Structural and Functional Characterization of a Cross-Reactive Dengue Virus Neutralizing Antibody that Recognizes a Cryptic Epitope

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Mapping and Characterization of a Sequential Epitope on the Rabies Virus Glycoprotein Which Is Recognized by a Neutralizing Monoclonal Antibody,RG719

We have established a murine hybridoma cell line RG719 which produces a rabies virus-neutralizing IgM-type monoclonal antibody (referred to as MAb RG719). Immunoblot analysis indicated that the antibody recognized a sequential epitope of G protein. Among four rabies virus strains tested, the antigenicity to MAb RG719 was absent from the Nishigahara strain, while the other three strains (HEP, ERA and CVS) reacted to the MAb. Studies with deletion mutants of the G protein indicated that the epitope was located in a middle region of the primary structure of G protein, ranging from position 242 to 300. By comparing the estimated amino acid sequence of the four strains, we found in this region two amino acids (at positions 263 and 291) which are common to three of those strains but are not shared by the Nishigahara strain. The site-directed point mutagenesis revealed that replacement of phenylalanine-263 by leucine destroyed the epitope of the HEP G protein, while the epitope was generated on the Nishigahara G protein whose leucine-263 was replaced by phenylalanine. These observations suggest that phenylalanine-263 is essential for constructing the epitope for MAb RG719. The synthetic 20-mer peptide produced by mimicking the amino acid sequence (ranging from amino acid positions 249 to 268) of the presumed epitope region was shown to bind specifically to MAb RG719 and also to raise the virus-neutralizing antibodies in rabbits. Vaccination with the HEP vaccine produced in Japan induced in humans and rabbits production of significant amounts of the antibodies which reacted with the 20-mer peptide.     

Characterization of a Discontinuous Neutralizing Epitope on Glycoprotein B of Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and newborn infants infected in utero. The viral envelope glycoprotein B (gB) is an attractive molecule for active vaccination and passive immunoprophylaxis and therapy. Using human monoclonal antibodies (MAbs), we have recently identified antigenic region 4 (AD-4) on gB as an important target for neutralizing antibodies. AD-4 is formed by a discontinuous sequence comprising amino acids 121 to 132 and 344 to 438 of gB of HCMV strain AD169. To map epitopes for human antibodies on this protein domain, we used a three-dimensional (3D) model of HCMV gB to identify surface-exposed amino acids on AD-4 and selected juxtaposed residues for alanine scans. A tyrosine (Y) at position 364 and a lysine (K) at position 379 (the YK epitope), which are immediate neighbors on the AD-4 surface, were found to be essential for binding of the human MAbs. Recognition of AD-4 by sera from HCMV-infected individuals also was largely dependent on these two residues, indicating a general importance for the antibody response against AD-4. A panel of AD-4 recombinant viruses harboring mutations at the crucial antibody binding sites was generated. The viruses showed significantly reduced susceptibility to neutralization by AD-4-specific MAbs or polyclonal AD-4-specific antibodies, indicating that the YK epitope is dominant for the AD-4-specific neutralizing antibody response during infection. To our knowledge, this is the first molecular identification of a functional discontinuous epitope on HCMV gB. Induction of antibodies specific for this epitope may be a desirable goal following vaccination with gB.     

Structural Basis of Differential Neutralization of DENV-1 Genotypes by an Antibody that Recognizes a Cryptic Epitope

We previously developed a panel of neutralizing monoclonal antibodies against Dengue virus (DENV)-1, of which few exhibited inhibitory activity against all DENV-1 genotypes. This finding is consistent with reports observing variable neutralization of different DENV strains and genotypes using serum from individuals that experienced natural infection or immunization. Herein, we describe the crystal structures of DENV1-E111 bound to a novel CC′ loop epitope on domain III (DIII) of the E protein from two different DENV-1 genotypes. Docking of our structure onto the available cryo-electron microscopy models of DENV virions revealed that the DENV1-E111 epitope was inaccessible, suggesting that this antibody recognizes an uncharacterized virus conformation. While the affinity of binding between DENV1-E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC′ loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development.     

Monoclonal Antibody #5-2-26 Recognizes the Phosphatase-Sensitive Epitope of Rabies Virus Nucleoprotein

We prepared monoclonal antibodies (MAbs) against the rabies virus N protein, among which one antibody (MAb 5-2-26) was shown to lack reactivity with the phosphatase-treated N protein. The MAb was able to recognize the sodium dodecyl sulfate (SDS)-denatured N protein. The MAb did not recognize the N-protein analogues produced in Escherichia coli (E. coli), indicating that the N-gene products were not normally processed in E. coli after translation. On the other hand, the MAb reacted normally with N-gene products produced in COS-7 cells, but not with those produced in the presence of K-252a (a protein kinase inhibitor of a broad spectrum). The MAb displayed weak cross-reactivity with the Triton-insoluble network structures composed of several components, while another phosphoprotein (M1) of the virus was not recognized at all. These results suggest that MAb 5-2-26 preferentially recognizes a phosphatase-sensitive linear epitope of N protein, which may enable further investigations to be conducted on the mechanism of N-protein phosphorylation and its role(s) in virus replication.     

Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils

Amyloid fibrils are associated with many maladies, including Alzheimer’s disease (AD). The isolation of amyloids from natural materials is very challenging because the extreme structural stability of amyloid fibrils makes it difficult to apply conventional protein science protocols to their purification. A protocol to isolate and detect amyloids is desired for the diagnosis of amyloid diseases and for the identification of new functional amyloids. Our aim was to develop a protocol to purify amyloid from organisms, based on the particular characteristics of the amyloid fold, such as its resistance to proteolysis and its capacity to be recognized by specific conformational antibodies. We used a two-step strategy with proteolytic digestion as the first step followed by immunoprecipitation using the amyloid conformational antibody LOC. We tested the efficacy of this method using as models amyloid fibrils produced in vitro, tissue extracts from C. elegans that overexpress Aβ peptide, and cerebrospinal fluid (CSF) from patients diagnosed with AD. We were able to immunoprecipitate Aβ_1–40 amyloid fibrils, produced in vitro and then added to complex biological extracts, but not α-synuclein and gelsolin fibrils. This method was useful for isolating amyloid fibrils from tissue homogenates from a C. elegans AD model, especially from aged worms. Although we were able to capture picogram quantities of Aβ_1–40 amyloid fibrils produced in vitro when added to complex biological solutions, we could not detect any Aβ amyloid aggregates in CSF from AD patients. Our results show that although immunoprecipitation using the LOC antibody is useful for isolating Aβ_1–40 amyloid fibrils, it fails to capture fibrils of other amyloidogenic proteins, such as α-synuclein and gelsolin. Additional research might be needed to improve the affinity of these amyloid conformational antibodies for an array of amyloid fibrils without compromising their selectivity before application of this protocol to the isolation of amyloids.     

A Monoclonal Antibody which Recognizes a Glycosaminoglycan Epitope in Both Dermatan Sulfate and Chondroitin Sulfate Proteoglycans of Human Skin

Studies have been initiated to identify various cell surface and matrix components of normal human skin through the production and characterization of murine monoclonal antibodies. One such antibody, termed PG-4, identifies both cell surface and matrix antigens in extracts of human foetal and adult skin as the dermatan sulfate proteoglycans, decorin and biglycan, and the chondroitin sulfate proteoglycan versican. Treatment of proteoglycans with chondroitinases completely abolishes immunoreactivity for all of these antigens which suggests that the epitope resides within their glycosaminoglycan chains. Further evidence for the carbohydrate nature of the epitope derives from competition studies where protein-free chondroitin sulfate chains from shark cartilage react strongly; however, chondroitin sulfate chains from bovine tracheal cartilage fail to exhibit a significant reactivity, an indication that the epitope, although present in some chondroitin sulfate chains, does not consist of random chondroitin 4- or 6-sulfate disaccharides. The presence of the epitope on dermatan sulfate chains and on decorin was also demonstrated using competition assays. Thus, PG-4 belongs to a class of antibodies that recognize native epitopes located within glycosaminoglycan chains. It differs from previously described antibodies in this class in that it identifies both chondroitin sulfate and dermatan sulfate proteoglycans. These characteristics make PG-4 a useful monoclonal antibody probe to identify the total population of proteoglycans in human skin.     

A Cryptic Polyreactive Antibody Recognizes Distinct Clades of HIV-1 Glycoprotein 120 by an Identical Binding Mechanism

Polyreactive antibodies play an important role for neutralization of human immunodeficiency virus (HIV). In addition to intrinsic polyreactive antibodies, the immune system of healthy individuals contains antibodies with cryptic polyreactivity. These antibodies acquire promiscuous antigen binding potential post-translationally, after exposure to various redox-active substances such as reactive oxygen species, iron ions, and heme. Here, we characterized the interaction of a prototypic human antibody that acquires binding potential to glycoprotein (gp) 120 after exposure to heme. The kinetic and thermodynamic analyses of interaction of the polyreactive antibody with distinct clades of gp120 demonstrated that the antigen-binding promiscuity of the antibody compensates for the molecular heterogeneity of the target antigen. Thus, the polyreactive antibody recognized divergent gp120 clades with similar values of the binding kinetics and quantitatively identical changes in the activation thermodynamic parameters. Moreover, this antibody utilized the same type of noncovalent forces for formation of complexes with gp120. In contrast, HIV-1-neutralizing antibodies isolated from HIV-1-infected individuals, F425 B4a1 and b12, demonstrated different binding behavior upon interaction with distinct variants of gp120. This study contributes to a better understanding of the physiological role and binding mechanism of antibodies with cryptic polyreactivity. Moreover, this study might be of relevance for understanding the basic aspects of HIV-1 interaction with human antibodies.     

狂犬病毒糖蛋白的克隆表达及其在中和抗体检测中的应用

目的:制备基因工程表达的狂犬病毒糖蛋白优势表位抗原,并评价其在疫苗免疫后中和抗体检测中的应用价值。方法:TRIzol法从狂犬病疫苗中提取总RNA,经RT-PCR获得糖蛋白目的基因片段,构建相应的原核表达质粒,转化大肠杆菌HB101,诱导表达获得纯化重组蛋白,以重组蛋白作为包被抗原,初步建立检测糖蛋白中和抗体的ELISA方法。结果:获得狂犬病毒糖蛋白优势表位区段抗原,建立了糖蛋白中和抗体ELISA检测方法。该检测方法对59例健康献血员血浆样本检测特异性为98.31%(58/59),接种狂犬疫苗免疫个体血浆样本抗体阳性率为98.95%(94/95)。结论:基因工程表达的狂犬病毒糖蛋白优势表位抗原可用于人接种狂犬疫苗后疫苗免疫效果评价。     

Monoclonal Antibody Tor 23 Recognizes a Determinant of a Presynaptic Acetylcholinesterase

A significant proportion of the acetylcholinesterase that is present in the electric organ of Torpedo californica exists as a presynaptic membrane molecule. The monoclonal antibody Tor 23 binds the Torpedo presynaptic nerve membrane where it recognizes a polypeptide of 68,000 daltons. Our present studies indicate that Tor 23 identifies acetylcholinesterase. From the homogenates of Torpedo nerve terminals, Tor 23 immunoprecipitates measurable esterase activity. Esterase precipitation was not observed with no Tor 23 added; nor was it observed with any other test antibodies, including other Tor antibodies, in particular, Tor 70, which binds, as does Tor 23, to the presynaptic nerve membrane. The esterase activity was specific for acetylcholinesterase. Our studies indicate the molecule defined by Tor 23 has the solubility properties described for that of presynaptic acetylcholinesterase: it is soluble in detergent-treated electroplax homogenates and insoluble in high-salt extractions. In sections of Torpedo back muscle, both nerve and endplate acetylcholinesterase can be detected histochemically. Tor 23 localizes to the nerve and is not clustered at the endplate. The utility of the antibody Tor 23 thus includes biochemical and histological analyses of the multiple forms of acetylcholinesterase.     

Partial Purification and Characterization of a Glycoprotein Cell Fusion Hormone from Griffithsia pacifica, a Red Alga

In filaments of the red alga Griffithsia, dead intercalary cells are replaced by the process of cell repair by cell fusion. This process is coordinated by a morphogenetic cell fusion hormone, rhodomorphin, which accelerates cell division and induces the production of a specialized repair cell. We have isolated rhodomorphin from Griffithsia pacifica Kylin and have purified it by concanavalin A affinity chromatography, hydrophobic interaction chromatography, and gel filtration chromatography. This molecule binds specifically to concanavalin A, is proteinase sensitive, and is inactivated by short treatments at temperatures of 50°C or above. It therefore appears that rhodomorphin from G. pacifica is a glycoprotein; its molecular weight, as estimated by gel filtration, is approximately 14,000.     

Mapping Paratope on Antithrombotic Antibody 6B4 to Epitope on Platelet Glycoprotein Ibalpha via Molecular Dynamic Simulations

Binding of platelet receptor glycoprotein Ibα (GPIbα) to the A1 domain of von Willebrand factor (vWF) is a critical step in both physiologic hemostasis and pathologic thrombosis, for initiating platelet adhesion to subendothelium of blood vessels at sites of vascular injury. Gain-of-function mutations in GPIbα contribute to an abnormally high-affinity binding of platelets to vWF and can lead to thrombosis, an accurate complication causing heart attack and stroke. Of various antithrombotic monoclonal antibodies (mAbs) targeting human GPIbα, 6B4 is a potent one to inhibit the interaction between GPIbα and vWF-A1 under static and flow conditions. Mapping paratope to epitope with mutagenesis experiments, a traditional route in researches of these antithrombotic mAbs, is usually expensive and time-consuming. Here, we suggested a novel computational procedure, which combines with homology modeling, rigid body docking, free and steered molecular dynamics (MD) simulations, to identify key paratope residues on 6B4 and their partners on GPIbα, with hypothesis that the stable hydrogen bonds and salt bridges are the important linkers between paratope and epitope residues. Based on a best constructed model of 6B4 bound with GPIbα, the survival ratios and rupture times of all detected hydrogen bonds and salt bridges in binding site were examined via free and steered MD simulations and regarded as indices of thermal and mechanical stabilizations of the bonds, respectively. Five principal paratope residues with their partners were predicted with their high survival ratios and/or long rupture times of involved hydrogen bonds, or with their hydrogen bond stabilization indices ranked in top 5. Exciting, the present results were in good agreement with previous mutagenesis experiment data, meaning a wide application prospect of our novel computational procedure on researches of molecular of basis of ligand-receptor interactions, various antithrombotic mAbs and other antibodies as well as theoretically design of biomolecular drugs.     

Human OS-9, a Lectin Required for Glycoprotein Endoplasmic Reticulum-associated Degradation, Recognizes Mannose-trimmed N-Glycans

In the endoplasmic reticulum (ER), lectins and processing enzymes are involved in quality control of newly synthesized proteins for productive folding as well as in the ER-associated degradation (ERAD) of misfolded proteins. ER quality control requires the recognition and modification of the N-linked oligosaccharides attached to glycoproteins. Mannose trimming from the N-glycans plays an important role in targeting of misfolded glycoproteins for ERAD. Recently, two mammalian lectins, OS-9 and XTP3-B, which contain mannose 6-phosphate receptor homology domains, were reported to be involved in ER quality control. Here, we examined the requirement for human OS-9 (hOS-9) lectin activity in degradation of the glycosylated ERAD substrate NHK, a genetic variant of α1-antitrypsin. Using frontal affinity chromatography, we demonstrated that the recombinant hOS-9 mannose 6-phosphate receptor homology domain specifically binds N-glycans lacking the terminal mannose from the C branch in vitro. To examine the specificity of OS-9 recognition of N-glycans in vivo, we modified the oligosaccharide structures on NHK by overexpressing ER α1,2-mannosidase I or EDEM3 and examined the effect of these modifications on NHK degradation in combination with small interfering RNA-mediated knockdown of hOS-9. The ability of hOS-9 to enhance glycoprotein ERAD depended on the N-glycan structures on NHK, consistent with the frontal affinity chromatography results. Thus, we propose a model for mannose trimming and the requirement for hOS-9 lectin activity in glycoprotein ERAD in which N-glycans lacking the terminal mannose from the C branch are recognized by hOS-9 and targeted for degradation.Recognition and sorting of improperly folded proteins is essential to cell survival, and hence, an elaborate quality control system is found in cells. ER⁴ quality control is well characterized with respect to the N-linked oligosaccharides regulating the folding and degradation of newly synthesized proteins in the ER (1). Immediately after polypeptides enter the ER, Glc₃Man₉GlcNAc₂ (G3M9) precursor oligosaccharides are covalently attached and subsequently processed. Terminally misfolded proteins are removed from the ER by the ERAD machinery (1–4). Aberrant conformers are recognized, retrotranslocated to the cytosol, and degraded by the ubiquitin-proteasome system (5, 6). Processing of mannose residues from the N-linked oligosaccharides acts as a timer for the recognition of misfolded glycoproteins in the ER lumen (1, 7). ER α1,2-mannosidase I (ER ManI) in mammals and ER α-mannosidase in yeast preferentially trim mannose residues from the middle branch of N-glycans, generating the Man₈GlcNAc₂ (M8) isomer B (M8B) (8). In mammals, further mannose processing is required as a signal for degradation (1, 9, 10), whereas the presence of M8B is sufficient to signal degradation in yeast (11). The postulated lectin EDEMs in mammals, their yeast homolog Htm1p/Mnl1p, and the yeast MRH domain-containing lectin Yos9p have all been proposed to recognize glycoproteins targeted for degradation (12).The role of Yos9p in glycoprotein ERAD was identified using a genetic screen in Saccharomyces cerevisiae (13). Yos9p, a homolog of hOS-9, contains an MRH domain (14) and functions as a lectin. Yos9p recognizes substrates of the ERAD-lumenal pathway (15–17), generating a large ER membrane complex containing the Hrd1p-Hrd3p ubiquitin ligase core complex (18–20). The M8B and Man₅GlcNAc₂ (M5) N-glycans are predicted to function as ligands for Yos9p (17). Bipartite recognition of both glycan and polypeptide by Yos9p has also been reported (15).Recent studies revealed that two mammalian MRH domain-containing lectins, OS-9 and XTP3-B, are ER luminal proteins involved in ER quality control and form a large complex containing the HRD1-SEL1L ubiquitin-ligase in the ER membrane (21–24). The components of the complex are similar to yeast, suggesting evolutionary conservation, although the molecular mechanisms underlying the role of OS-9 and XTP3-B remain elusive. Studies using lectin mutants have suggested that the MRH domains are required not for binding to ERAD substrates but for interactions with SEL1L (21), which has multiple N-glycans (25, 26). Additionally, lectin activity appears to be dispensable for hOS-9 binding to misfolded glycoproteins (21, 24). Thus, to understand the role of hOS-9 in the ER quality control pathway, the specific carbohydrate structures recognized by the hOS-9 MRH domain need to be identified, and the requirement of the lectin domain in substrate recognition needs to be determined.In the present study we demonstrate that the lectin activity of hOS-9 is required for enhancement of glycoprotein ERAD. We identified the N-glycan structures recognized by the recombinant hOS-9 MRH domain in vitro by frontal affinity chromatography (FAC). Using a model ERAD substrate, NHK (27), we show that the ability of hOS-9 to enhance ERAD in vivo depends on the oligosaccharides present on NHK, consistent with the FAC results.     

抗人呼吸道合胞病毒融合糖蛋白单克隆抗体的制备及体外鉴定

目的:获得能稳定分泌抗人呼吸道合胞病毒(human respiratory syncytial virus, RSV)融合糖蛋白(fusion glycoprotein, F)单克隆抗体(monoclonal antibody, mAb)的杂交瘤细胞株,以期用于RSV感染的早期诊断和被动免疫治疗研究。方法:通过杂交瘤技术制备可特异性识别RSV F的单抗,体外鉴定生物学特性。结果:获得了可分泌抗RSV F蛋白的杂交瘤细胞株F8,体外连续传代培养2个月,能稳定分泌抗体F8,培养上清效价为1∶1000,亲和常数(Ka)为6.8×10⁸ L/mol。F8属IgG1型抗体,可特异性识别RSV F1亚单位的AA 205-222。免疫酶法蚀斑减少中和实验证实F8具有体外中和活性及融合抑制活性。结论:获得具有中和活性的抗RSV F蛋白的单克隆抗体,为RSV感染的早期诊断及被动免疫治疗等奠定了基础。     

Oligosaccharide-Related Epitope Specific for a Brain-Specific Glycoprotein, 1D4 Antigen

The characteristics of glycosylation of a brain-specific glycoprotein, 1D4 antigen, and the epitope recognized by its monoclonal antibody were studied. Removal of high-mannose and hybrid types of N-linked oligosaccharides by treatment with endoglycosidase H converted the molecular mass of the 1D4 antigen from 89 kDa to 78 kDa, but did not affect its reactivity with the 1D4 monoclonal antibody. Removal of all types of N-linked oligosaccharides by treatment with glycopeptidase F or removal of both N- and O-linked oligosaccharides by chemical treatment caused both reduction of the molecular mass of the antigen to 63 kDa and loss of its reactivity with the monoclonal antibody. These results suggest that the 1D4 monoclonal antibody recognizes a complex-type oligosaccharide-related epitope specific for the 1D4 antigen. Results also showed that N-linked glycosylation was not responsible for the charge heterogeneity of the 1D4 antigen. The oligosaccharide chain-related epitope was detected in rat brain but not in mouse, rabbit, or bovine brain, but the 1D4 antigen was recognized in rat and mouse brains with antiserum (polyclonal antibodies). These findings indicate that the oligosaccharide-related epitope is species specific. Furthermore, results with neuraminidase-treated 1D4 antigen indicated that sialic acids were not involved in the oligosaccharide-related epitope. These findings suggest that the 1D4 antigen may have the oligosaccharide structure specific for rat brain and itself.     

Epitope Characterization of an Aromatase Monoclonal Antibody Suitable for the Assessment of Intratumoral Aromatase Activity

Immunohistochemistry is one of the most suitable methods for the detection of intratumoral aromatase in order to identify patients who may respond to aromatase inhibitor therapy in hormone-dependent breast cancer. Previous studies showed statistically significant correlation between results of immnuohistochemistry and biochemical analysis in carcinoma components stained by aromatase monoclonal antibody 677. In this study, determination of the antigenic peptides recognized by aromatase antibodies through epitope mapping, combined with the new knowledge on aromatase-reductase interaction, provide insights for understanding various immunostaining patterns using different aromatase antibodies. Our studies on aromatase-reductase interaction also provided critical information on how aromatase and reductase interact with each other on the endoplasmic reticulum membrane, and identified key residues, including K108 of aromatase, that are involved in the interaction with reductase. Through epitope mapping and taking into consideration the interference with aromatase immunohistochemical staining by NADPH-cytochrome P450 reductase, we demonstrated that monoclonal antibody 677 is a suitable antibody for an assessment of intratumoral aromatase activity in breast cancer patients for making clinical management decisions. These results also provide valuable information to identify new aromatase antibodies for immunohistochemical diagnosis of hormone-dependent breast cancer in future.