Structural Basis for Antibody Recognition of Lipid A: INSIGHTS TO POLYSPECIFICITY TOWARD SINGLE-STRANDED DNA*

Septic shock is a leading cause of death, and it results from an inflammatory cascade triggered by the presence of microbial products in the blood. Certain LPS from Gram-negative bacteria are very potent inducers and are responsible for a high percentage of septic shock cases. Despite decades of research, mAbs specific for lipid A (the endotoxic principle of LPS) have not been successfully developed into a clinical treatment for sepsis. To understand the molecular basis for the observed inability to translate in vitro specificity for lipid A into clinical potential, the structures of antigen-binding fragments of mAbs S1–15 and A6 have been determined both in complex with lipid A carbohydrate backbone and in the unliganded form. The two antibodies have separate germ line origins that generate two markedly different combining-site pockets that are complementary both in shape and charge to the antigen. mAb A6 binds lipid A through both variable light and heavy chain residues, whereas S1–15 utilizes exclusively the variable heavy chain. Both antibodies bind lipid A such that the GlcN-O6 attachment point for the core oligosaccharide is buried in the combining site, which explains the lack of LPS recognition. Longstanding reports of polyspecificity of anti-lipid A antibodies toward single-stranded DNA combined with observed homology of S1–15 and A6 and the reports of several single-stranded DNA-specific mAbs prompted the determination of the structure of S1–15 in complex with single-stranded DNA fragments, which may provide clues about the genesis of autoimmune diseases such as systemic lupus erythematosus, thyroiditis, and rheumatic autoimmune diseases.     

Regulation of Phosphorylation of tau by Protein Kinases in Rat Brain

Microtubule associated protein tau is abnormally hyperphosphorylated in Alzheimer disease (AD) brain. To investigate the role of protein kinases involved in this lesion, metabolically active slices made from brains of adult rats were treated with or without various specific kinase activators in oxygenated artificial cerebrospinal fluid. The basal kinase activities of protein kinase-A (PKA), CaM Kinase II and GSK-3 were stimulated more than two-fold by isoproterenol, bradykinin and wortmannin, respectively. We found that cdk5 activity was co-stimulated with PKA by isoproterenol. Sequential activation of PKA (+cdk5), CaM Kinase II and GSK-3 produced hyperphosphorylation of tau at Ser-198/Ser-199/Ser-202, Ser-214, Thr-231/Ser-235, Ser-262, Ser-396/Ser-404 and Ser-422 sites. Like AD P-tau, the P-tau from brain slices bound to normal tau and its binding to tubulin was inhibited. These studies suggest that PKA, cdk5, CaM Kinase II and GSK-3 are involved in the regulation of phosphorylation of tau and that AD-type phosphorylation of tau is probably a product of the synergistic action of two or more of these kinases.     

Structural Basis for Neutralization and Protection by a Zika Virus-Specific Human Antibody

1. Download high-res image (354KB)
2. Download full-size image

     

Structural Basis of Epitope Recognition by Heavy-Chain Camelid Antibodies

Truncated versions of heavy-chain antibodies (HCAbs) from camelids, also termed nanobodies, comprise only one-tenth the mass of conventional antibodies, yet retain similar, high binding affinities for the antigens. Here we analyze a large data set of nanobody–antigen crystal structures and investigate how nanobody–antigen recognition compares to the one by conventional antibodies. We find that nanobody paratopes are enriched in aromatic residues just like conventional antibodies, but additionally, they also bear a more hydrophobic character. Most striking differences were observed in the characteristics of the antigen's epitope. Unlike conventional antibodies, nanobodies bind to more rigid, concave, conserved and structured epitopes enriched with aromatic residues. Nanobodies establish fewer interactions with the antigens compared to conventional antibodies, and we speculate that high binding affinities are achieved due to less unfavorable conformational and more favorable solvation entropy contributions. We observed that interactions with antigen are mediated not only by three CDR loops but also by numerous residues from the nanobody framework. These residues are not distributed uniformly; rather, they are concentrated into four structurally distinct regions and mediate mostly charged interactions. Our findings suggest that in some respects nanobody–antigen interactions are more similar to the general protein–protein interactions rather than antibody–antigen interactions.     

Structural Insights into the Neutralization Properties of the Fully Human,Anti-interferon Monoclonal Antibody Sifalimumab

We report the three-dimensional structure of human interferon α-2A (IFN-α2A) bound to the Fab fragment of a therapeutic monoclonal antibody (sifalimumab; IgG1/κ). The structure of the corresponding complex was solved at a resolution of 3.0 Å using molecular replacement and constitutes the first reported structure of a human type I IFN bound to a therapeutic antibody. This study revealed the major contribution made by the first complementarity-determining region in each of sifalimumab light and heavy chains. These data also provided the molecular basis for sifalimumab mechanism of action. We propose that its interferon-neutralizing properties are the result of direct competition for IFN-α2A binding to the IFN receptor subunit 1 (IFNAR1) and do not involve inhibiting IFN-α2A binding to the IFN receptor subunit 2 (IFNAR2).     

Structural Basis for the Recognition of Human Cytomegalovirus Glycoprotein B by a Neutralizing Human Antibody

Human cytomegalovirus (HCMV) infections are life-threating to people with a compromised or immature immune system. Upon adhesion, fusion of the virus envelope with the host cell is initiated. In this step, the viral glycoprotein gB is considered to represent the major fusogen. Here, we present for the first time structural data on the binding of an anti-herpes virus antibody and describe the atomic interactions between the antigenic domain Dom-II of HCMV gB and the Fab fragment of the human antibody SM5-1. The crystal structure shows that SM5-1 binds Dom-II almost exclusively via only two CDRs, namely light chain CDR L1 and a 22-residue-long heavy chain CDR H3. Two contiguous segments of Dom-II are targeted by SM5-1, and the combining site includes a hydrophobic pocket on the Dom-II surface that is only partially filled by CDR H3 residues. SM5-1 belongs to a series of sequence-homologous anti-HCMV gB monoclonal antibodies that were isolated from the same donor at a single time point and that represent different maturation states. Analysis of amino acid substitutions in these antibodies in combination with molecular dynamics simulations show that key contributors to the picomolar affinity of SM5-1 do not directly interact with the antigen but significantly reduce the flexibility of CDR H3 in the bound and unbound state of SM5-1 through intramolecular side chain interactions. Thus, these residues most likely alleviate unfavorable binding entropies associated with extra-long CDR H3s, and this might represent a common strategy during antibody maturation. Models of entire HCMV gB in different conformational states hint that SM5-1 neutralizes HCMV either by blocking the pre- to postfusion transition of gB or by precluding the interaction with additional effectors such as the gH/gL complex.     

Structural Basis for Antigen Recognition by Transglutaminase 2-specific Autoantibodies in Celiac Disease

Antibodies to the autoantigen transglutaminase 2 (TG2) are a hallmark of celiac disease. We have studied the interaction between TG2 and an anti-TG2 antibody (679-14-E06) derived from a single gut IgA plasma cell of a celiac disease patient. The antibody recognizes one of four identified epitopes targeted by antibodies of plasma cells of the disease lesion. The binding interface was identified by small angle x-ray scattering, ab initio and rigid body modeling using the known crystal structure of TG2 and the crystal structure of the antibody Fab fragment, which was solved at 2.4 Å resolution. The result was confirmed by testing binding of the antibody to TG2 mutants by ELISA and surface plasmon resonance. TG2 residues Arg-116 and His-134 were identified to be critical for binding of 679-14-E06 as well as other epitope 1 antibodies. In contrast, antibodies directed toward the two other main epitopes (epitopes 2 and 3) were not affected by these mutations. Molecular dynamics simulations suggest interactions of 679-14-E06 with the N-terminal domain of TG2 via the CDR2 and CDR3 loops of the heavy chain and the CDR2 loop of the light chain. In addition there were contacts of the framework 3 region of the heavy chain with the catalytic domain of TG2. The results provide an explanation for the biased usage of certain heavy and light chain gene segments by epitope 1-specific antibodies in celiac disease.     

Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody

Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.     

Epitope mapping and structural basis for the recognition of phosphorylated tau by the anti‐tau antibody AT8

Microtubule‐associated protein tau becomes abnormally phosphorylated in Alzheimer's disease and other tauopathies and forms aggregates of paired helical filaments (PHF‐tau). AT8 is a PHF‐tau‐specific monoclonal antibody that is a commonly used marker of neuropathology because of its recognition of abnormally phosphorylated tau. Previous reports described the AT8 epitope to include pS202/pT205. Our studies support and extend previous findings by also identifying pS208 as part of the binding epitope. We characterized the phosphoepitope of AT8 through both peptide binding studies and costructures with phosphopeptides. From the cocrystal structure of AT8 Fab with the diphosphorylated (pS202/pT205) peptide, it appeared that an additional phosphorylation at S208 would also be accommodated by AT8. Phosphopeptide binding studies showed that AT8 bound to the triply phosphorylated tau peptide (pS202/pT205/pS208) 30‐fold stronger than to the pS202/pT205 peptide, supporting the role of pS208 in AT8 recognition. We also show that the binding kinetics of the triply phosphorylated peptide pS202/pT205/pS208 was remarkably similar to that of PHF‐tau. The costructure of AT8 Fab with a pS202/pT205/pS208 peptide shows that the interaction interface involves all six CDRs and tau residues 202–209. All three phosphorylation sites are recognized by AT8, with pT205 acting as the anchor. Crystallization of the Fab/peptide complex under acidic conditions shows that CDR‐L2 is prone to unfolding and precludes peptide binding, and may suggest a general instability in the antibody. Proteins 2016; 84:427–434. © 2016 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

微小RNA在tau蛋白过度磷酸化中的作用

阿尔茨海默病(Alzheimer’s Disease, AD)是一种慢性神经系统退行性疾病,AD的主要病理表现为脑组织中的老年斑和神经纤维缠结,老年斑的主要成分是异常积聚的β-淀粉样蛋白,过度磷酸化的tau蛋白是神经纤维缠结的主要成分。研究发现AD患者脑内微小RNA表达异常,且证据表明微小RNA参与β-淀粉样蛋白过量生成和tau蛋白过度磷酸化等Alzheimer样病理机制,在AD的发病中起着重要作用。本文就微小RNA在tau蛋白过度磷酸化中的作用及机制进行概述。     

Involvement of Both the N-Glycan-relevant and N-Glycan-irrelevant Structural Elements in the Recognition of Human Milk Lactoferrin by the 1CF11 Monoclonal Antibody

Monoclonal antibody 1CF11 has been suggested to specifically recognize a certain carbohydrate epitope shared by glycoproteins in human external secretions. We examined the effect of cleaving the polypeptide backbone and removing N-linked oligosaccharides on the reactivity with 1CF11 of human milk lactoferrin (hLf) to elucidate the structural features of the 1CF11 epitope. We reveal by treating hLF with trypsin and/or N-glycosidase that both the N-glycan-relevant and N-glycan-irrelevant structural elements were involved in the recognition of hLf by 1CF11.     

Structural Basis for Cell-Wall Recognition by Bacteriophage PBC5 Endolysin

1. Download : Download high-res image (208KB)
2. Download : Download full-size image

     

Neuroglobin attenuates Alzheimer-like tau hyperphosphorylation by activating Akt signaling

Neuroglobin (Ngb) is a recently identified member of hemoglobin family, distributed mainly in central and peripheral nervous systems. Recent studies suggest that Ngb can protect neural cells from β-amyloid-induced toxicity in Alzheimer disease (AD). Hyperphosphorylation of tau is another characterized pathological hallmark in the AD brains; however, it is not reported whether Ngb also affects tau phosphorylation. In this study, we found that the level of Ngb was significantly reduced in Tg2576 mice (a recognized mouse model of AD) and TgMAPt mice, and the level of Ngb was negatively correlated with tau phosphorylation. Over-expression of Ngb attenuates tau hyperphosphorylation at multiple AD-related sites induced by up-regulation of glycogen synthase kinase-3β (GSK-3β), a crucial tau kinase. While Ngb activates Akt and thus inhibits GSK-3β, simultaneously inhibition of Akt abolishes the effects of Ngb on GSK-3β inhibition and tau hyperphosphorylation. Our data indicate that Ngb may attenuate tau hyperphosphorylation through activating Akt signaling pathway, implying a therapeutic target for AD.     

Structural Basis for Antigenic Peptide Recognition and Processing by Endoplasmic Reticulum (ER) Aminopeptidase 2

Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias.     

Structural Basis for the Initiation of Glycosaminoglycan Biosynthesis by Human Xylosyltransferase 1

1. Download high-res image (206KB)
2. Download full-size image

     

抗人肝素酶单克隆抗体的制备与鉴定

目的:肝素酶在白细胞游走和恶性肿瘤转移的过程中发挥重要作用,肝素酶抗体的制备对于自身免疫病和肿瘤的良恶性鉴别诊断具有重要意义。制备抗人肝素酶单克隆抗体,用于肝素酶的研究及临床恶性肿瘤的鉴别诊断。方法:通过杂交瘤技术将分泌抗人肝素酶单抗的小鼠B细胞与小鼠骨髓瘤细胞Sp2/0融合,获得稳定分泌抗人肝素酶单抗的杂交瘤细胞;用有限稀释法获得单克隆,以重组人肝素酶及含肝素酶的血小板裂解液对抗体进行Western印迹检测。结果:Western印迹结果显示制备的单抗与人肝素酶具有特异性免疫识别特性。结论:获得了能够特异性免疫识别人肝素酶的分泌性抗人肝素酶单克隆抗体。     

Unique Occurrence of the 1CF11 Carbohydrate Epitope in Primate Saliva

We examined a large number of individual human and animal saliva samples for the reactivity with 1CF11, a mouse monoclonal antibody previously produced for the characterization of human milk mucin and apparently recognizing a certain carbohydrate antigenic structure shared by various human glycoproteins in secretions. The results obtained here confirm the unique occurrence of 1CF11 epitope in each and every saliva sample from humans and Old world monkeys as well, though a vast variety was observed among individual saliva samples in the immunological reactivity with 1CF11.     

人源单克隆抗人免疫缺陷病毒1型抗体Fab段基因的获得

应用噬苏体抗体库技术有效地筛选出了多株抗ＨＩＶ－１人源单克隆抗体。以逆转录聚合酶链反应（ＲＴ－ＰＣＲ）从ＨＩＶ－１感染者外周血淋巴细胞中扩增抗体轻重链可变区基因,插入载体ｐＣＯＭＢ３,建立噬菌体抗体库。分别以ＨＩＶ－１ｇｐ１２０和ｇｐ１６０为固相抗原,经过多轮筛选,从中获得了多株抗ＨＩＶ－１ｇｐ４１、ｇｐ１２０和ｇｐ１６０的单克隆抗体Ｆａｂ段基因。抗ＨＩＶ特异性噬菌体抗体随抗体库的筛选高度富集,抗     

Structural characterization of monoclonal antibodies targeting C-terminal Ser404 region of phosphorylated tau protein

Targeting tau with immunotherapies is currently the most common approach taken in clinical trials of patients with Alzheimer’s disease. The most prominent pathological feature of tau is its hyperphosphorylation, which may cause the protein to aggregate into toxic assemblies that collectively lead to neurodegeneration. Of the phospho-epitopes, the region around Ser³⁹⁶/Ser⁴⁰⁴ has received particular attention for therapeutic targeting because of its prominence and stability in diseased tissue. Herein, we present the antigen-binding fragment (Fab)/epitope complex structures of three different monoclonal antibodies (mAbs) that target the pSer⁴⁰⁴ tau epitope region. Most notably, these structures reveal an antigen conformation similar to a previously described pathogenic tau epitope, pSer⁴²², which was shown to have a β-strand structure that may be linked to the seeding core in tau oligomers. In addition, we have previously reported on the similarly ordered conformation observed in a pSer³⁹⁶ epitope, which is in tandem with pSer⁴⁰⁴. Our data are the first Fab structures of mAbs bound to this epitope region of the tau protein and support the existence of proteopathic tau conformations stabilized by specific phosphorylation events that are viable targets for immune modulation.     

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.