombinant forms of the antigens and a panel of monoclonal antibodies (mAbs) commonly believed to discriminate between core and e-antigen. Surface plasmon resonance (SPR) was used to measure the affinities, in contrast to previous studies that used more error-prone and less sensitive plate-type assays. Four of the six mAbs did not discriminate between core and e-antigen, nor did they discriminate between e-antigen and dimers of dissociated core antigen capsids. One mAb (3120) was specific for assembled capsids and one (e6) was specific for unassembled dimers. Epitope valency of the e-antigen was also studied, using a sandwich SPR assay where e-antigen was captured with one mAb and probed with a second. The e-antigen is often considered to be a monomeric protein on the basis of monovalent reactivity with antibody pairs specific for either an α or β epitope (in a prior nomenclature for e-antigen specificity). This model, however, is incorrect, because recombinant e-antigen is a stable dimer and its apparent monovalency is due to steric blockage. This was proven by the formation of a 2:1 Fab e6-e-antigen complex. These results suggest new approaches for the isolation of the authentic e-antigen, its biological assay, and its stabilization as an immune complex for structural studies.     

Structural and immunologic characterization of Ara h 1, a major peanut allergen

Allergic reactions to peanuts and tree nuts are major causes of anaphylaxis in the United States. We compare different properties of natural and recombinant versions of Ara h 1, a major peanut allergen, through structural, immunologic, and bioinformatics analyses. Small angle x-ray scattering studies show that natural Ara h 1 forms higher molecular weight aggregates in solution. In contrast, the full-length recombinant protein is partially unfolded and exists as a monomer. The crystal structure of the Ara h 1 core (residues 170-586) shows that the central part of the allergen has a bicupin fold, which is in agreement with our bioinformatics analysis. In its crystalline state, the core region of Ara h 1 forms trimeric assemblies, while in solution the protein exists as higher molecular weight assemblies. This finding reveals that the residues forming the core region of the protein are sufficient for formation of Ara h 1 trimers and higher order oligomers. Natural and recombinant variants of proteins tested in in vitro gastric and duodenal digestion assays show that the natural protein is the most stable form, followed by the recombinant Ara h 1 core fragment and the full-length recombinant protein. Additionally, IgE binding studies reveal that the natural and recombinant allergens have different patterns of interaction with IgE antibodies. The molecular basis of cross-reactivity between vicilin allergens is also elucidated.     

A dimer of the lymphoid protein RAG1 recognizes the recombination signal sequence and the complex stably incorporates the high mobility group protein HMG2.

RAG1 and RAG2 are the two lymphoid-specific proteins required for the cleavage of DNA sequences known as the recombination signal sequences (RSSs) flanking V, D or J regions of the antigen-binding genes. Previous studies have shown that RAG1 alone is capable of binding to the RSS, whereas RAG2 only binds as a RAG1/RAG2 complex. We have expressed recombinant core RAG1 (amino acids 384-1008) in Escherichia coli and demonstrated catalytic activity when combined with RAG2. This protein was then used to determine its oligomeric forms and the dissociation constant of binding to the RSS. Electrophoretic mobility shift assays show that up to three oligomeric complexes of core RAG1 form with a single RSS. Core RAG1 was found to exist as a dimer both when free in solution and as the minimal species bound to the RSS. Competition assays show that RAG1 recognizes both the conserved nonamer and heptamer sequences of the RSS. Zinc analysis shows the core to contain two zinc ions. The purified RAG1 protein overexpressed in E.coli exhibited the expected cleavage activity when combined with RAG2 purified from transfected 293T cells. The high mobility group protein HMG2 is stably incorporated into the recombinant RAG1/RSS complex and can increase the affinity of RAG1 for the RSS in the absence of RAG2.     

Crystal structures of protein glutaminase and its pro forms converted into enzyme-substrate complex

Protein glutaminase, which converts a protein glutamine residue to a glutamate residue, is expected to be useful as a new food-processing enzyme. The crystal structures of the mature and pro forms of the enzyme were refined at 1.15 and 1.73 ? resolution, respectively. The overall structure of the mature enzyme has a weak homology to the core domain of human transglutaminase-2. The catalytic triad (Cys-His-Asp) common to transglutaminases and cysteine proteases is located in the bottom of the active site pocket. The structure of the recombinant pro form shows that a short loop between S2 and S3 in the proregion covers and interacts with the active site of the mature region, mimicking the protein substrate of the enzyme. Ala-47 is located just above the pocket of the active site. Two mutant structures (A47Q-1 and A47Q-2) refined at 1.5 ? resolution were found to correspond to the enzyme-substrate complex and an S-acyl intermediate. Based on these structures, the catalytic mechanism of protein glutaminase is proposed.     

Generation of Biologically Active Multi-Sialylated Recombinant Human EPOFc in Plants

Hyperglycosylated proteins are more stable, show increased serum half-life and less sensitivity to proteolysis compared to non-sialylated forms. This applies particularly to recombinant human erythropoietin (rhEPO). Recent progress in N-glycoengineering of non-mammalian expression hosts resulted in in vivo protein sialylation at great homogeneity. However the synthesis of multi-sialylated N-glycans is so far restricted to mammalian cells. Here we used a plant based expression system to accomplish multi-antennary protein sialylation. A human erythropoietin fusion protein (EPOFc) was transiently expressed in Nicotiana benthamiana ΔXTFT, a glycosylation mutant that lacks plant specific N-glycan residues. cDNA of the hormone was co-delivered into plants with the necessary genes for (i) branching (ii) β1,4-galactosylation as well as for the (iii) synthesis, transport and transfer of sialic acid. This resulted in the production of recombinant EPOFc carrying bi- tri- and tetra-sialylated complex N-glycans. The formation of this highly complex oligosaccharide structure required the coordinated expression of 11 human proteins acting in different subcellular compartments at different stages of the glycosylation pathway. In vitro receptor binding assays demonstrate the generation of biologically active molecules. We demonstrate the in planta synthesis of one of the most complex mammalian glycoforms pointing to an outstanding high degree of tolerance to changes in the glycosylation pathway in plants.     

Cell-associated activation of latent transforming growth factor-β by calpain

Transforming growth factor-β (TGF-β) is normally secreted in a latent form, and plasmin-mediated proteolytic cleavage of latency-associated peptide (LAP), a component of latent TGF-β complex that makes the complex inactive, activates latent TGF-β. In the present study, we investigated the possible involvement of calpain, one of the cysteine proteases, in the activation of latent TGF-β. When recombinant latent TGF-β was incubated with calpain (1–10 u/ml) in a test tube, calpain cleaved LAP and released mature TGF-β from the latent complex. When calpain was applied to cultured bovine capillary endothelial (BCE) cells, a low concentration of calpain (0.05–0.1 u/ml) inhibited the migration and proliferation of the cells, and these inhibitory effects were abrogated by anti-TGF-β antibody as well as by calpain inhibitor peptide, but not by α₂-antiplasmin, a specific inhibitor of plasmin. Active TGF-β was detected in the conditioned medium of BCE cells collected in the presence of calpain. Chemical cross-linking of ¹²⁵I-calpain to BCE cells followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that calpain bound to the cell surface through chondroitinase ABC-sensitive proteoglycan. In addition, treatment of the BCE cells with chondroitinase ABC abrogated the inhibitory effect of calpain on the migration of these cells. Our data thus suggest that calpain is able to activate latent TGF-β through a mechanism independent of plasmin. This activation is efficient in the presence of cells, and calpain binds to the cell surface via proteoglycan and activates latent TGF-β, which is targeted to the same surface. J. Cell. Physiol. 174:186–193, 1998. © 1998 Wiley-Liss, Inc.     

Histones H3/H4 form a tight complex with the inner nuclear membrane protein LBR and heterochromatin protein 1

We have recently shown that heterochromatin protein 1 (HP1) interacts with the nuclear envelope in an acetylation-dependent manner. Using purified components and in vitro assays, we now demonstrate that HP1 forms a quaternary complex with the inner nuclear membrane protein LBR and a sub-set of core histones. This complex involves histone H3/H4 oligomers, which mediate binding of LBR to HP1 and cross-link these two proteins that do not interact directly with each other. Consistent with previous observations, HP1 and LBR binding to core histones is strongly inhibited when H3/H4 are modified by recombinant CREB-binding protein, revealing a new mechanism for anchoring domains of under-acetylated chromatin to the inner nuclear membrane.     

X-ray structure of the PHF core C-terminus: insight into the folding of the intrinsically disordered protein tau in Alzheimer's disease

The major constituent of Alzheimer's disease paired helical filaments (PHF) core is intrinsically disordered protein (IDP) tau. In spite of a considerable effort, insoluble character of PHF together with inherent physical properties of IDP tau have precluded so far reconstruction of PHF 3D structure by X-ray crystallography or NMR spectroscopy. Here we present first crystallographic study of PHF core C-terminus. Using monoclonal antibody MN423 specific to the tertiary structure of the PHF core, the in vivo PHF structure was imprinted into recombinant core PHF tau. Crystallization of the complex led to determination of the structure of the core PHF tau protein fragment 386TDHGAE391 at 1.65A resolution. Structural analysis suggests important role of the core PHF C-terminus for PHF assembly. It is reasonable to expect that this approach will help to reveal the structural principles underlying the tau protein assembly into PHF and possibly will facilitate rationale drug design for inhibition of Alzheimer neurofibrillary changes.     

Three-dimensional structural analysis of recombinant rotavirus-like particles with intact and amino-terminal-deleted VP2: implications for the architecture of the VP2 capsid layer.

Rotaviruses are the leading cause of severe infantile gastroenteritis worldwide. These viruses are large, complex icosahedral particles consisting of three concentric capsid layers enclosing a genome of eleven segments of double-stranded RNA (dsRNA). The amino terminus of the innermost capsid protein VP2 possesses a nonspecific single-stranded RNA and dsRNA binding activity, and the amino terminus is also essential for the incorporation of the polymerase enzyme VP1 and guanylyltransferase VP3 into the core of the virion. Biochemical and structural studies have suggested that VP2, and especially the amino terminus, appears to act as a scaffold for proper assembly of the components of the viral core. To locate the amino terminus of VP2 within the core, we have used electron cryomicroscopy and image reconstruction to determine the three-dimensional structures of recombinant virus-like particles that contain either full-length or amino-terminal-deleted forms of VP2 coexpressed with the intermediate capsid protein VP6. A comparison of these structures indicates two significant changes along the inner surface of VP2 in the structure lacking the amino terminus: a loss of mass adjacent to the fivefold axes and a redistribution of mass along the fivefold axes. Examination of the VP2 layer suggests that the proteins are arranged as dimers of 120 quasi-equivalent molecules, with each dimer extending between neighboring fivefold axes. Our results indicate that the amino termini of both quasi-equivalent VP2 molecules are located near the icosahedral vertices.     

Influence of an ER-retention signal on the N-glycosylation of recombinant human α-l-iduronidase generated in seeds of Arabidopsis

Processes associated with late events of N-glycosylation within the plant Golgi complex are a major limitation to the use of plant-based systems to produce recombinant pharmaceutical proteins for parenteral administration. Specifically, sugars added to the N-glycans of a recombinant protein during glycan maturation to complex forms (e.g. β1,2 xylose and α1,3 fucose) can render the product immunogenic. In order to avoid these sugars, the human enzyme α-L-iduronidase (IDUA, EC 3.2.1.76), with a C-terminal ER-retention sequence SEKDEL, was expressed in seeds of complex-glycan-deficient (cgl) mutant and wild-type (Col-0) Arabidopsis thaliana, under the control of regulatory (5'-, signal-peptide-encoding-, and 3'-) sequences from the arcelin 5-I gene of Phaseolus vulgaris (cgl-IDUA-SEKDEL and Col-IDUA-SEKDEL, respectively). The SEKDEL motif had no adverse effect on the specific activity of the purified enzyme. Surprisingly, the majority of the N-glycans of Col-IDUA-SEKDEL were complex N-glycans (i.e. contained xylose and/or fucose) (88 %), whereas complex N-glycans comprised a much lower proportion of the N-glycans of cgl-IDUA-SEKDEL (26 %), in which high-mannose forms were predominant. In contrast to the non-chimeric IDUA of cgl seeds, which is mainly secreted into the extracellular spaces, the addition of the SEKDEL sequence to human recombinant IDUA expressed in the same background led to retention of the protein in ER-derived vesicles/compartments and its partial localization in protein storage vacuoles. Our data support the contention that the use of a C-terminal ER retention motif as an effective strategy to prevent or reduce complex N-glycan formation, is protein specific.     

Ferredoxin-dependent glutamate synthase moonlights in plant sulfolipid biosynthesis by forming a complex with SQD1

UDP-sulfoquinovose synthase, SQD1, catalyzes the transfer of sulfite to UDP-glucose giving rise to UDP-sulfoquinovose, which is the head group donor for the biosynthesis of the plant sulfolipid sulfoquinovosyldiacylglyerol. The native SQD1 enzyme of spinach exists as a 250 kDa heteroprotein complex with much higher affinity for the substrate sulfite than the recombinant SQD1 protein itself. The SQD1 protein co-purified with nine proteins. Likely binding partners included rubisco activase, HSP70, and ferredoxin-dependent glutamate synthase (FdGOGAT). While the first two proteins are known to interact with many other proteins, the identification of FdGOGAT was most intriguing because this 160kDa protein contains an FMN cofactor known to bind sulfite in vitro. Using different constructs expressing recombinant forms of the multidomain protein FdGOGAT, it was demonstrated that the FMN-binding domain of FdGOGAT is essential for specific binding of the protein to SQD1. A model suggests that FdGOGAT could channel sulfite to SQD1.     

Expression analysis of the nucleocytoplasmic lectin 'Orysata' from rice in Pichia pastoris

The Oryza sativa lectin, abbreviated Orysata, is a mannose-specific, jacalin-related lectin expressed in rice plants after exposure to certain stress conditions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin-related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS/PAGE and western blot analysis showed that the recombinant lectin exists in two molecular forms. Far western blot analysis revealed that the 23 kDa lectin polypeptide contains an N-glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high-mannose structures, Man9-11 glycans being the most abundant. Glycan array analysis showed that Orysata interacts with high-mannose as well as with more complex N-glycan structures. Orysata has potent anti-human immunodeficiency virus and anti-respiratory syncytial virus activity in cell culture compared with other jacalin-related lectins.     

Low-molecular-mass proteins in cyanobacterial photosystem II: identification of psbH and psbK gene products by N-terminal sequencing

The O2-evolving photosystem II core complex was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus Copeland. Analysis by SDS-polyacrylamide gel electrophoresis revealed that the complex contained at least seven low-molecular-mass proteins in addition to the well characterized CP47 apoprotein, CP43 apoprotein, 33 kDa extrinsic protein, D1 protein, D2 protein and large subunit of cytochrome b-559. The separation of these low-molecular-mass proteins were very similar between cyanobacterial and higher plant PS II. N-terminal sequences of the 6.5 kDa and 3.9 kDa proteins of cyanobacterial core complex were determined after blotting to a polyvinylidene difluoride membrane. The sequence of the 6.5 kDa protein showed high homology with an internal sequence of plant psbH gene product, so-called 10 kDa phosphoprotein, but did not conserve the Thr residue which is specifically phosphorylated in plants. The sequence of the 3.9 kDa protein corresponded to the K protein of higher plants (mature form of psbK gene product). These results indicate that the products of both psbH and psbK genes are present in cyanobacterial PS II as well as being associated with the O2-evolving core complex.     

猪流行性腹泻病毒流行株S蛋白高变区B细胞线性表位肽的鉴定

【背景】对猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)经典毒株与流行毒株S蛋白氨基酸序列比对显示,变异位点主要集中在S蛋白的N端。【目的】鉴定PEDV流行毒株S蛋白高变区(S10A,1-496 aa)的B细胞线性表位肽,特别是流行毒株特异性B细胞表位肽。【方法】以SDS-PAGE割胶纯化大肠杆菌表达的PEDV流行株SD2014 S蛋白高变区片段(S10ASD2014),免疫新西兰大白兔制备多克隆抗体;在E. coli中谷胱甘肽巯基转移酶(Glutathione S-transferase,GST)融合表达覆盖S10ASD2014序列全长且彼此重叠8个氨基酸残基的系列16肽。以制备的抗S10ASD2014多抗为一抗,通过Western blot筛选系列16肽中的阳性反应性16肽,鉴定S10ASD2014上的B细胞线性表位肽;利用抗PEDV经典毒株(CV777)S蛋白高变区片段(S10ACV777)多抗血清鉴定S10ASD2014上的保守性B细胞线性表位肽。【结果】重组表达的S10ASD2014相对分子质量约为72 kD;纯化的S10ASD2014表位肽能被PEDV阳性血清识别。制备的抗S10ASD2014多抗可以识别PEDV DR13弱毒株。利用抗S10ASD2014血清和抗S10ACV777血清从61个GST融合表达的16肽中鉴定到28个阳性反应16肽,其中13个为2种血清共同识别16肽。对24株PEDV不同亚群代表毒株的对应区域进行同源性分析表明2个阳性16肽为保守性表位肽。【结论】确定了PEDV流行株SD2014 S蛋白高变区的B细胞线性表位肽,B细胞线性表位肽,特别是流行毒株特异性表位肽的确定有助于了解S蛋白的结构与功能,为建立以表位为基础的PEDV感染检测方法打下良好基础。     

电子显微镜观察SNARE核心复合体的结构

作为神经突触中介导囊泡融合的核心蛋白,SNARE(soluble NSF attachment protein receptors)复合体由VAMP、syntaxin和SNAP25三种蛋白组成。而由其可溶性部分组成的SNARE核心复合体是发挥融合功能的关键部分。通过表达SNARE蛋白,应用分子筛和离子交换柱等纯化方法,重组得到了高纯度的SNARE核心复合体,并分析了其在水溶液中的稳定性。最后,采用负染电镜观察方法得到了该复合体的二维平均结构。在水溶液中,SNARE复合体核心部分为2.5 nm宽、12 nm长的棒状结构。     

Crystal Structure of the Human Short Coiled Coil Protein and Insights into SCOC-FEZ1 Complex Formation

The short coiled coil protein (SCOC) forms a complex with fasciculation and elongation protein zeta 1 (FEZ1). This complex is involved in autophagy regulation. We determined the crystal structure of the coiled coil domain of human SCOC at 2.7 Å resolution. SCOC forms a parallel left handed coiled coil dimer. We observed two distinct dimers in the crystal structure, which shows that SCOC is conformationally flexible. This plasticity is due to the high incidence of polar and charged residues at the core a/d-heptad positions. We prepared two double mutants, where these core residues were mutated to either leucines or valines (E93V/K97L and N125L/N132V). These mutations led to a dramatic increase in stability and change of oligomerisation state. The oligomerisation state of the mutants was characterized by multi-angle laser light scattering and native mass spectrometry measurements. The E93V/K97 mutant forms a trimer and the N125L/N132V mutant is a tetramer. We further demonstrate that SCOC forms a stable homogeneous complex with the coiled coil domain of FEZ1. SCOC dimerization and the SCOC surface residue R117 are important for this interaction.     

Visualization of an FtsZ ring in chloroplasts of Lilium longiflorum leaves.

FtsZ is a bacterial division protein which forms a ring at the leading edge of the cell division site. To date, a hypothesis that the plant FtsZ forms the same structure in chloroplast division is proposed, but has not been demonstrated yet. In this study, recombinant LlFtsZ (Lilium longiflorum FtsZ) protein was produced from a previously isolated ftsZ cDNA clone [Mori and Tanaka (2000) Protoplasma 214: 57] and used to raise polyclonal anti-LlFtsZ antibodies in rabbits. In immunoblot analysis with the total protein extracted from L. longiflorum leaves, purified antibodies specifically recognized LlFtsZ whose molecular mass was approximately 43 kDa. This size corresponded to that of the recombinant LlFtsZ protein lacking N-terminal sequence, which suggests that the full-length LlFtsZ translation product has a putative N-terminal signal peptide. Moreover, fluorescent and electron microscopy revealed that the anti-LlFtsZ antibodies recognized ring structures at stromal side of the constriction point of dividing chloroplasts. Here, we show direct evidence that FtsZ ring is involved in chloroplast division.     

Characterization of subunit-specific interactions in a double-stranded RNA virus: Raman difference spectroscopy of the phi6 procapsid

The icosahedral core of a double-stranded (ds) RNA virus hosts RNA-dependent polymerase activity and provides the molecular machinery for RNA packaging. The stringent requirements of dsRNA metabolism may explain the similarities observed in core architecture among a broad spectrum of dsRNA viruses, from the mammalian rotaviruses to the Pseudomonas bacteriophage phi6. Although the structure of the assembled core has been described in atomic detail for Reoviridae (blue tongue virus and reovirus), the molecular mechanism of assembly has not been characterized in terms of conformational changes and key interactions of protein constituents. In the present study, we address such questions through the application of Raman spectroscopy to an in vitro core assembly system--the procapsid of phi6. The phi6 procapsid, which comprises multiple copies of viral proteins P1 (copy number 120), P2 (12), P4 (72), and P7 (60), represents a precursor of the core that is devoid of RNA. Raman signatures of the procapsid, its purified recombinant core protein components, and purified sub-assemblies lacking either one or two of the protein components have been obtained and interpreted. The major procapsid protein (P1), which forms the skeletal frame of the core, is an elongated and monomeric molecule of high alpha-helical content. The fold of the core RNA polymerase (P2) is also mostly alpha-helical. On the other hand, the folds of both the procapsid accessory protein (P7) and RNA-packaging ATPase (P4) are of the alpha/beta type. Raman difference spectra show that conformational changes occur upon interaction of P1 with either P4 or P7 in the procapsid. These changes involve substantial ordering of the polypeptide backbone. Conversely, conformations of procapsid subunits are not significantly affected by interactions with P2. An assembly model is proposed in which P1 induces alpha-helix in P4 during formation of the nucleation complex. Subsequently, the partially disordered P7 subunit is folded within the context of the procapsid shell.